y | lef Bo THE CONTEMPORARY “SCIENCE SERIES Cornell Mniversity Library BOUGHT WITH THE INCOME FROM THE SAGE ENDOWMENT FUND THE GIFT OF Henry W. Sage 1891 A... £6.0L€. ee fp | Bacteri id their products. THE CONTEMPORARY SCIENCE SERIES. Epitep sy HAVELOCK ELLIS. BACTERIA AND THEIR PRODUCTS. BACTERIA AND THEIR PRODUCTS. EOP TI PPIVT REIT Y boLES EY BY GERMAN SIMS WOODHEAD, M.D. (EDIN.), Director of the Laboratories of the Conjoint Board of the Royal Colleges of Physicians (Lond.) and Surgeons (Eng.); Formerly Research Scholar of the Honourable Grocers’ Company. WITH 20 PHOTO-MICROGRAPHS AND AN APPENDIX GIVING A SHORT ACCOUNT OF BACTERIOLOGICAL METHODS, AND A DIAGNOSTIC DESCRIPTION OF THE COMMONER BACTERIA. LONDON: WALTER SCOTT, Lrp., 24 WARWICK LANE. CHARLES SCRIBNER’S SONS, 743 & 745 BROADWAY, NEW YORK. PREFACE. oo N the following pages an attempt is made to give some account of the main facts in Bacteriology, and of the life-history of Bacteria and closely allied organisms, and also to discuss the more important theories as to the part played by them in Nature’s Economy ; especially in their relation to the commoner fermentative, putrefactive, and disease pro- cesses. It may be held by some of my readers that too much pro- minence is given to certain questions, whilst others have been relegated to a comparatively obscure position, and that some points have been accentuated, perhaps at the expense of others; but to such criticism it may be answered that whilst some facts carry conviction along with them, others can only be properly appreciated when seen by stage lights. On the other hand, many points have of set purpose been lightly touched upon because experience is constantly bringing home the fact that what is new to-day may be out of date to-morrow. I have, therefore, thought it better, in most instances, to confine myself to an exposition of well- accredited facts and to a discussion of some of the more stable theories. Being privileged to hold a Sanitary Research Scholarship of the Honourable Grocers’ Company for some years, I was enabled to devote considerable time to the study of the relations of Bacteria to Disease, especially in the case of tuberculosis, and many of the interpretations of facts here mentioned are based on observations then made; the re- vi PREFACE. mainder are drawn chiefly from the works enumerated at the end of each section. These lists, however, represent but a small part of the enormous mass of literature dealing with Bacteria and their Products, which has accumulated during the last decade. To Professor Léffler’s admirable work, ‘ Vorlesungen tiber die geschichtliche Entwickelung der Lehre von den Bacterien,” and to Mr. Watson Cheyne’s ‘“ Antiseptic Surgery,” I am specially indebted for much information and guidance in my search for facts and papers dealing with the earlier history of the subject. It may be said of the Appendix that it is given, not with the object of supplying intending workers with every known method of research, and with full descriptions of every organism with which they may have to deal, but simply to enable them to commence work, and to recognize the commoner forms of Bacteria, of which about .one hundred and forty are here described. I take the opportunity of expressing my thanks to Dr. Cartwright Wood, who, whilst revising the proof sheets, has made several valuable suggestions; to my former as- sistant, Mr. Coghill, now of the Royal Veterinary College, and to Mr. Andrew Pringle, both of whom have made for me beautiful photo-micrographs, which, however, can be but imperfectly reproduced by any photo-mechanical process now available. G. S. W. CONTENTS. CHAPTER I. PAGE INTRODUCTION. - . : 1-23 Growth of Subject—Place of Bacteria in Nature—Pure Cuttures —Morphology—Physiology—Protoplasm governed by same laws whether in higher or lower Plants or Animals—Relation of Bacteria to everyday Processes—Fermentations, Butyric, Lactic, Colour, &c.—Brewing—Baking—Kephir making— Flax preparation — Digestion — Putrefaction — Nitrification, Mineralization—Relation of Bacteria to Water Supply—Fil- tration—Sewage—Modes of Transference of Pathogenic Bac- teria from Patient to Patient, or from Water or Earth to Patient. CHAPTER II. WHAT ARE BACTERIA? - : 24-48 Structure — Myco-protein — Limiting Membrane — Gelatinous Capsule—Special Cell Contents—Oxide of Iron—Sulphur— Colouring Material—Flagella—Modes of Multiplication and Development—Division—Rate of Vegetative Multiplication —Endospores—Arthrospores—Classifications of Cohn, Van Tieghem, Zopf, Winter and Rabenhorst, De Bary and Hueppe, Fliigge, Baumgarten, &c. CHAPTER IIL Tue HisToRY OF BACTERIOLOGY ss - 49-74 Earliest Workers—Kircher’s Contagium Animatum—Bacteria in Fermentation, Putrefaction, and Disease—Early Classifica- tions — Miiller — Abiogenesis — Needham — Biogenesis — Bonnet—Spallanzani—Schultz’s Experiments—Schwann— Later Experiments— Pasteur — Bastian—Colour and Fer- mentation — Cohn and Naegeli’s Classification — Henle’s Researches and Postulates—Pasteur’s Researches on Fer- vill CONTENTS. PAGE mentation and Putrefaction—‘* Flower of Wine ”—‘‘ Flower ‘of Vinegar ”—Bacteria as Scavengers—Pasteur on Silkworm Disease, and Wine Disease—Germs killed by Carbolic Acid— Origin of Antiseptic Treatment. CHAPTER IV. BACTERIA AS THE CAUSES OF DISEASE : 75-86 Anthrax — Pollender—-Davaine—Rayer—Laplat and Jaillard’s Observations controverted by Davaine—Pyzmia and Sep- ticeemia— Salisbury on Bacteria as the Cause of certain Fevers — Johanna Liiders’ and Hallier’s Observations on Pleomorphism or Polymorphism—Burdon Sanderson, Hoff- man, and others, state that there is no Connection between Bacteria and the Higher Fungi—Demonstration of Infective Element in Anthrax Blood—Bacteria found in Organs in certain Diseases—Sarcina in Stomach—Specific Organisms— Specific Activities. CHAPTER V. FERMENTATION - 87-114 Fermentation, a Key to the Whole Position—Chemical Fer- mentations — Illustrations — Organic Fermentations — Al- coholic Fermentation—Result of Activity of Living Proto- plasm—Lactic and Butyric Fermentations—Cagniard Latour ——Schwann— Reess—Hansen—Liebig’s Theory of Fermenta- tioh—High Beer—Low Beer—Methed of obtaining Pure Yeasts—Hansen’s Classification of the Saccharomyces—Spore Formation—Film Formation—Character of Species of Sac- charomyces—Metschnikoft's Monospora—Torulz. CHAPTER VI. FERMENTATION (conéznued ) 115-144 Soluble Constituents of Yeast—Action of these: upon Sugar— Conversion of Glycogen in the Liver and other Tissues— Growth of Yeast-Cells in Organic and Inorganic Fluids— Fermentation of Fruit Juice—A®Zrobic and Anzrobic Fer- mentations—Effect of Free Oxygen on Yeast-Cells—Fer- mentation not necessarily equal to Growth of Yeast — Enzyme or Unorganized Ferment a Secondary Function— Function depends partly on Organism, partly on Medium in which it is Growing—Peptonizing Function usually requires presence of Oxygen—Various kinds of Fermentation: Lactic, Urea, Butyric, Ammoniacal, Acetic—Formation of Fatty Acids—Mycoderma Aceti—General Processes of Fermenta- tion —Hoppe-Seyler’s Classification. CONTENTS. CHAPTER VII. PARASITES AND SAPROPHYTES Putrefactive Bacteria — Pigment-forming Bacteria — Enzyme- forming Bacteria—Non-pathogenic, Saprophytic, Parasitic, Pathogenic Bacteria—Sarcina Ventriculi—Leptothrix Buccalis — Facultative Saprophytes — Facultative Pathogenic Para- sites—Anthrax Bacillus—‘‘ Pathogenic,” ‘‘ Parasitic,” ‘* Sa- prophytic,” merely Relative Terms. CHAPTER VIII. CHOLERA : Cholera a Parasitic Disease—Early Observations on the Comma Bacillus — Characters—Methods of Staining— Methods of Isolation—Use of Plate Method in Practical Public Health Work—Tube Cultures—Motility of Cholera Bacilli—Potato, Blood-serum, and Milk Growths—Behaviour in Water and Sewage—Infection of Man through Agency of Bacillus— Early Inoculation Experiments—Koch—Nicati and Rietsch —Macleod—Difficulties—Cholera in Guinea-pigs—Position of Bacilli in Tissues—Presence of Spores doubtful. CHAPTER IX. CHOLERA (continued ) . Pettenkofer’s researches —Saprophytic and Parasitic Stages of Cholera Bacillus — Temperature Conditions — Relation to Epidemics —Moisture—Ground Water — Flushing—Cholera in Shanghai Endemic but Intermittent—Cholera Endemic at the Mouth of the Ganges — Vitality of Cholera in Ohl Cultures—Relation of this to Quiescent Periods during Parts of the Year—Gastro-intestinal Irritations prepare for Cholera —Chinese Vegetables—Cholera Poison formed in the In- testine, absorbed into Body—Inoculation against Cholera —Gamaleia’s Experiments—Germicides useful in attacking the Cholera Bacilli—Water Supply, Pilgrimages, Feasts pre- disposing to Cholera—Quarantine except in Harbours Use- less—Time and Place Dispositions. CHAPTER X. TYPHOID FEVER - - . : ig “ Typhoid Fever a Bacterial Disease—Recklinghausen’s Observa- tions — Klein — Eberth — Klebs—Coats— The Bacillus — Method of Staining—Position in Tissues—Gaffky’s Observa- tions-—Pure Cultures—Excretory Products—Experiments on Animals—Mixed Infections—Action of Light and Heat on Typhoid Bacilli—Pseudo-typhoid Bacilli. PAGE 145-149 150-170 171-193 194-204 x CONTENTS. CHAPTER XI. TUBERCULOSIS Tuberculosis a widespread Disease —- The Tubercle Bacillus — Koch—Baumgarten—Spores seen by Watson Cheyne—Re- lation of Organisms to Tissues—Bacillus in Tuberculosis of Animals—Tubercle Bacilli as Saprophytes—Bacilli cultivated outside the Body by Koch—Methods—Temperature Rela- tions—Cultivation on Different Media—Channels of In- fection — Ransome—Williams—Cornet—Conditions of In- fection—Methods of Disinfection—Tuberculosis at Different Ages—Tubercle in Milk—Diagnosis of Tuberculosis in Cattle — Tuberculous Meat — Koch’s Method of Treatment — Nature of Virus and Mode of Action—Is Immunity con- ferred ?—Koch’s Method a New Departure—Sterile Products cause Marasmus ; Maffucci-—Indications for Treatment. CHAPTER XII. LEPROSY - Distribution of Leprosy—Similarity to Tuberculosis—Description of Disease—“ Tubercular,” ‘* Anzesthetic,”’ “‘ Mixed "—The Leprosy Bacillus—Method of Staining — Position—Leprosy Cells— Bacilli Resistant and Grow Slowly—Cultivation Ex- periments mostly Negative—Theories of Cause of Leprosy. CHAPTER XIII. ACTINOMYCOSIS : Nature. of the Disease—Differences in Cattle, Man, Pig, and Horse—Methods of Preparation and Examination of the Fungus—Microscopic Appearances of Fungus in different Animals — Nature of Clubs — Cultivation Experiments — History of Actinomyces—The Actinomyces a Streptothrix. CHAPTER XIV. GLANDERS : : 3 Glanders — Farcy — Clinical Appearances of the Disease — Chauveau’s observations on Glanders Poison—Léffler and Schiitz—Method of Demonstrating the Glanders Bacillus— The Bacillus—Methods of Cultivation—Glanders in Various Animals—Farcy in Man—Temperature relations of Bacillus— Desiccation—Germicides. PAGE 205-243 244~252 253-261 262-270 CONTENTS. xi CHAPTER XV. PAGE ANTHRAX - 271-286 The Bacillus Anthracis — Early Observations —- Pollender — Davaine—Koch—Pasteur— Methods of Examination—Ap- pearances of Bacillus under Different Conditions — Spore Formation—Non-spore Bearing Bacilli—The Vitality of the Bacillus and of the Spores—Cultivation Experiments—Cover- glass Preparations—Inoculations into Animals—Methods of Infection—Anatomical Characters of Malignant Pustule— Animals Affected—Spores not formed in the Living Body— The Disposal of Anthrax Carcases—Various Disinfectants— Pathogenic and Saprophytic Anthrax—Buchner’s Experi- ments on Anthrax Bacillus and Bacillus Subtilis—Hueppe and Wood’s Experiments. CHAPTER XVI. TETANUS - - 286-206 Tetanus a Specific Infective Disease—A Wound Fever—Organism found by Nicolaier in the Soil taken from Streets and Fields —Experiments on Animals—Symptoms of Disease—Pure Cultivations Obtained—Description of Organisms—Charac- teristic Shape— Spore Formation — Organism Anzrobic — Cultivations—Kitasato’s Method of Cultivating the Organism —The Bacillus found only at the Seat of Inoculation—-Wide Distribution of Spores—Bossano’s Examination of Earth— Vaillard and Vincent’s Observations—Tetanus Bacillus a Facultative Saprophyte—Conditions under which Tetanus is Contracted—Poisoned Arrows. CHAPTER XVII. DIPHTHERIA 297-313 Diphtheria an Infective Disease—The Organism found in the False Membrane in its Deeper Parts — Method of Staining the Bacillus—Characters of the Bacillus—Involution Forms— Cultivation Methods — Appearance of Colonies—Nutrient Media—Results of Inoculation Experiments—Klein’s Bacilli differ somewhat from Léffler’s — Streptococci found in Diphtheria Poison — Extreme Virulence—:Resemblance to snake-bite poison — Toxicity — Predisposing Conditions— Conditions fatal to the Bacillus—Roux and Yersin’s Observa- tions—Fraenkel’s Observations—Attenuated Diphtheria Virus —Increase of Virulence, xii CONTENTS. CHAPTER XVIII. PAGE HYDROPHOBIA. - 7 314-336 Pasteur’s Experiments—Attempts to Demonstrate Micro-organisms —Hydrophobia does not arise Spontaneously—Disease not Confined to Man or Canine Animals—Pasteur’s Early Ex- periments with Saliva Unsuccessful—Successful Experiments —Symptoms of the Disease—Position in which Virulent Material is Found—Different Animals Differently Affected— _ Alteration of Virulence—Method of Preparing Inoculation Material—Description of Experiments—Joseph Meister the First Patient Treated—Method of Treatment now Adopted— Treatment of Wolf Bites—The Time Factor in the Disease— Rationale of Inoculation Method — Filtered Virus Non- Virulent—Method of Inoculation—Description of Depart- er Apparatus and Methods of Working in the Pasteur nstitute. CHAPTER XIX. BACTERIA OF THE MOUTH ~ 336-346 The Mouth a Good Forcing Ground for Bacteria—Food Material —Kinds of Bacteria found in the Mouth by Miller and Vignal —Bacteria in the Teeth in Caries, in Milk Teeth, and in Abscesses—Combination of Lactic Acid with Lime Salts— Organisms Attack Decalcified Base Substance — Artificial Decay—Mode of Invasion of Teeth by Bacteria—Poisonous Saliva-—Micrococcus of Sputum Septiceemia—Pneumococcus —Other Organisms found in the Mouth—Septicemia fol- lowing Slight Operations in the Mouth. CHAPTER XX. Tue BACTERIA OF COLOUR AND PHOSPHORESCENCE > 347-355 Colour-Forming Bacteria— Micrococcus Prodigiosus— Magenta Micrococcus—Beggiotoa Roseopersicina— Bacillus of Blue Milk — Sulphur Pigments — Iron Pigments — Bacillus Fluorescens Putidus—Phosphorescent Bacteria—Six Species — Method of Cultivation— Conditions under which they produce Light. CHAPTER XXI. PoIsoNnous ALKALOIDS AND ALBUMINOIDS~ - - 356-367 Early Observations—Burrows, Kerner, Panum, &c.—Ptomaines Leucomaines—Brieger’s Work—The Alkaloids, Poisonous and Non-Poisonous — General and Local Reactions — Structure and Composition—Sketch of Chemistry—Cholera CONTENTS. xill PAGE Poisons and other Members of this Group—Mussel Poisons : two Classes, viz., those Without Oxygen and those Con- taining Oxygen—Ptomaines the Result of the Activity of Micro-organisms — Léffler’s Experiments on Diphtheria Poison, an Enzyme—Roux and Yersin’s Diastase—Hankin, Albumose—Brieger and Fraenkel, Toxalbumen— Method of Preparation of Albumoses and of Toxalbumens—Near Relationship of Proteid Poisons and the Ptomaines—Martin’s Observations. CHAPTER XXII. VACCINATION s Natural Immunity—Ingrafting of Small-pox—Jenner’s Discovery —Klebs—Pastetr—Chauveau—Grawitz’s Theory—Buchner’s Theory — Metschnikoff’s Work—Greenfield’s Observations on Anthrax —Toussaint’s Vaccinal Fluid—Pasteur’s Classical Experiments—Other Methods of Diminishing Virulence— Chauveau’s Soluble Poison Experiments — Protection by Alkaloids, Albumoses and Toxalbumens — Saprophytic ‘© Anthrax ’—Hueppe and Wood—Antagonism by Blue-pus Products—Immunity not the same as Antagonism—Sum- mative Action of Bacteria. 368-380 CHAPTER XXIII. BACTERIA IN AIR, EARTH, AND WATER 381-396 Spores in the Airin Hospitals—Effects of Currents and Altitude— Direction of Wind—Nature of Country over which it passes —Frankland’s, Carnelley’s, Haldane’s and Petri’s Experi- ments — Few Bacteria in the Air of Sewers — Tyndall’s Glycerine Chamber—Examination of Air—Koch’s Method— Miquel’s Method—Hesse’s Apparatus — Modified Hesse’s Apparatus—Miquel’s Sugar Method—Bacteria in Water— Elect of Standing—Sluggish Movement—Oxidation of Or- ganic Matter in Water—Organisms carried by Sewage— Number of Organisms in Water—Method of Procedure in Analysing Water — Pfuhl’s Flasks — Petri's Dishes — Pe- truschky’s Flask—Plate Cultivations—Cooling Apparatus— Von Esmarch’s Tube Cultures—lffect of Rains, Frosts and Thaws—Relation of Bacteria to Ammonia—Basis on which to determine whether Water is fit for Drinking or not— Filtration—Methcd of Examination of Soil. APPENDIX 397-442 INDICES - . < s : : - 443-459 BACTERIA AND THEIR PRODUCTS. CHAPTER I. INTRODUCTION. Growth of Subject—Place of Bacteria in Nature—Pure Cultures—Morph- ology—Physiology—Protoplasm governed by same laws whether in higher or lower Plants or Animals—Relation of Bacteria to everyday Processes—Fermentations, Butyric, Lactic, Colour, &c.—Brewing— ' Baking—Kephir making—Flax preparation—Digestion—Putrefaction —Nitrification, Mineralization—Relation of Bacteria to Water Supply —Filtration—Sewage—Modes of Transference of Pathogenic Bacteria from Patient to Patient, or from Water or Earth to Patient. ITHIN the last decade bacteria have laid a very strong hold on the thought and imagination of the scientific world, and have come to be looked upon as playing a most important part, not only in the production of disease and in fermentation, but also in many everyday processes hitherto supposed to be dependent on very different causes. In consequence of this, bacteriology has been raised to the dignity of a science, and its ramifications have become so numerous and so wide-spreading that many of the other sciences, and even some of the arts, have been freely pressed into the service of one or other of its branches. The evolution of the science for long went on but slowly ; the study of bacteria remained almost entirely in the hands of the botanists, although now and again scientific medical men, whose powers of observation and deduction were far superior to the methods of experimentation that had been at their command, made shrewd guesses at the causal relationship between the growth of certain bacteria 2 2 BACTERIA. ‘within and without the body and the etiology of certain infective diseases, and certain processes of putrefaction and fermentation. At first bacteria were claimed for both the animal and vegetable kingdoms by the workers in each, and the fortunes of war seemed for long to favour the zoologists ; but after a sharp struggle the fission fungi, as they are called, were relegated to the domain of botany, and now for many years the morphology of these organisms has been an object of most careful study by scientific botanists. Bacteria were, however, so minute that it was impossible to study them very accurately with the older microscopes, and for long the group contained very few species, whilst in addition to the fallacies connected with actual observation of size, form, and methods of reproduction, there were others that resulted from the fact that it was impossible, until com- paratively recent years, to obtain what are now known as pure cultivations; forms that were utterly dissimilar in morphological characters were supposed ‘to be stages of the developmental cycle of the same organism, although they might be, and probably were, really nothing but different organisms that were capable of growing in a common medium. When at length more or less pure cultivations of certain organisms were obtained, botanists: were able to study, in some cases very completely, the life-history and morphology of various forms of bacteria, and great ad- vances “in bacteriological science were made. Still no very accurate observations on the functions or physiological chemical processes associated with the life-history of bacteria and allied forms were made, and many of the biological questions, some of which had already been answered as regards animal and the higher vegetable protoplasm, still remained as in a sealed book so far as this lowly vegetable protoplasm was concerned. In the last ten or twelve years, however, owing to the vast improvements that have been made in, the methods of culti- vation of these organisms, and especially of obtaining what are known as pure cultures, z.¢., cultures that contain a single species of organism only, most valuable data as to the func- tions and biological chemistry of these minute ‘specks of vegetable protoplasm have been rapidly accumulated. Pasteur’s wonderful observations on yeasts first opened up the way for future workers. His practically pure cul- INTRODUCTION. 3 tures were obtained by a kind of physiological separation, to obtain which he fed his yeasts on food specially suited to their nutrition—more suited to their wants, in fact, than to those of any micro-organisms with which they are usually found associated— and they were thus enabled to develop so luxuriantly that they prevented the growth of almost all other organisms, which might have found their way into the nutrient medium along with them. These cultures were comparatively pure, the presence of the small number of other organisms not being sufficient to vitiate the main general results, Klebs (1873) tried to obtain pure cultures by his fractional method, which was simply the removal of those organisms that vegetated most luxuriantly in any special fluid to another .flask, making special cultures of these, and so on until com- paratively pure cultures were obtained. The difficulty, how- ever, was that only the commoner forms could thus be separated and even then they could not be relied upon as being absolutely pure. This method (the ‘‘maas” method) is even now sometimes employed to obtain pure cultivations of cholera bacillus. A drop of the discharge from a cholera patient containing a large number of organisms besides the specific cholera bacillus, when placed in broth which is so prepared as to be specially suited to the nourishment of the cholera organism, soon becomes almost a pure culture, as the cholera bacilli are able to multiply in an extraordinary degree at their ‘‘optimum” temperature and to far outnumber all the other organisms present, and it becomes a much easier matter to obtain from the broth culture a pure culture of the ‘‘comma” bacillus than directly from the cholera discharge itself. It may be said to correspond to the passing of a mixed culture through an animal. Certain organisms—those that are ‘‘ pathogenic *—can grow luxuriantly whilst the others make a much weaker struggle for existence. As soon as it became evident that no further steps of any very great importance could be taken unless pure cultiva- tions were used, various workers set themselves to devise methods by which single organisms might be isolated, and from which a “pure-bred stock” might be raised. Then Roberts (1874) and Cohn (1876) obtained pure cultures of different forms of spore-bearing bacilli, especially the bacillus subtilis, by subjecting the fluid to the heat of 100°C., by which all non-spore-bearing forms were killed off. This method, however, has a very limited application, though combined with other methods, it enabled Kitasato to obtain 4 BACTERIA, pure cultures of the tetanus bacillus, Salomonsen (1876) observing that the dark colour which makes its appearance in defibrinated ox blood always commences as minute points, concluded that each of these patches was due to the changes set up by micro-organisms growing at these points, He therefore drew such defibrinated blood into a capillary tube, and by carefully noting the appearances presented in the tube was able to follow the development of a variety of organisms which, when few in number, were separated by considerable intervals of bright red blood in which no organisms could be seen, though in time these gradually ran together. Lister (1878) and Naegeli (1879) obtained their pure cultivations by a kind of fractionating method, in which they distributed a number of organisms into large quantities of fluid by making greater and greater dilutions of their organisms in broth, until the dilution was such that a single drop did not con- tain more than a single organism, and a series of such “drops introduced into a number of flasks containing sterilized broth gave a large proportion of pure cultivations. With the pure cultivations so obtained the first really accurate ex- periments in connection with the physiological chemistry of bacteria were carried on. This method, however, though a very great advance on any that had hitherto been devised, was somewhat complicated, and it was open to certain other objections (though it is still most useful in many investiga- tions). It was not until Koch, perfecting Klebs’ and Brefeld’s gelatine method, was able to “ fix” the organisms zm szfu as it were, in the nutrient. medium, that the bacteria could be kept isolated, the resulting colonies studied and then removed to other media for further examination. The method was so simple and so reliable that it was eagerly seized upon by those who were interested in the solution, not of -botanical problems merely, but of many questions con- nected with putrefaction, fermentation, and disease that had cropped up so frequently, but which had to be left without a satisfactory answer, and which, in consequence of Davaine’s and Pasteur’s researches, were again assuming such prominence. In the first place, experimenters were able to put to the test the many observations that had been made on both the structure and the function of micro-organisms. Once a pure culture became available, it was an easy matter to determine INTRODUCTION. 5 the conditions under which a special bacterium would grow. It could be transferred from medium to medium ; it could be placed under erobic and anzrobic conditions; or it could be plied with antiseptic or other special reagents, and its behaviour under varying conditions or in the presence of these material could be accurately observed. There was no longer any danger of one form being mistaken for the de- velopmental stage of a totally different organism, and much confusion as regards classification was gradually done away with, though the effects of the observations made on impure cultivations still make themselves felt in the obscurity that enshrouds certain groups which are still met with even in some of the better known classifications. As these pure cultivations were obtained it gradually became more apparent that, for practical purposes, it would be necessary to base our classification on certain specific cha- racters of the organism ; and as in all processes in which bacteria play a part they are usually met with in a single and special form, it was originally found advisable to take these special forms as the diagnostic features in making such classification. The features of form and size, however, were merely superficial characters, and as a more careful study of bacteria was made, especially by the French school, it was found that the biological characters afforded a much more satisfactory basis of classification, especially, however, when taken in conjunction with the morphological features ; so that the media on which they grow, the products to which they give rise, the methods in which they cause the breaking down of dead or living protoplasm, the actions and reactions that take place between them and living animal and vegetable cells, and the effects upon them of organic and inorganic antiseptic substances, are now all taken into account in drawing up any classification. Thus we have yeasts that cause fermentation and yeasts that do not, whilst nearly related to them are ascomycetes that are essentially parasitic in their character ; we have micrococci that set up urea fermentation, micrococci that form pigment, others that give rise to suppuration or that apparently assist in the production of diphtheria ; and so on, throughout the whole of these low vegetable forms. The appearance under the microscope can, of course, give us no information on many of these points, and it is only by a most careful study of the 6 BACTERIA. life-history of these organisms that any accurate information has been obtained. Within quite recent years it has been observed that very marked modifications of certain of the features may be ob- tained by subjecting the micro-organisms to conditions different from those under which they are ordinarily met with ; and as the outcome of these observations the way has been opened up for the study of the evolution, perhaps not of the micro-organisms themselves, but of their specific functional characteristics. It has been found, for instance, that under certain conditions, yeasts that ordinarily set up active alcoholic fermentation are no longer able to do so ; that by continued cultivation outside the body and in certainspecial media the anthrax bacillus loses much of its virulence ; whilst the tubercle bacillus, the cholera bacillus, the organisms met with in diphtheria and in other diseases, may, under certain conditions of cultivation, lose their disease-producing power in a most remarkable degree. On again being placed under conditions of a more favourable nature these organisms resume their pathogenetic or disease-producing properties: The same variations are met with in the. colour-forming species of bacteria, according as the conditions under which they exist are favourable or unfavourable to the accentuation of certain functions of the protoplasm. It will be evident from a consideration of all these facts that bacteria must be looked upon as being governed by much the same laws which govern other plants and animals ; that they are composed of protoplasm, the functions of which may be modified in various ways, and the forms of which may become more fully differentiated, and that where greater differentia- tion takes place the modification of function is always in the direction of greater specialization ; that the conditions suit- able for the existence of the more lowly organized and functionally less specialized organisms need to be less specialized than do those that are necessary for the growth of the more highly developed fungi ; and that, consequently, the bacteria of disease and putrefaction are, with few excep- tions, of a comparatively low form, although the functions of the protoplasm of these less specialized bacteria become more specialized as they dwell for a longer and longer period under any special set of conditions, one phase or power of the protoplasm being drawn out and developed in INTRODUCTION. ” excess and frequently at the expense of, some of the others, sometimes even at the expense of the living or resisting power itself of the organism. In all cases, however, it should be remembered that the differences in protoplasm are differences in degree rather than in kind, and that the laws which govern animal and other vegetable protoplasm govern the proto- plasm of bacteria, and that the results of all investigations that have been carried on in connection with them must conform as strictly to physiological laws as any work that has hitherto been done in the domain of animal or vegetable biology ; and that where discrepancies of any kind, or appa- rent departures from ordinary physiological laws, are met with, the facts must be carefully revised, and if the dis- crepancies still continue, then so much the worse for either the facts or the laws. The “facts” are still incorrect or the laws must be revised. There has grown, then, and still continues to grow, a science dealing with microbiology in all its morphological and physiological phases. Facts have gradually been accu- mulated, observations have succeeded observations, patient work and powerful concentration have played their part in elaborating our knowledge of the habits of micro-organisms without and within the higher plants and animals; the conditions under which the modes of life of micro-organisms may be altered have been investigated ; the effects that they or their secretions exert on living and dead proto- plasm have been most conscientiously examined ; the conditions that-are essential in order that these organisms may stimulate the. activity of living protoplasm, and the phases through which this protoplasm passes, from the stimulated condition to degeneration and death, have all been carefully studied. The organisms that have been found in certain diseases have been identified and classified, their modes of propagation and the channels by which they are conveyed from one host to another, sometimes through intermediate saprophytic or non-parasitic stages, have been determined, and the whole subject has been so prepared, that the great epoch-making minds of such men as Pasteur, Chauveau, Lister, and Koch, being brought to bear on these questions have found sufficient material at their disposal on which to generalize, and have placed before the world theories that appear to be more like fairy tales 8 BACTERIA. than deductions made as the results of sober scientific investigation and thought. ‘These men have also, how- ever, been able to obtain many new facts, and to point out the gaps that still remain to be filled in before the theories so admirably expounded can be proved to demon- stration. The French can boast of their Davaine and Pasteur, who, assisted by Chauveau and others, have given to us the germ theory of disease and the theories of fermentation and protective inoculation. In Germany, Klebs, Cohn, Koch, and their followers, have made marvellous contributions to the study of bacteria, and additions to our knowledge of their relation to disease. The Danes have furnished O. F. Miller, Warming, Panum, Salomonsen, Bang, and Chr. Hansen, who, seizing most of what was good in both the French and German schools, have succeeded in making most valuable contributions to various branches of the subject ; whilst, in this country, Burdon Sanderson, Greenfield, Klein, Watson Cheyne, and others, have all contributed their share, though Lister’s name must always stand pre-eminent for the mag- nificent work which he has done in the domain of antiseptic surgery, by the evolution and perfecting of which he has. done more, both directly and indirectly, to ameliorate the suffering, and to diminish the mortality in surgical cases, than has been achieved by the most brilliant operators the world has produced during the last century. Many of the- processes of everyday life are intimately associated with the specific activities of micro-organisms ; we are constantly meeting with these organisms, and it is now proved, beyond all dispute, that their presence is not merely accidental but is absolutely essential to the carrying on of, one might almost say, the most commonplace operations. Take, for example, those that are associated with the different processes of fermentation. Leaving for the moment the yeasts or saccharine ferments, I may refer first to the butyric acid ferment—Clostridium butyricum—which plays a most important part in interfering with the ordinary course ‘of the saccharine fermentation. It decomposes starch and cellulose without requiring the presence of oxygen ; it, like other micro-organisms, appears to require for its nutrition, nitrogenous material, and, especially in the presence of the lactic acid ferment, it brings about the conversion of sugar into butyric acid; it is, as might be anticipated, one of the INTRODUCTION. 9 bacteria most frequently met with in some of the forms of putrefaction. It must be remembered however that this is not the only organism that gives rise to the butyric acid fermentation ; several other bacteria, differing most markedly in many of their features from the Clostridium butyricum, generally, however, like that organism, carrying on their work in the absence of free oxygen, have the power of setting up alone, or in conjunction with the Clostridium butyricum, the butyric fermentation. In ripened cheese, part of the flavour at any ‘rate is due to the products formed in the ripening curd in the presence of this organism. The lactic acid fermentation so frequently met with in milk, is also the result of the vital activity of several organisms, Pasteur and Lister both describing lactic acid bacteria and micrococci. Hueppe also describes a special bacterium which he says has the power of breaking up milk sugar and saccharose into lactic acid and carbonic acid ; whilst from material taken from the mouth and teeth he succeeded in separating two micrococci, both of which had the power of converting sugar into lactic acid, and bya series of experiments he also proved that certain of the pigment-forming bacteria, such as Bacillus prodigiosus— the organism that gives rise to what is known as bleed- ing bread—supply so much lactic acid as a result of their metabolic processes, that in their presence milk is curdled, the casein being precipitated. Further, even a pathogenic form of micro-organism—micrococcus osteomye- litis —is said by Krause to set up the same reaction. Jérgensen mentions that “ Delbruck found that in a mash prepared from dry mould and water, lactic acid was first formed at a temperature of 50° C., and from this he draws the conclusion that in this case the active lactic acid ferment has its maximum temperature at this degree of heat.” This is an exceedingly interesting fact, for as Schéttelius and Wood have pointed out, as the temperature rises the Bacillus prodigiosus loses its power of forming a pigment, and if it is grown on potato or bread paste for example, in an incubator at blood heat instead of at the temperature of the room, the colour is gradually lost and the culture no longer smells of herring brine, but the power of forming lactic acid from milk sugar, with the accompanying precipitation To BACTERIA... of the casein, is frequently considerably increased ; so that it would appear that the energy required for the building up of the pigment substance was in this case diverted into another channel, and laetic acid, and perhaps other sub- stances, are produced in-place of the usual pigment. This example may afford some idea of the complexity of the problems that have to be grappled with by bacterio- logists, and it may also help to explain how such different results have been obtained by different observers, and how it still may be possible to reconcile statements which at first sight appear to be in direct opposition. It is a well-known fact that if the lactic acid fermentation once obtains a footing, in a solution of sugar for example, many of the other putrefactive bacteria find it impossible’ to’ develop. On the other hand, whilst the lactic acid organism cannot grow beyond a certain point, yeasts are perfectly able to develop where there is a certain quantity of acid present. This has: been adduced as an explanation of the fact that comparatively pure yeast fermentation may go on even in the presence of the lactic acid organism, when it is unable to make further head- “way.and when other putrefactive organisms are unable to . grow at all. Another fermentation, the results of the careful studies of which have been utilized in the commercial production of vinegar; is that due to the acetic acid ferments Mycoderma aceti and Mycoderma Pastorianum, by the action of either of which alcohol is converted into acetic acid. In all the works on brewing, lists of micro-organisms that give rise to such conditions as bitterness, muddiness, various colorations — red, yellow, green, etc.—are given, and it is pointed out at the same time that all appear to give rise to some form of acid. It is supposed, for example, that certain species of sarcinz are responsible for the sour and bitter tastes which are sometimes developed in beer ; a similar organism is also described as giving rise to the red colour which is sometimes developed in white beer, though it may be remarked that in this case a rod-shaped bacterium is also frequently present, and may be answerable for the presence of the substances that so seriously alter the taste of this. beverage. A series of other organisms, which Lindner speaks of as Pediococci, all produce acid, and give rise to INTRODUCTION. Il tnsoundness of beer, but as they are readily killed at a temperature of 60° C., they may easily be got rid of. In the process of baking, as carried on in this country, there is a regular conversion of some of the starch of the flour into sugar by the yeast used —+ this sugar being in turn converted into alcohol and carbonic acid gas, to e setting free of which the “rising” of bread is due. ‘he baking that follows serves three purposes ; it kills the ee Shia Grab caer starchy matter in posi- tion, and it drives off the alcohol and the carbonic acid. In the baking of other kinds of bread certain other organisms are said to play a part; for instance, the Saccharomyces minor (Engel) was supposed to be the active fermenting agent in the manufacture of rye bread, but more recently this organism has been superseded, and the work of fermen- tation has been assigned to a bacillus—Bacillus Panificans (Laurent)—which in pure cultivations was found to be capable of setting up all the characteristic fermentation changes in the dough of black bread. This organism is made up of short motile rods, from which threads may be formed, these threads (in which spores are sometimes found) interlacing to form a film, especially when it is grown on the surface of nutrient liquids. Its spores are almost as resistant as those of the hay bacillus, and can only be killed by being subjected to boiling heat for a period of at least ten minutes. Jorgensen, summing up Laurent’s investigation, says: “The bacillus usually dissolves the gluten substance of the dough, grows in starch paste, and in mixtures of saccharose and mineral substances. It is found in an active state in large quantities in bread, and according to the author's researches, it can withstand for twenty hours the action of an artificial gastric juice. In the excreta it is found still more abundantly, and it appears to be generally distributed in plants and in various substances.” More recent researches, however, have thrown some discredit even on the Bacillus Panificans ; Dinnenberger maintaining that these bacteria are merely an impurity, and that in all cases the fermenta- tion of bread is due to an alcoholic-forming organism—a saccharomyces in all cases proving the best agent for bring- ing about this fermentation. Certain forms of unsoundness of bread are also due to micro-organisms. In addition to bleeding-bread (caused by: 12 BACTERIA. the growth of the Bacillus prodigiosus), which has served the purpose of the miracle-monger before to-day, sticky reddish- brown patches have been described as occurring in unsound bread, in which various bacilli, such as the ordinary potato bacillus, Bacillus liodermos, Bacillus mesentericus vulgatus, have been found, and on analysis of these patches dex- trine, dextrose, starch, sugar, and even a small quantity of peptone, have been separated ; moulds of other fungi are also found in unsound bread, In 1882, Kern described the peculiar ferment known as kephzy grains, by means of which the Caucasians set up a double alcoholic and acid fermentation in milk. These kephir grains, says De Bary, in the fresh living state are “white bodies, usually of irregular roundish form, equal to or exceeding a walnut in size. They have their surface roughened with blunted projections, and furrowed like. a cauliflower ; they are of a firm, tough, gelatinous consistence, becoming gradually cartilaginous, and are of a yellow colour when dried ; they are chiefly composed of a rod-shaped bacte- rium,” many of these being united to form long threads, arranged in a kind of felt or network, the meshes of which are filled with a tough gelatinous membrane, which binds the organisms together into a kind of zoogloea mass. This rod-shaped organism is known as Dispora caucasica as at the end of each rod is a rounded spore. Along with these may usually be found a small proportion of a, yeast-like fungus which, however, is merely entangled in the gelatinous mass, although it certainly undergoes development by sprouting. There is also present the ordinary Bacterium lactis which, with a number of other impurities, adheres to the kephir grains; this also occurs in the milk itself. To prepare the specially fermented milk, one volume of these kephir grains is moistened and added to about six or seven volumes of fresh milk, the whole is protected from the dust, but is exposed to the air for about twenty-four hours at the ordinary temperature of the room, and is frequently shaken ; the milk is then poured off and a fresh quantity added ; the milk that is poured off is mixed with double its quantity of fresh milk, put into bottles, well corked, and frequently shaken. This bottled sour milk soon becomes sparkling and effervescent, and is ready for use after it has been bottled for a day or two. It then con- INTRODUCTION. 13 tains lactic acid, a considerable quantity of carbonic acid, which varies “ according to the temperature and the duration of the fermentation, but is sometimes sufficient to burst the bottle or drive out the corks.” The liquid contains about one per cent. of alcohol. _ We have already seen that the kephir grains and the sour milk, together, contain (1) a yeast fungus which is capable of bringing about the fermentation of grape sugar ; (2) the bacillus of lactic acid ; and (3) the rod-shaped bacteria that predominate in the gelatinous mass of the tough grains. Now, though this yeast fungus can set up the fermentation of inverted milk sugar, it cannot affect the milk sugar itself, so that we must look elsewhere for the inverting power. This appears to be contained in an enzyme, or ferment, that is produced by a large number of bacteria, amongst * others by the rod-shaped organisms already referred to, and by the lactic acid bacillus, and these were supposed to prepare the milk sugar for the action of the yeast fungus. This appeared to be a perfectly satisfactory explanation, until De Bary found that, by violent agitation of the souring milk to which no kephir grains have been added, alcoholic fermentation may still be induced ; so that it would appear that by freely oxygenating the milk during the process of lactic acid fermentation, when in fact the molecules are being re-arranged, oxygen is taken up into chemical combina- tion, and alcohol and carbonic acid are generated; water, in all probability, being formed as this goes on. This is adduced as one of the processes of fermentation by free oxidation, and is an example of a chemical fermentation giving results similar to those yielded by biological fer- mentation. Until, however, it can be demonstrated that De Bary was working with material in which all impurities were excluded, these results can scarcely be accepted as abso- lutely reliable. In the kephir, which is a perfectly fluid mass, we have a quantity of lactic acid. Now, it is a well-known fact that, under ordinary circumstances, when milk turns sour, there is a precipitation of the curd which forms a more or less solid coagulated mass. What has become of this coagulum in the kephir? If a piece of meat be exposed to the action of putrefactive germs, it will be found that after a time it is reduced to a soft, pulpy, almost liquid mass. In the same 14 BACTERIA, way, a number of the nitrogenous foods taken into the stomach are softened and otherwise very considerably altered, These materials have in both cases undergone what is known as a process of peptonization: from coagulable or colloid albumen they have been converted into a much more solubleand diffusible albuminoid, and are thus prepared for fur- ther changes as they are acted on directly by living animal or vegetable protoplasm. This peptonization appears, however, to be also brought about by an enzyme which is elaborated by certain bacteria, especially by such as are associated with putrefaction and with the digestive processes. In the case of the kephir it was thought that the peptonization took place’ as a result of the action of an enzyme, formed by the rod-shaped organisms of the zoogloea mass of the kephir grains, and it was argued that this must be a soluble ferment which could diffuse from the gelatinous mass in which the grains were embedded into the milk; it could there act on the casein as it was gradually precipitated, or possibly even before precipitation took place; for it was observed that these rod-shaped organisms were never found outside the gelatinous masses, and therefore could not be acting directly on the casein. This is well enough in theory, but as equally good kephir can be produced by the oxidation effected by free movement of the sour milk, the peptonization cannot be solely due to these organisms. It may be urged that other organisms may, in the presence of a large quantity of oxygen, supply the peptonizing function in an acid fluid in which, under ordinary conditions, they would not be able to exist, the large amount of free oxygen driven into the milk enabling them to obtain a supply of energy, to live and carry on their function even in the presence of the special lactic acid bacteria, which seize with avidity the whole or a part of the oxygen contained in the milk, according to the quantity present, in this instance a part only, as there is such a large supply under the above conditions. Tt has already been mentioned that the Clostridium * For the further description of the kephir grains the reader is referred to E. Kern, “Bot. Ztg.,” 1882, p. 264; Alexander Levy, ‘‘ Deutsche Medi- cinal Zeitung,” 1886, p. 783 ; De Bary, ‘‘ Lectures on Bacteria,” translation, Oxford, 1887. INTRODUCTION. 15 butyricum or Bacillus amylobacter plays an important part in determining the butyric acid fermentation of the vegetable acids, and that it plays an active part in the ripening of cheese. Because of its action on cellulose, and then of its further action on dextrine and glucose, it also has much to do with the decomposition and destruction of the cellulose of fleshy and juicy plant tissues, and its aid is requisitioned in the separation of these parts from the tougher fibre of hemp, flax, and similar materials, as in the process of macera- tion the enzyme converts the cellulose into butyric acid. Van Tieghem holds that much the same processes go on in the stomachs of ruminant animals, and that the Bacillus amylobacter, which is usually found there, thus does a very large part of the work which, otherwise, would have to be performed by the epithelial cells of the stomach itself. This bacillus, however, acts not only upon cellulose and starch paste, but it also exerts a most important action on nitro- genous substances. Fitz and Hueppe have both pointed out that the casein of milk is first coagulated in the presence of this organism, is then peptonized and liquefied by the action of its enzyme, and that the products thus formed are afterwards converted into certain lower compounds, such as leucine, tyrosine, and even ammonia, all of which are constantly met with during the processes of both digestion and decomposition. Duclaux also showed by experiment that an organism, which resembles the bacterium amylobacter very closely in many respects, sets up a series of similar changes in the casein of milk, and in casein that has been converted into cheese ; and he showed that the process of “ripening” brought about in the presence of the Tyrothrix bacillus, is due to the peptonization and liquefaction of some of the substances of which unripe cheese is composed ; certain secondary or ultimate products similar to those above mentioned being found during the ripening process. That bacteria are general scavengers is now generally acknowledged, and almost innumerable observations have been made with the object of proving that the presence of certain definite organisms is essential for the perfect breaking down of dead or effete animal and vegetable matter. It has already been mentioned that some species have the power of breaking up cellulose and of converting it into much simpler substances, both externally and in the alimentary canal. 16 BACTERIA. Bienstock went so far as to describe a particular bacillus which, he considered, was able by itself to produce the whole series of changes that occur in the contents of the intestine and in putrefying albumen or fibrine. This organism he describes as being somewhat smaller than the Bacillus subtilis; it is rod-shaped, but at one end there is usually a small enlargement, in the centre of which a clear round spore may be seen. It is from this feature that the organism derives its name of “Drum-stick” bacillus. Cultivated on fibrine it disintegrates it and gives rise to the formation of leucine, tyrosine, carbonic. acid, water, and ammonia, and of traces of other putrefactive products. The process does not however stop at this point ; the bacillus still continues to act on the leucine and tyrosine, and decomposes them into still simpler compounds; whilst, if it be introduced at once into a prepared solution of one of these earlier decomposition products, tyrosine for example, it continues the breaking-down process just as if it were still acting on the tyrosine which had been formed during the process of ordinary putrefaction. As De Bary points out, however, this bacillus cannot claim a monopoly of the work connected with putrefaction. If any putrefying fluid be examined, countless organisms will be found, and amongst these very different species may be observed. There are rods of different sizes both as regards thickness and length, spirals of different forms, micrococci of different sizes and arranged in different groupings, one or other organism predominating according to the nature of the putrefaction process, of the material that is being broken down and the stage at which the breaking- down process has arrived. It would appear in fact as though there were developed special organisms for the setting up of special fermentations, and also that after the breaking down has been carried a certain length by one organism, the aid of another is invoked to complete the process more thoroughly and more expeditiously. We have in this, as in the case of the process of digestion, an exemplification of the fact that nature economizes her resources as much as possible ; she does not call on the animal cells of the alimen- tary tract to do work that can be equally well done by micro-organisms, nor does she demand the exercise of more than one or two functions from each of the simple proto- plasmic spécks that we call bacteria. To each one is assigned INTRODUCTION. 17 its special work and, though it is possible that many of them started with certain powers in common, it seems that through the exercise of some of these common powers under special conditions they have become gradually so differentiated functionally, that, as amongst organisms more highly developed, each is able to carry on its own work best at those special stages of the putrefactive process at which it is found. It might at first sight appear that all this can have but little bearing on any practical work in which we are engaged, or in which we take an interest, but on more careful consideration it will be found that these putrefac- tive organisms really keep up the circulation of matter, utilizing the excretions of living beings and the carcases of dead animals and plants, after breaking them down into their simplest constituents, to supply those elements that are necessary for the nutrition of plants, allowing them to present themselves in their most assimilable forms, and in the proportions most suitable for the nutrition of the growing, highly organized vegetable protoplasm. Bacteria in fact serve to transform inert organic matter into inorganic substances, This transformation, or “ Mineral‘zation,” in most cases, com- mences only after protoplasm has lost its vitality, and most micro-organisms are capable of attacking this dead protoplasm only ; though, as we shall find later, a certain number of bacteria have acquired the faculty of being able to attack even living protoplasm. The processes of decomposition may be divided into two kinds : first, those going on as the result of the activity of organisms that are capable of taking up their oxygen from the air, and, second, those the result of the activity of organisms that so break up and rearrange the organic molecules containing oxygen, that not only do they, the bacteria, take up oxygen themselves but they allow of its being handed on to the products to which in their processes of metabolism they give rise. It is probable that here we have to do, not only with nascent oxygen, but that we have certain products set free during the proces of decomposition which seize upon oxygen with very great avidity. This decomposition or rearrangement is spoken of as a process of nitrification, or a conversion of the nitro- genous elements into ammonia, nitrous and nitric acids, carbonic acid and water, or, speaking more generally, it may be said to be a process of mineralization of the organic 3 18 BACTERIA. forms of nitrogen, phosphorus, carbon, and hydrogen, during which they become finally oxidized or mineralized to nitric acid (HNO,), phosphoric acid (H,PO,), carbonic acid, CO,, and water, H,O. In nature this process goes on in the superficial layers of the earth or in the presence of the atmosphere. That it takes place much more readily near the surface of the ground and in porous earth can easily be understood if what takes place in the oxidation that goes on in spongy platinum is borne in mind. In the earth we have a spongy material, the upper surface of which is well supplied with air, and usually also with moisture ; there is also a certain amount of organic matter present on which micro-organisms feed, and any additional organic matter brought to this spongy mass is rapidly seized upon by the micro-organisms, is oxidized, and thus prepared for the nutrition of the plants that are growing in this soil. So necessary is this whole process of oxidation by micro- organisms that Duclaux insists that soil rendered sterile (as regards micro-organisms), and supplied only with sterilized water and air, is incapable of supplying sufficient nutrient material to plants to enable them to flourish even moder- ately well. All the organisms found in these superficial layers under ordinary conditions are zrobes ; in the deeper layers of the soil are micro-organisms that give rise to the second kind of decompositions. These bacteria, which are anerobic (that is, they can flourish without being supplied with free oxygen) in character, have a special power of taking up the oxygen that is contained or combined in the products which have filtered down from the surface where the decom- positions by direct oxidation are going on. Living away from the atmosphere and being unable to obtain or to utilize free oxygen, these organisms have developed the faculty of being able to wrest oxygen, by force, as it were, from the oxygen-containing bodies that come down to them from nearer the surface, sometimes however using part only of the combined oxygen and setting free another part by which further oxidation may go on; in doing this they carry the process of decomposition a stage further, and after the altered organic materials have been attacked by these anzrobic organisms they appear to contain little that will provide nutrition for micro-organisms of any kind ; so that, after we come to a certain depth, bacteria are not INTRODUCTION. 19 to be found in the soil. This depth, usually reckoned at about twelve feet, varies of course according to the nature of the soil, its moisture, porosity and temperature, and also according to the amount of organic matter that lies on the surface; cultivated ground always containing more organisms and to a greater depth than fallow ground having the same geological characters. The relation of all this to our water supply is obviously one of paramount importance. If water be taken from near the surface of soil in which there is a large-quantity of organic matter present, there must necessarily be a large number of putrefactive organisms in it, especially those of an erobic nature ; if however we take water directly from the deeper layer, these putrefactive organisms are usually absent, but a number of “ water” organisms are now present, which under special conditions, especially if the water be kept perfectly quiet and unoxygenated, and a high tempera- ture be maintained, can develop in the water, from which they may in turn be cultivated by certain well-known methods. If water be taken from a much deeper layer, micro-organisms are found to be almost or altogether absent, and not only micro-organisms, but organic matter, although in some cases in which micro-organisms are almost entirely absent, organic matter may still be present in appreciable quantities. The superficial layers of earth in this case act, not only as a mechanical, but also as a biological, filter ; the water, with its contained organic matter, passes through the successive layers in which bacteria can grow, and gradu- ally percolates to those layers of earth where there are no organisms. It has been demonstrated that even deep natural water contains facultative zrobic organisms, unless it is obtained at once after it has undergone natural filtration— that is water that has not stood in an underground basin— when it may be almost germfree. The organisms cannot go down with the water; first, because they are held back mechanically, the soil acting as a porous filter, by which solid particles, extremely minute as they are, are kept back ; but, in addition, and quite apart from this purely mechanical effect, the bacteria (many of which are unable to develop without oxygen) cannot leave the surface with impunity, and such as are carried down by the action of the water die as their supply of oxygen is gradually cut off ; for, in consequence 20 BACTERIA, of the rapid oxidation that is going on at the surface, very little free oxygen is left for the use of bacteria at even a short distance from the surface. It will, of course, be objected that this does not apply to the anzrobic bacteria, but in their case it must be remembered that only a certain definite proportion of oxidized material can reach them from above, most of the organic matter having already been converted into inorganic material and used up by growing plants ; the supply is very rapidly cut off, the reduction of what remains after the plants are satisfied being com- pleted, and the bacteria cannot continue to live, because they can no longer obtain any material for their nutrition. All the knowledge that has been gained concerning the pro- cess of putrefaction has not been collected in a day, and it was only bya careful marshalling of facts as they were accumulated, and by filling in,sometimes merely with suggestions, gaps that still remained, that the theory of the filtration and biological and chemical purification of water has been built up. But to the practical hygienist it is not sufficient to know that water coming from springs deep down in the earth is free from organisms, and that it may be drawn and consumed with impunity as it rises from the ground. He has something more to consider ; he has to consider under what conditions it can be kept fit for drinking purposes, and by what means it can be most readily and safely distributed to those who use it. He has to remember that water at rest containing organic matter, and exposed to the hot rays of the sun, soon teems with organisms that may or may not be perfectly harmless ; he has to bear in mind the nature of the ground from which water is collected, whether it comes from cultivated areas or from regions in which there is sewage of any kind; for upon these factors depend the absence or presence of bacteria from the water supply. Further, he may be faced with the problem of how to transform a supply of biologically impure water into a supply fit for drinking purposes. The mere chemical analysis of water will not give suffi- cient information to guide a sanitary engineer on these points, though it will indicate to him the lines on which he will have to work. For example, it is quite possible to have a water containing a considerable quantity of organic material which, through its treatment by sand filters, or by INTRODUCTION, 21 some similar process, may contain no micro-organisms, and may be perfectly fit to drink so long as it is fresh, although, if it were allowed to stand in a warm place and exposed to dust and germs of various kinds, there would soon develop in it such an enormous number of organisms that it would be looked upon as totally unfit for drinking purposes. On the other hand, water containing little or no organic matter might be so contaminated by certain disease germs that it would be absolutely dangerous to health, and even life, if used for domestic purposes. The relation of bacteria to sewage need not be insisted upon, as it is evident that with such a large quantity of organic matter, in which putrefactive changes must be rapidly going on, bacteria of many kinds must necessarily appear, and it is easy to conceive that an enormous number of different species might be present, especially where the sewage is diluted as it flows into comparatively pure water. Let us observe how a disease-producing organism may find its way into water that is used for drinking or other purposes, and thence may attack a comparatively healthy individual. In typhoid fever, a germ, which will after- wards be described, has been found. It has special charac- teristics, and may be separated asa pure culture. A patient has typhoid fever ; the bacilli which we find specially in the walls of the alimentary tract pass out along with the excreta, and, under ordinary circumstances, would be killed by the addition of disinfectant fluid to the stool ; but, as frequently happens, some of the excreta without the addition of a disinfecting fluid by chance finds its way into the drains, or, worse still, on to the surface of the soil near a well or some other source of water supply. The typhoid bacillus con- tinues to multiply in the water or in the organic matter of the sewage, from which it ultimately finds its way into the water, and although such water may appear pure enough (it is often beautifully clear and sparkling), as soon as it is taken into a slightly disordered stomach and intestinal canal, the bacillus gains a foothold, and another patient is attacked with typhoid fever. This may happen simply through the rinsing out of a milk pail. A patient partaking of milk (a most admirable nutrient medium for the growth of the typhoid bacillus) from such a pail is struck down with ‘ Typhoid.” Another example: there 22 BACTERIA, is a small drum-stick shaped bacillus, similar to the putre- factive bacillus described by Bienstock, which, on making its way into a surgical wound, sets up a series of changes in the tissues, accompanied by the production of a most virulent poison, which, acting apparently on the nervous system, gives rise to reflex spasms and convulsions, and a ‘condition known as lock-jaw or tetanus is set up. This organism is found on the manure heap, in cultivated soil, and even in water that comes from such soil ; it is also found in the dust of hay, straw, and even in the harness and cloths used for equipping the horse. If water containing these organisms be used for the purpose of washing a con- tused wound, or if any of the above dirt or dust should obtain access to such a wound—often merely a most insignificant bruise—tetanus or lock-jaw is set up with terrible certainty, and the patient very frequently succumbs to the disease. Innumerable other instances might be given in proof of the statement that the knowledge, first, of the forms, and, secondly, of the biological and physiological characteristics of the various micro-organisms, is now absolutely necessary for a thorough understanding of even many everyday pro- cesses. The colour formed in “ blue milk” is due to the action of a micro-organism ; as also are the phenomena of bleeding bread, the Cape meal orange ferment, and the characteristic appearance of green cheese. Asa result of the knowledge gained through the study of the life history of septic organisms, thousands of valuable lives have been saved in our surgical wards alone; large industries have, through Pasteur’s indefatigable exertions, been preserved from almost absolute ruin ; as a result of the observations of numerous investigators, our knowledge of certain classes of diseases is gradually becoming more precise and accurate, and the time has now arrived when we may look forward to a system of medicine in which, by preventive and curative inoculation, we shall be able to grapple successfully with some of the deadliest forms of disease with which we have at present helplessly and almost hopelessly to contend. LITERATURE. The following works may be consulted : BrensTock.—Zeitschr. f. Klin. Med. Bd. vii. p. 1, 1884. Coun.—Beitr. z. Biologie der Pflanzen, Bd. ., 1876. INTRODUCTION. 23 Duciaux.—Chimie. Biologique Encyclop. Chimique, Paris, 1883. DUNNENBERGER.—Bakt. chem. Unters. Ueber d. Beim. Aufgehen des Brodteiges wirkenden Ursachen.—Bot. Centralbl., 1888. Firz.—Ber. d. Deutsche chem. Gesellschaft, 1876-1884. Hansen.—Meddel fra. Carlsberg Laborat., Copenhagen. Hueppre.—Der Bakt. Forsch., 1889. JoRGENSEN.—The Micro-organisms of Fermentation (transl.), 1889. K.iess.—Beitr. z. Kenntn. d. Mikrokokken.—Arch. f. Exp. Path., Bd. 1., 1873. Kocu.—Mitt. a. d. k. Gesundheitsamt, Bd. 1., 1881. Lister.—Trans. of the Path. Soc. Lond., vol. xxix., p. 425, 1878. NaEGELI.—Nageli u. Schwendener.—Das Mikroskop, 1877 ; Theorie der Gahrung, 1879. Pasteur.—Etudes sur le vin, 1866 ; Etudes sur le Vinaigre, -1868 ; Etudes sur le Biere, 1876. Rogerts.—Phil. Trans., vol. clxiv., p. 457, 1874. SALOMONSEN.—Bakteriologisk Teknik., Copenhagen, 1890. Van TIEGHEM.—Bull de la Soc. Bot. de France, p. 128, 1877 ; p. 25, 1879. CHAPTER II. : WHAT ARE BACTERIA? Structure — Myco-protein — Limiting Membrane—Gelatinous Capsule— Special Cell Contents-——Oxide of Iron—Sulphur—Colouring Material —Flagella—Modes of Multiplication and Development—Division— Rate of Vegetative Multiplication —Endospores — Arthrospores— Classifications of Cohn, Van Tieghem, Zopf, Winter and Rabenhorst, De Bary and Hueppe, Fliigge Baumgarten, &c. UnpDER the general term Bacteria may be included those minute, rounded, ellipsoid, rod-shaped, thread-like, or spiral forms of vegetable organisms sometimes spoken of as be- longing to the class of lower fungi. They are also known as Fission fungi (the Schizomycetes of the Germans) ; a term applied to them because of their multiplication by a process of division, transverse to the longitudinal axis in the case of the rod-like forms, but varying somewhat in the case of the rounded organisms. Each single organism consists of a small speck of protoplasm or vegetable albumen, to which may be given the name of cell, as although these specks are so minute that they can be seen and studied only with the aid of the very best optical appliances at our command, it is found that they are by no means simple in structure, nor are they in all cases even similar. This proto- plasmic speck is differentiated into certain well-defined parts. The round cells or micrococci, the simplest of all forms, are seldom more than 1-25,oooth part of an inch in diameter ; the elongated cells have, on-an average, the same diameter, or they may be a little more, and are from 1-12,000th part to 1-6,0ooth part of an inch in length, though there are marked deviations from these dimensions in certain forms. Accepting the above figures as being accurate, it would require 25,000 of these spherical cells, placed in a row, or the same number of the longer ones, placed side by side, to make up a chain or band one inch in length. The vegetable WHAT ARE BACTERIA ? 25 albuminoid or protoplasmic substance of bacteria -was first analysed and described by Nencki, who, because of the nitrogen contained in it, and because of the similarity of its structure to animal and vegetable protoplasm, looked upon it as a proteid material; he gave to it the name of myco-protein or fungus protein.t* During certain periods of the existence of these micro-organisms, especially in cer- tain species, this white of egg or jelly-like substance, which invariably occupies the central part of the cell, is perfectly transparent and is slightly more refractile than water ; at other periods, or in other species, it may be finely or coarsely granular ; under certain conditions still more marked and characteristic changes may occur, vacuoles or clear spaces making their appearance. It is usually extremely resistant to the action of acids and even of alkalies. That this myco-protein cannot always be of the same composition is evident from the fact that minute granules of chlorophyll or of fat may be made out lying in the substance of the protoplasm in special species, whilst in others small starch or sulphur granules, or particles of different kinds of pigment may be observed. This speck of vegetable albumen is really the active part of the cell, and it is in this that we obtain those histo-chemical colour or staining reactions that are so characteristic of the protoplasm of the higher vegetable and animal cells which, as is well known, seem to have a peculiar affinity for certain dyes or stains, Carmine, for example, is taken up most voraciously by the nucleus or central portion of the cell which then assumes a brilliant carmine hue, not by any means equal throughout; in consequence of this inequality the minute structure of the nucleus may be readily and even accurately studied. The surrounding protoplasm, which appears to be less active, is much less vividly stained, whilst the cell wall, which is the least active part of the cell and serves as little more than a boundary wall of formed material, if present at all, remains, in the majority of cases, quite unstained. Logwood, the aniline dyes, or iodine may be substituted for carmine, with the general result that the same staining reactions as regards the different parts of these cells are almost invariably obtained. ® Analysis of myco-protein: Water, 84.81; albumen, 13.207; fat, 1.198; ash, 0.638; extractives, 0.327. 26 BACTERIA, Exactly the same reactions are given when one of the above colouring reagents or stains is added to a mass of micro-organisms. The individual cells or organisms are brought prominently into view, the central jelly-like speck of protoplasm is most vividly stained in each, and reasoning from analogy, certain authors have looked upon this active part as the nucleus of the micro-organismal cell. Sur- rounding the central protoplasm is a dense, sometimes lamellated, thin skin or sheath which acts as a limiting or protecting membrane. It very frequently contains a sub- stance known as cellulose, almost, if not quite, identical in composition with that forming the hard covering of the vegetable cells that are found in the higher plants. In other bacteria, in fact in the majority of them, the limiting membrane seems to consist of a firm layer composed of gelatinous material, sometimes stiff and rigid, or it may be more or less elastic or pliable. Between the central stained portion and the limiting sheath there sometimes exists a narrow unstained area which by some is said to be a space produced artificially by the chemical action on the protoplasm of staining and other reagents ; others, however, hold that it is a kind of modified protoplasm which, surrounding the more active nuclear protoplasm, divides it from the limiting membrane. Outside this limit- ing membrane, or more strictly speaking, continuous with it, there may usually be seen a mass of gelatinous or muci- laginous matter which does not take on any special stain, and which serves to separate the individual cells, or, to speak more accurately, to bind them together into little groups. It is due to the presence of this gelatinous material that we have those frog-spawn masses or jelly-like lumps that are met with in certain germ fermentative processes. These masses, in which the organisms are embedded, as it were, in the softened jelly-like part of their sheaths, are known as zoogloea masses or masses of living glue. If the cells remain isolated when the membrane becomes gelatinous, capsules or highly refractile areas are seen around each cell, when examined under the microscope. These may sometimes be very delicately stained by certain methods (Gram’s method, see Appendix). As examples, may be taken the capsule that surrounds the bacillus of pneumonia (Fried- lander), the false Diplococcus of pneumonia and certain WHAT ARE BACTERIA ? 27 other organisms such as the Leuconostoc mesenteroides, which is not pathogenic, and the Actinomyces or Ray fungus. It is rather a curious fact, as Friedlander pointed out, that this gelatinous capsule is formed only under certain conditions, and he noted that in the case of the bacillus of pneumonia the capsule could not always be demonstrated ; that it occurred in the bacilli found in the lung tissue or in the prune juice sputum so characteristic of the disease, but that it could not be made out in the organisms grown on such cultivation media as peptonized beef jelly. In the case of the Ray fungus or Actinomyces, the organism that causes wooden-tongue or Actinomycosis in cattle, this swelling of the capsule at one end of the organism and not at the other gives rise to a very peculiar club-shaped appearance of the threads, and as these are frequently arranged so that the thin ends of the rods are grouped together, whilst the swollen .ends are placed at the periphery of the radius, a most peculiar and characteristic radiate arrangement of wedge or club-shaped rods is the result. In some cases bacteria first divide and multiply whilst they are embedded in this common gelatinous mass, and it is only after they have undergone a certain development that each organism becomes invested with its own capsule and is allowed to lead an independent life. It appears that in most cases in which the organisms con- tain natural colouring matter, it is deposited in the capsule and not in the protoplasm itself, and that the red, magenta, blue, and yellow colouring particles that are met with in this position give rise to the naked eye colour that is seen where large masses of these organisms are growing. The beautiful brown that is seen in Crenothrix and Cladothrix, not only in the capsule, but in the surrounding cultivation medium, is due to the presence of oxide of iron which the organism is able to separate from water, or from other media in which that substance is held in suspension or com- bination. The composition of the limiting or external membrane, as already pointed out, varies somewhat in different organ- isms ; in many cases it appears to consist merely of an altered myco-protein, but in others, as, for example, in Bacillus anthracis, it is composed of a material analogous to the casein of plants, combined with a substance which 28 BACTERIA, has many points of resemblance to the mucine found in the tissues, especially the embryonic tissues of animals. This capsule of the B. anthracis, unlike myco-protein, contains no sulphur, is soluble in dilute alkalies, but insoluble in water and acetic acid. Dallinger some time ago pointed out that at each pole of an organism which he had studied most thoroughly (the Bac- terium termo, a small oval organism not now recognized as a species) there was developed an extremely delicate flagellum which, he assumed, had something to do with propelling the organism through the fluid in which it was growing, as this organism exhibited most active move- ments in such fluid. As a considerable number of other organisms also possess this same motion in fluid media, it was argued that as in the case of the above organism and of other cells that have an extreme degree of motility, there would also be found flagella or cilia such as are met with in the swarm cells of certain alge, where cilia or flagella are continued directly fror: the protoplasm, through the cell membrane, if present, and so to the out- side of the organism. Although the existence of these flagella was suspected in many bacteria, they could actually be demonstrated in the case of some of the larger organisms only. Now, however, the number of bacteria in which flagella have been seen has been gradually but surely increased, and lately Léffler has described-and photographed most exquisite lash or thread-like filaments, single or in little groups, in Koch's cholera bacillus, in the bacillus associated with the causation of typhoid fever, and in a considerable number of other motile bacteria. Whether these cilia and flagella are developed from the protoplasm of the organism, or whether they are merely secondary modifications of the external membrane, remains as yet a doubtful point. It is quite possible that in many, even of the motile forms, flagella are entirely absent ; the organism in such case relying for its motile power on con- traction of the protoplasm within an elastic or not too rigid membrane. In a straight rod-like organism an un- dulating movement may often be observed, whilst’ both rotary and undulating movements are met with in spiral organisms, though these latter are very frequently supplied with well-formed flagella. It is possible that in certain species, WHAT ARE BACTERIA ? 29 in which motion is very slight, if not entirely absent, the flagella serve to set up currents round the organism by which food is brought to, and excretions removed from, the bacillus. These flagella may be met with as a single pair, or we may have three or four pairs attached to the same organism. They stain like the membranes, and appear to develop only in those Photo-micrograph of Spirillum Undala, with pair of well-marked flagella at each end. X 1000. organisms that have special affinity for oxygen, for as soon as the ciliated forms reach the surface of a fluid, they lose their cilia or they become much less active. This latter, however, seems to-be the more probable explanation, for Léffler has shown that many organisms are provided with cilia, which at one time were supposed to possess nothing of 30 BACTERIA. the kind. Up to the present, however, micrococci, the Bacillus anthracis, and many other organisms, cannot be said to be supplied with flagella or cilia, and in many organisms in which there seems to be independent movement, really nothing but the so-called Brownian movement can be dis- tinguished when they. are examined in fluid, a movement that may be observed equally well when particles, of Photo-micrograph of Cladothrix Dichotoma with pseudo-branching filaments, with well-marked tranverse divisions. x 1000. inorganic colouring matter are suspended in a fluid medium. and examined under the microscope. The simplest form of division or multiplication met with in bacteria is that known as vegetative multiplication, in which, taking as an example a short rod-shaped bacillus, there is first an increase in the length of the rod, then a zone of constriction in the middle of this lengthened rod, and then WHAT ARE BACTERIA ? 31 division, complete or partial, of this lengthened rod into two shorter daughter-cells. Under suitable conditions these two again increase in length, each again is divided into two, and so regular chains or swarms of bacteria are developed. The first sign of division is a delicate transverse mark or line across the middle of the bacterium in a plane at right angles to the longitudinal axis of the cell. By staining, as De Bary recommends, with an alcoholic solution of iodine and causing the young protoplasm to retract, it may be made out that this line is due to the ingrowth of the cell membrane from the periphery towards the centre so as to form a septum, more or less com- plete, between the little rods of protoplasm, which are thus gradually cut off from one another by the constricting, and growing in, cell membrane. In the rod-shaped bacteria this division takes place in one plane only—at right angles to the longitudinal axis of the rod—and when it is imperfect or incomplete it gives rise to chain-bacteria or Strepto-bacteria.? The individual bacteria of which the chain consists are held together in series by the constricted but incompletely divided membranous portion. Similar divisions take place in the rounded bacteria or micrococci, and we have then chain-cocci or strepto-cocci formed, but instead of going on to form or remaining in long chains, they may be arranged in pairs, and are then spoken of as diplococci. There is sometimes division taking place in two dimensions of space, the one at right angles to the other, a good example of which is seen in the Bacterium Merismopcedioides described by Zopf, in which the lines of divisions are placed at right angles to one another, but in one surface plane, z\e., at the surface of a fluid. In other cases there appear to be division, multiplication, and growth in three directions. To obtain an idea of what occurs, suppose that a two-inch cube is divided into single cubes, each one inch in diameter : and that these single-inch cubes then grow until each reaches the size of the original two-inch cube, when it, in turn, is again divided into one- : A long spiral may divide into short curved rods, as in the case of the Cholera bacillus. In Cladothrix, where there is a false branching, the terminal cell before branching, instead of dividing transversely divides vertically, and then the division again goes on transversely. 32 BACTERIA, inch cubes. This mode of division was first demonstrated by Goodsir in the Sarcina ventriculi. We have already seen that these minute organisms are endowed with great power of movement and locomotion, a fact on which stress has been laid in connection with their rapid diffusion through fluids; but an equally im- portant factor in the bringing about of this rapid diffusion is their extreme prolificness. If they can obtain sufficient food, and if the food is of exactly the right nature, the rate at which bacteria grow is marvellous. From actual experi- ment it has been found that if in a cubic centimetre of any specimen of water, we find, say, a couple of hundred organisms ; on standing for twenty-four hours the number will have risen to about 5,000 per c.c. ; at the end of another twenty-four hours, to 20,000 ; and on the fourth day they are uncountable. Cohn calculated that a single germ could produce, by simple fission, as above described, two of its kind in one hour ; in the second hour these would be multiplied to four, and in three day they would, if their surroundings were ideally favourable, form a mass which can scarcely be reckoned in numbers—or if reckoned, could scarcely be imagined— 4,772 billions. If we reduce this number to weight, we find that the mass arising from this single germ would, in three days, weigh no less than 7,500 tons. Fortunately for us, they can seldom get food enough to carry on this appalling rate of development, and a great number die both for want of food, and because of the presence of other conditions un- favourable to their existence. Vegetative multiplication only takes place when the conditions are extremely favour- able to the growth of the organism. If nutrition is in- terfered with in any way, or if the removal of excretory products is obstructed, or if there be a large amount of oxygen present marked changes may at once be observed in the appearance of the protoplasm of the micro-organism. It becomes granular, then a small bright point appears in each cell ; this point gradually increases in size until its diameter may be greater than that of the original organism. This large, clear, rounded, ovoid, or rod-shaped node is known asa spore, or resting-spore. It is really the seed or egg formed by the bacterium, by which the species may be continued although the parent should perish. The shape varies slightly in different species, but in every case it has a dark limiting out- WHAT ARE BACTERIA ? 33 line ; it is devoid of colour, and is highly refractile. The dark outline of the spore is usually surrounded by a pale, soft, gelatinous envelope, the substance of which may, in some cases, be accumulated in rather larger quantity near the two poles of the refractile body. As soon as these glistening Photo-micrograph of Bacillus subtilis, with well-developed free endospores, ‘Those in focus are seen to have clear centre and dark outline. x 1000. bodies make their appearance, degeneration of the protoplasm of the bacteria in which they are found invariably follows, but the period at which the death of the protoplasm actually takes place varies in different cases. Where the spores are small they may lie for some time embedded in the protoplasm 4 34 BACTERIA. of the cell, which, only as it degenerates or dies, leaves the resting-spore free to be carried about from place to place ‘by currents of air or water, to be developed when the conditions of moisture, temperature, and food supply again become suffi- ciently favourable. Where the diameter of the spore exceeds that of the bacterium, it may be situated in the centre, giving rise to a spindle-shaped organism, or it may be at one end, when the organism becomes clubbed or pendulum-shaped. The spore in this case appears. to escape moré readily, and before the complete disintegration of the bacterium has taken place ; the side or end of the swollen organism gives way and allows of the spore being set free. This is only one kind of spore formation, and is spoken of by De Bary as endospore formation. It is met with in some varieties of vibriones, in many of the bacilli and in certain of the spirilla (Cornil and Babes) ; whilst Zopf describes similar spore formation as occurring in certain micrococci, and Escherich points out that he obtained undoubted spores in sarcina pulmonum, z.e., bodies that admitted of double staining. A second kind of spore, to-which is given the name of Arthrospore, is also described by De Bary, Hueppe and others. In this there is a combination of spore formation and of fission ; the mother-cell undergoes division into aseries of daughter-cells, a few of which differ from the rest in very important and essential points. There appear to be two kinds of arthrospores ; one form, met with in Leuconostoc, for example, where simple vegetative division of small round bacteria goes on regularly, so long as the conditions are favourable, and a regular chain is formed. In this chain there appear at intervals, micrococci, which differ from the remainder of the elements of the chain in the following points: As soon as the conditions of nutrition are altered they do not, like the other parts of the chain, die off, but they become “somewhat larger than the rest, acquire a more distinct outline, become thicker-walled, and their protoplasm grows darker. Eventually they become free by the deliquescence of the gelatinous envelopes, and may claim the name of spores, because, when placed in the fresh nutrient solution, they develop into new rows of beads like those of the mother plant.” Here is a body which has most of the characteristics of the resting-spore or seed, but it is not formed within the protoplasm of the vegetative organism, WHAT ARE BACTERIA ? 35 but by a process of fission, and as a result of the vegetative division of the organism. It is quite possible, however, that there is just as much differentiation of the protoplasm in this case, as there is where the spore is formed within the cell ; the only distinction being that the separation between the spore and the vegetative element of the chain takes place at an earlier stage, and more completely than in endosporous reproduction. The reverse takes place, however, in the Bacterium Zopfii, which during the vegetative or fission stage consists of short rods, then of motionless filaments, and, if the temperature be lowered from 30° C. to 25° C. of short motile rods. As soon, however, as the conditions become unfavourable, especially when the nutrient material in which they are growing is exhausted, the rods, apparently, by a simple process of fission are divided up into short roundish cells, which retain their vitality for a considerable time, and which, when again placed under favourable conditions, act as spores, z.¢., they develop into the original characteristic rod-shaped bacteria. In organisms in which spores are not found, the con- ditions for their existence and propagation must always remain favourable or they die out very rapidly, having no specially resistant phase to enable them to tide over their period of adversity; and had we to deal with asporous organisms only, they could, probably, by an organized attack, be rapidly and completely exterminated. The vege- tative organisms, as distinguished from their spores, cannot survive the prolonged action of heat ;—a comparatively low temperature, 60° C. or less, being usually sufficient to kill them, whilst the weaker chemical germicidal reagents are quite sufficient to render them altogether innocuous and inactive. The spores, on the other hand, can withstand the action of a temperature anything short of 100° C..for a considerable length of time. Cold and dessication have no effect upon them, for after being submitted to any of these, spores will, if placed under suitable conditions, develop into the more characteristic vegetative forms. It must be borne in mind, however, that certain organisms may, by careful acclimatization, be accustomed to exist and even develop at extreme temperatures. Globig hasshown that certain bacteria found in the soil can only grow at a tempera- ture of 50° C. or more, and that they can flourish up to 36 BACTERIA. 70° C., whilst certain forms of “ Light” bacilli can, accord- ing to Fischer, grow luxuriantly at a temperature of 0° C. © It should be remembered, however, that arthrospores are much less resistant to all germicidal reagents than are endospores; indeed, they are able to withstand a temperature only about as high as that at which the vegetative forms succumb ; whilst some of the endospores are capable of withstanding dry heat of 105°, 110°, and even 130°C, They are certainly more resistant to the action of some of the weaker germicidal reagents, but they withstand for a short time only, or not at all, the action of the more powerful ones. From the nature of the dense membrane that surrounds these spores, their staining has always been a matter of extreme difficulty. They stood out most distinctly as clear spaces in the deeply stained protoplasm of the cell, but could not be stained. By subjecting the spore-containing organisms to the action of dry heat at 110° C. for half an hour or an hour, as suggested by Buchner, or by exposing them to the action of concentrated sulphuric acid for fifteen seconds, or to a longer treatment with concentrated caustic potash, the membrane is so altered that the staining reagents are enabled to penetrate into the substance of the spore and act on its protoplasm, and impart to it a characteristic colour. If this heating be excessive the protoplasm of the bacillus may be destroyed when it in turn refuses to take on the stain, although the spore itself may, under these circumstances, be stained most beautifully, this fact also indicating that the spore is more resistant to the action of heat than is the vegetative cell. Hueppe, Babes, and Neisser have all described arthrospores as making their appéarance at the end of Koch’s cholera bacillus, which may become free, says Hueppe, and from which, he thinks, he has seen the bacillus being developed.* t In order to stain endospores, the best fluid to use is probably Ehrlich’s aniline water fuchsin solution. Sections are left in this for several days, they are then decolorised with 25°/, solution of nitric acid, washed thoroughly in water and alcohol, to which a trace of ammonia has been added; a con- trast stain is obtained by treating for a few minutes with a dilute solution of methylene blue. In some cases the acid removes the fuchsin from the spores also; it is then well to wash simply with alchohol and stain with methylene blue as a contrast stain. Instead of leaving cover glass preparations for so long a period in the fuchsin they may be heated along aed 4 WHAT ARE BACTERIA ? 37 These so-called arthrospores of the cholera bacillus do not, however, give the ordinary reactions of spores, nor are they any more resistant to the action of heat and germicidal agents than the vegetative forms themselves : they must therefore still be looked upon as pseudo-spores. True spores, then, appear to be special protoplasmic cells, which are first developed in the mother cells and are then surrounded by a very thin, but hard and dense membrane. It is this dense covering that protects the delicate proto- plasm within, against the action of the numerous destructive influences to which the spore is exposed. If this structure be borne in mind, it becomes evident that ‘dry heat must necessarily be less efficacious than moist, in determining the destruction of the spore. Dry heat causes no swelling of the protoplasm within, whilst moist heat causing swelling brings about rupture of the softened membrane by pressure from within, and the unprotected protoplasm, exposed under most unfavourable conditions, is at once rendered inert. It appears probable that this process of expansion from within also comes into play whenever spores are placed in conditions favourable to their development, ze., when they are placed in a warm medium in which are present both nutriment and moisture. The first change then noticed is that the clear, strongly refractile protoplasm becomes cloudy or granular, the dark outline is not quite so prominent and the clear boundary line or limiting membrane appears to swell somewhat, and the spores gradually assume the form of an ordinary vegetative cell. In some cases, however, as soon as the spore begins to swell, the delicate outer sheath may be seen to split either longitudinally as in the Bacillus amylobacter, or across the middle as in Bacillus subtilis. In either case there is a swelling of the softened gelatinous layer which causes the removal of the frm membrane. This thin, firm, delicate membrane may come away in the form of two separate cups, or there may be simply a transverse slit through which the germinating spore escapes. Having once made its way from the membrane, vegetative division at once sets in, the segmentation always taking place at with the fluid almost to boiling point for ten or fifteen minutes. The after procedure is as above. The spores most difficult to stain are those of the bacilli of tuberculosis, and of typhoid fever. 38 BACTERIA, right angles to the long axis of the young organism, as has already been described. It was at one time thought that in the case of Bacillus subtilis division at first went on longitu- dinally, but it has now been demonstrated that the general rule is not departed from, the well-known appearance being due to the fact that after rupture of the membrane the two halves remaining attached by a kind of hinge are thrown outwards, the ends of the vegetative spore protoplasm remain- ing within these little cups, and the body growing rapidly before division takes place ; a kind of loop is thus formed which gradually becomes longer and longer, and the two limbs which were supposed to be the result of a longitudinal division are nothing more than the two ends of the same rod. After a time transverse divisions may be seen in the different parts of the loop, the ends escape from the cups formed by the halves of the opened-up spore capsule, the rods straighten out and assume the regular straight form. There are other modifications of the same process of development from the spore, but the above are the essential or most important forms. CLASSIFICATION. Our present classification of Bacteria is based uponthat given by Cohn. He arranged these organisms into four groups, taking as his characterising feature the form that was most commonly assumed by each organism. His first group con- sisted of rounded bacteria or cocci—the Sphzro-bacteria ; the second group was made up of short rods, or cylinder-shaped bacteria—Micro-bacteria ; longer rods or thread-like organ- isms—the Desmo-bacteria—were placed in the third group ; and to the fourth group were assigned screw-shaped or spiral bacteria—the Spiro-bacteria. According to this author the cocci consist of rounded or ellipsoid bodies, .5 to 2m? in diameter, the smaller ones being spoken of as Micrococci, the larger as Mega- or Macro-cocci. ‘They are found singly, or may be grouped in pairs or in longer chains. The micro-bacteria or short rods are more variable in their size, measuring Ip in diameter and a little more than that in length ; but when the length of an organism reaches more 1 Ip = 0.001 mm, = zoohoooth part of a metre = zz 55th part of an inch. WHAT ARE BACTERIA ? 39 than about twice its diameter, it is usually spoken of as a Bacillus :—the length of a bacillus may be eight or ten times the diameter, which ranges from 1# to 2n. In some cases these bacteria take the shape of short stout spindles or lemons, especially during the stage of spore formation, when they are Photo-micrograph of Proteus Vulgaris Bacillus, in the form of shortrods, x 1000. spoken of as Clostridium forms, of which an exceedingly good example is the spore-bearing Bacillus butyricus. Bacilli increase in length, and, becoming more or less jointed as vegetative division takes place, form the long delicate jointed threads which are spoken of as Lepto- thrix forms when there is no apparent branching, but if pseudo-ramifications, such as are found in Cladothrix dicho- toma and are due to vertical division taking place in one of the terminal cells, are present, we have the Cladothrix form. 40 BACTERIA, The Spiro-bacteria vary very much, not only in size but also in length and in general appearance. é In some instances other isolated features have determined a nomenclature. For example, the appearance of sulphur in one series of forms has determined a species of Ophido- monades ; whilst the form and length of curves have also been used asa distinguishing feature. ‘The organism is spoken of as a Vibrio when the curves are slightly marked ; if the curves Photo-micrograph of Bacillus Figuraus in which Leptothrix form is well seen, X 150, are short and slightly pronounced and the organism is thin, it is known as a Spirocheta ; a ribbon-shaped spiral is a Spiromonad, and a spindle-shaped spiral, a Spirulina. COHN’S CLASSIFICATION. Schrzophytes, Thallophytes that develop by division or by ; endogenous germinating cells, : e WHAT ARE BACTERIA ? 41 TRIBE I. A. Free cells arranged in pairs or fours. Cells round—chroococcus (Naegeli). Cells cylindrical—synechococcus (Naegeli). B. Cells united into zoogloea masses by homogeneous gelatinous sub- stance. a. Cellular membrane shading off into the intercellular substance. Cells round—micrecoccus (Hallier). Cells cylindrical—bacterium (Dujardin). 4. Intercellular substance arranged in concentric layers. Cells round—glzocapsa. Cells cylindrical—glzothece. C. Cells forming circumscribed zoogloea masses with a definite shape. a. Families arranged in flat layers in a single plane—merismopedia. b. Cells rounded, arranged in a zoogloea mass forming a network— clathrocystis. ¢. Cells cylindrical or wedge-shaped, the families divided by con- strictions—ccelospherium. d. Cells forming families, dividing in several planes, colourless cubical cells with a quadrate arrangement—sarcina. e. A large and indefinite number of colourless cells—ascococcus. TRIBE 2. Filamentus forms in which the cells are thread-shaped. A. Without branching. 1. Colourless cylinders with little sign of division, very delicate, when short—bacillus, when long—leptothrix. 2. Similar filaments, but thicker and longer—beggiatoa. 3. Filaments deeply divided at intervals with colourless spore-bearing tissue and a well developed sheath—crenothrix. 4. Spiral filaments. ~ : Short and undulating—vibrio. Short with rigid spirals—spirillum. pone with flexible spirals and containing phycochrome—spiro- cheete. Filaments long and spirals flexible—spirulina. 5. Filaments in chains without phycochrome—streptococcus (strepto- bacteria). Zoogloea masses or cylindrical cells. ‘When colourless—myconostoc. In chains—nostoc. . Filaments thinner at one end——rivolaria. B. Filaments with pseudo ramifications—cladothrix. Cylindrical colourless filaments—streptothrix. It may be of interest to some to glance over a few of the classifications suggested by different authors, and to compare -the bases on which these classifications are made. Cohn, as we have seen, classifies entirely according to the elementary form of the.organism, the nature of the membrane and the mode of division, Wan Tieghem founded his classification s 42 BACTERIA, on much the same features, but he also takes into considera- tion some of the physiological and biological characters, the nature of the processes set up by them, the resulting products and the nature of the division. VAN TIEGHEM’S CLASSIFICATION. Van Tieghem places all the schizomycetes in a family which Photo-micrograph of Bacillus Figuraus Leptothrix and rod forms. x ro000. he calls bacteria, a family closely related to the nostocaceze and the oscillaria of the Alge; he divides them into micro- cocci; bacteria or short rods; bacilli or short threads; longer threads, without any definite sheath, being called leptothrix ; with a sheath, crenothrix ; with a sheath and undergoing division, cladothrix. WHAT ARE BACTERIA ? 43 The genus vibrio consists of spiral filaments which break up into short fragments. The spirillum consists of longer filaments that have a helicoid arrangement. The spirochete, of greater length, are spirilla with more numerous turns of the spiral. > Micrococci, arranged in zoogloea masses and held together by a thick layer of gelatinous material, he called ascococcus ; when held together, but without or with less of this gelatinous material, He gave them the name of punctula. Bacteria united by this gelatinous material are called ascobacteria ; without the gelatinous envelope, polybacteria ; bacteria in the form of spiral threads, and massed together, -form the myconostoc. These bacteria are again divided according to their pro- perties of forming colouring matter, chromogenes ; of setting up fermentation, the zymogenes; and of giving rise to disease in animals or plants, the pathogenes. He gave a further classification based on the mode of division of the primary cells : 1. The bacteria in which the division takes place in one axis only. This includes micrococcus, bacterium, bacillus, leptothrix, crenothrix, cladothrix, vibrio, spirillum, spiro- cheete, ascococcus, punctula, ascobacteria, polybacteria, and myconostoc. 2. The meristz, in which a membranous thallus divides in two directions but on one plane only, giving rise to the formation of characteristic tetrads. 3.. The sarcinz, in which there is division in three direc- tions, so that the resulting masses always remain cubical in form. Zopf's classification rests (Fligge, ‘Etiology of Infective Diseases,” p. 180) on the doctrine of pleomorphism, which cannot be accepted as in any way proved except in the case of a few well-known non-pathogenic forms ; but his classifica- tion may be accepted asa basis from which to work in bring- ing proof or disproof of the theory of pleomorphism, al- though it is not at present, at any rate, founded on a large number of facts. Zopf, who has studied most carefully the subject of pleo- morphism, holds that several forms may occur in the cycle 44 BACTERIA, of development of any species, and he has determined that both form and developmental series must be used in drawing up a classification. ZOPF’S CLASSIFICATION. Group 1.—CoccacE —These are as yet only known in the coccus form. To these the following genera belong : 1. Streptococcus (cocci arranged in threads like strings of beads). 2. Merismopedia, tablet cocci (division in two directions, leading to the formation of tablet-like flat layers of cells). 3. Sarcina, packet cocci (division in three directions, leading to the formation of bale-like colonies). 4. Micrococcus (the cocci become aggregated in irregular heaps). 5. And ascococcus (the heaps of cocci accompanied by marked formation of gelatinous material). Group 2.—BACTERIACE#.—These possess chiefly coccus, rod, and thread forms; the former may be absent; in the latter there is no distinction between base and apex. Threads straight or screw-like. Genera: 1. Bacterium, forms cocci and rods, or only rods which are arranged in rows to form ordinary threads ; spore formation absent or unknown. 2. Spirillum, threads screw-like, formed only of rods, or of rods and cocci; spore formation absent or unknown. . Vibro, threads screw-like, spore formation in the longer or shorter joints . Leuconostoc, forms cocci and rods, spore formation in cocci. . Bacillus, cocci and rods, or only the latter in the form of simple or twisted threads ; spore formation present. 6. Clostridium, the bacillus form in which the spore formation occurs in peculiar enlarged rods. Group 3..—LEPTOTHRICHE.—Coccal, rod, and thread forms; the latter show a distinction between base and apex; threads straight or screw-like, spore formation not demonstrated. Genera: 1. Crenothrix, threads jointed and enclosed in a sheath, cells contain no sulphur granules ; inhabit water. . 2. Beggiatoa, threads thicker than Crenothrix, indistinctly articulated, cells contain sulphur granules ; inhabitants of water. 3. Phragmidiothrix, threads without sheaths, successive divisions very numerous ; cells contain no sulphur ; inhabit water. 4. Leptothrix, threads with or without sheaths, divisions not very numerous or well marked ; cells devoid of sulphur. Group 4.—CLADOTHRICHEA.—Show coccus, rod, thread, and spirillar forms. The thread form is provided with a sheath, well-marked segments and pseudo-branches. Spore formation not yet demonstrated. Genus: Cladothrix. Winter and Rabenhorst’s classification, though very con- venient, is far from scientific, but it serves especially well for the classification of micro-organisms that are found in disease, as these, as met with in their host, usually correspond to one stage only of Zopf’s developmental cycle of any special organism. 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Sper “+ sgzodsopua Suturejuoo “uorstatp [eurpnytSuo SuroSsepun ‘spor pedvys-aypurds ‘speo1y) ON ta ae Spor 1yS1exJs ay} Jo ‘yNs JO afixay ‘spraryy uoryerajfe Aq patueduiosoe ‘uoreuioy JO s[[ao epsurg *jutod -a1ods jo ssaooid oy} 10 padeys-arpuids pue astq uaemjoq 10 ae “*-uoryeurtoy orods jo ssao0id ay} spud OM} oY} UDsaM19q SuULINp spor WYSreI}s ay} Jo uoresaye ON ainjonis Jo souvivedde Ul SdUaTAYIp OU SI 919q} yoy ur ‘spvary} 40 sutvyd JeZao] 10 1931048 ‘s]99 [Burts 10 suoqqit SY *SUOISIAIP UJIM Speary, L *yurod *sa1odsopus y}IM ‘Kaem JO yYSsre1ys spvsiyy sozods -oryje YIM ynq ‘sarodsopua ou { yerds 10 ‘AaeMm ‘jyStes speory ‘+ sazodsoryjie Surureyugo ‘sarodsopua ou ‘Aavm 10 YSrer]s spearyL sasseul vao[sooz pepunod ur pasuely see ‘ oa he fas “ sades3 Jo soupung O%'T byemsomt ut eae oe a ae ae aoe juoulasuviue ojtuyep Aue nou, 1 four Pe v “sureyo sis co ee os ee **+sazodsopua jnoyjIM JO yA Sur you nq ‘s}ysIe JO sInoj ul pasueuy (2) sarodsomyyze ATU *suleyo Joys (2) seaodsopua jnoyjtA fur 10 samoy ul pasuBuy nee wee vee aB1r] AIoA ‘ond se nou, *3ZIS A aoe ‘NOILVOIMISSVID SadddNH AGNV AUVA Ad “suey UI pasuBIIe 19909 asUuIS n eye Och ae eze zag < nzg Mm na 4 > a " ‘SHDVLS TALLVLEDFA FHL ONINAG SHAY AAdVHS-dOY ‘ADVIS AAILVLADTA HL ONIUNG SWYOT SADD0D *a0}souIh a ‘ayzypomds *(ouqia) winqndg *eoyeissag: “xuqjojdarT “supe “untae “(eraqoD) sysfoomyeI[D *BUIDIeG “sna20200sy *SN90090I 91] “xEOpRID |... 2 xuyjojdais J sosseul snounepes ystpunod ul peSofous spevoly yy uOTJeWIOJ YURI as[e} YIM Spy], “ g[txag ‘BuoT *yerds 10 Aavm speaiyL, ‘spearq} WO} 0} paytun ‘qeotpuryAo * prsir Grogs sy “joursr 3 tpAo s{ya spear | , an etitiebe *sorpunq ‘raZu0T S[[2D TeoHPUrl’® St uty} Aaa ‘Suoy spearyy, ul Jo “1949501 sprog, poyyeur sa ee pajueuides ‘yysrexs *payouviqun JO ‘payey Apounsip ‘oys spray, sprary L, speoiyL. -OSI SPpealy : Sat[ruey snounrjes Jo paytun-Ajasooy May v WIJ 0} Jay}2T0} padnosd Jo apsuis ‘srapurfAo yzOYs s[[PD Aroyduad ye syjao Jo zoAe] apduns v yy satuo[o; sdnoad render ULIOF 0} SISqWUNU ayUyap 3nq yews ut JayJed0} punoq s[jaD ae ie ++ SgTUOTOD IP snail Uloy 0} sraquinu 931u -yapul pue adel ul payrun s]}aD ts att ae SaSSEUl SNOUT}Y[Id snoydiowe ut 10 SUTeYD UT payuN Jo “payelost s][ID *sat[TWey PaquOSUINIIIO cee Apayayap ur payun sy]2> “sT129 YIM ~padeys-339 petty A[artque “pros saruojoa Jo jeotayds s]jaD ‘NOLLVOIAISSVIO S\NHOD dO NOILVOISIGOW SADDNTA WHAT ARE BACTERIA. 47 Van Tieghem and De Bary, followed by Hueppe, laying great stress on the method of reproduction, have made two great divisions of bacteria—those which form endospores and those which form arthrospores; but as Fliigge points out, this division is of little service for practical purposes, as spore formation is at present so imperfectly understood, that it is most difficult to follow the exact method of sporogenous reproduction in a Schizophyte. Winter and Fliigge attempt to get rid of their objection to Cohn’s classification by speci- ally modifying it to contain only the pathogenic organisms. They take no account, as already stated, of any morphological developmental cycle or of any biological features, but they give a useful and practical classification. Quite recently it has been suggested that bacteria should be classified according to the number and arrangement of the flagella developed, but as such classification is necessarily based on a single characteristic, and that one of the least important, and as it will be some time before a complete examination can be made in order to determine the number and arrangement of these flagella, such a classification may for the present be left out of consideration. The same holds good as regards Baumgarten’s classification; he divides bacteria into two groups—those which appear to assume a single form only (the Monomorphic) and those in which pleomorphism is a well-developed characteristic. In each he has three genera. In the first: the coccus, the bacillus and the spirillum ; and in the second: the spirulina, the leptothrix and the cladothrix. A similar remark applies to these, however, that applies to Zopf's classification. It may be of some service as a provisional classification, especially to those who are engaged in the study of the morphology and life history of bacteria. To the pathologist, however, these classifications are of comparatively little value except in so far as by their aid the morphologist is able to supply him with information as to the life history of bacteria outside the body. LITERATURE. Author already quoted. Hueppe. Bucuner.—Centralbl. f. Bakt. u. Parasiteuk. Bd. iv., 1888. Coun.—Beitr. z. Biol. d. Pflanzen. Bd. 1, Heft. 2, 1872 ; Bd. 1., Heft. 3, 1875. 48 BACTERIA, *CorRNIL AND Basrs.—Les Bactéries. Paris, 1890. DALLINGER.— Monthly Journ. of Micro. Sct. Sept., 1875. bE Bary.—Morphologie u. Physiologie der Pilze Handbuch der physiol. Botanik. Bd. 1, Leipzig, 1866. Dujarpin.—Histoire Naturelle des Zoophytes. Paris, 1841. EsRENBERG.—Die Infusionsthierchen als vollk. organismen. Leipzig, 1838. Fiiiccr.—Die Mikro-organismen. Leipzig, 1886. (Trans. Watson Cheyne, New Syd. Soc. London, 1890). Goopsir.—Fdin. Med. and Surg. Journ., p. 430, 1842. *LOFFLER.—Vorlesungen, tiber, die Geschichtliche En- Stes lung der Lehre von den Bacterien. Leipzig, 1887. NEISSER.—Virchow’s Archiv. Bd. 84, 1881. Van TirGHEeM.—Traite de Botanique. 1883. Zopr.—Die Spaltpilze. Breslau. Those marked * contain full lists of papers and references. CHAPTER III. Tue History or BACTERIOLOGY. Earliest Workers—Kircher’s Contagium Animatum—Bacteria in Fermenta- tion, Putrefaction and Disease—Early Classifications—Miiller—Abio- genesis — Needham — Biogenesis— Bonnet — Spallanzani — Schultz’s Experiments — Schwann — Later Experiments— Pasteur — Bastian— Colour and Fermentation—Cohn and Naegeli’s Classification—Henle’s Researches and Postulates—Pasteur’s Researches on Fermentation and Putrefaction—‘' Flower of Wine ”—“ Flower of Vinegar ”—Bacteria as Scavengers—Pasteur on Silkworm Disease, and Wine Disease— Germs killed by Carbolic acid—Origin of Antiseptic Treatment. Since Athanasius Kircher mistook blood and pus corpuscles for small worms, and built up on his mistake a new theory of disease and putrefaction, and since Christian Lange, the ‘professor of Pathological Anatomy in Leipzig, in the preface to Kircher’s book (1671) expressed his opinion that the purpura of lying-in-women, measles, and other fevers were the result of putrefaction caused by worms or animalcule, a “Pathologia Animata” has, from time to time, been put forward to explain the causation of disease. Crude as was his theory, and imperfect as were the observations on which it was based, it is marvellous that Kircher, with the simple lenses that he had at his disposal, magnifying only some thirty-two diameters or one thousand times, was able to make out as much as he did. The observations that he made were, naturally enough, not generally credited ; and the theories he formulated were received with chilling in- credulity by most of his contemporaries. Remarkable as were Kircher’s observations, still more wonderful were those of Anthony van Leeuwenhoek, a native of Delft in Holland, who in his youth had learned the art of polishing lenses, and who was able, ultimately, to produce the first really good microscope that had yet been constructed. Not only did Leeuwenhoek make his micro- scope, but he used it to such good purpose that he was able 50 BACTERIA. to place before the Royal Society of London a series of most interesting and valuable letters giving the result of his researches on minute specks of living protoplasm. The results of these observations are fully detailed in his collected works. Towards the end of 1675 he discovered in water, in an infusion of pepper, in the intestinal canals of horses, flies, frogs, pigeons, fowls, and even in his own diarrhcea stools, small moving and living forms of such extreme minuteness that other observers with the best apparatus they had at command, even with the accurate and lucid description he gave, could not for long confirm his results. It was not, however, until 1683 that he actually described and depicted minute organisms in material taken from the teeth, that we can at present recognize from his descriptions and drawings as bacteria. Describing them, he says: “I saw with very great astonishment, especially in the material mentioned, that there were many extremely small animals which moved about in a most amusing fashion ; the largest of these” (represented by him in an admirable figure) “showed the liveliest and most active motion, moving through rain-water or saliva like a fish of prey darts through the water; this form, though few in actual numbers, was met with everywhere. A second form moved round, often in a circle, or in a kind of curve; these were present in greater numbers. The form of a third kind I could not distinguish clearly ; sometimes it appeared oblong, some- times quite round. They were very tiny, in addition to which they moved forward so rapidly that they tore through one another : they presented an appearance like a swarm of midges and flies buzzing in and out between one another. I had the impression that I saw several thousands in a single drop of water or saliva which was mixed with a small part of the above-named material not larger than a grain of sand, even when nine parts of water or saliva were added to one part of the material taken from the incisor or molar teeth. Further examination of the material showed that out of a large number which were very different in length, all were of the same thickness. Some were curved, some straight, lying irregularly and interlaced.” Since, he says, “I had seen minute living animalculz of the same shape in water, I endeavoured most carefully to observe whether THE HISTORY OF BACTERIOLOGY. 51 these also were living or not, but I was unable to recognize even the slightest movement as a sign of life.” - In the material taken from the teeth of an old man who never cleaned his teeth, Leeuwenhoek found an inconceivable number of living animalculz which darted about more quickly than any he had ever seen before ; ‘‘ the largest were present in very great numbers, and waved about by the locomotion of their bodies. Besides these, other animalculz were present in such large numbers that the whole water seemed to be alive.” This admirable account really contains the first accurate description of the rod-shaped bacteria, motile and motionless, of longer threads or bacilli, of the spiral threads or spirilla, and of rounded micro-organisms or micrococci. It was con- siderably improved upon in a letter to the Royal Society, dated October 1, 1692, in which he speaks of small rounded animalculz, the diameter of which is a thousand times less than that of a fine grain of sand ; of organisms having a somewhat greater diameter than the round ones, and being five or six times as long as they were broad, equally thick throughout their whole length, and which moved slowly backwards and forwards through.a bending of their bodies. Along with these he describes what are evidently spirilla, with their characteristic movements, “a few organisms about the same length or slightly longer, which moved their bodies in comparatively marked curves, swam forwards or backwards, or twisted themselves in an extremely lively fashion.” He also observed still longer and more sluggish organisms, sometimes straight and sometimes bent. Although Leeuwenhoek did not attempt to theorize as to the meaning of the presence of these organisms in the mouth, we find that, in 1713, after finding similar organisms in the greenish pellicle formed on the surface of the water in an aquarium, he came to the conclusion that the organ- isms seen on the teeth found their way into the mouth through the medium of the drinking-water that had been stored in barrels, and that some of these found there a nidus in which they might multiply. The world that Leeuwenhoek thus opened up so thoroughly was rapidly invaded by other observers and theorists. The thoughtful physicians of the time believed that at last they had found the fons et ortgo mai, and Nicolas Andry, reviewing Kircher’s “Contagium Ani- matum,” replaced his worms by these newly-described 52 BACTERIA. animalcule or germs, and pushing the theory to its legitimate and logical conclusion, he also evolved a germ theory of putrefaction and fermentation. He maintained that air, water, vinegar, fermenting wine, old beer, and sour milk, were all full of germs; that the blood and pustules of smallpox also contained them, and that other diseases, very rife about this period, were the result of the activity of these organisms. Such headway did he make, and such conviction did his arguments carry with them, that the mercurial treatment much in vogue at that time was actually based on the supposition that these organisms, the cause causantes of disease, were killed by the action of mercury and mercurial salts. With a kind of prophetic instinct, and certainly as the result of keen observation, Varro and Lancisi ascribed the dangerous character of marsh or swamp air to the action of invisible animalcul ; in fact the theory was so freely and forcibly propagated that even where no micro-organisms could be found their presence was inferred, with the inevi- table result, as Léffler points out, that these “inconceivable” worms became the legitimate butts for the shafts of ridicule ; and in 1726 there appeared in Paris a satirical work, in which these small organisms received the name of “fainter,” “body-pincher,” “ulcerator,” “ weeping fistula,” “ sensual- ist ;” the whole system was thus laughingly held up to satire, and the germ theory of disease completely dis- credited. Linnzus, however, with his wonderful powers of observation and deduction, considered that it was possible that there might be rescued from this “chaos” small living beings which were as yet insufficiently separated and ex- amined, but in which he firmly believed might lie not only the actual contagium of certain eruptive diseases, and of acute fevers, but also the exciting causes of both fermentation and putrefaction. The man, however, who of all workers earliest recognized the importance of Linnzeus’ observations was a Viennese doctor, Marcus Antonius Plenciz, who with great shrewd- ness recognized the prime importance of these organisms in connection with the etiology not only of contagious diseases, but also of putrefaction. He it was who, at this time, insisted upon the specific character of the infective agent in every case of disease: for scarlet fever there was a THE HISTORY OF BACTERIOLOGY. 53 scarlet fever seed or germ—a seed which could never give rise to smallpox. He showed that it was possible for this organism to become disseminated through the air, and for it to multiply in the body; and he explained the incubation stage of a febrile disease as dependent on the growth of a germ within the body during the period after its introduc- tion, when its presence had not yet been made manifest. He very rationally explained the differences in the character of the symptoms and the'severity of the same disease by refer- ring them to differences in the constitutions and surround- ings of the patients. As regards: putrefaction, having corroborated Linnzus’ observations and found countless animalculz in putrefying matter, he came to the conclusion that this process was the result of the development, multiplication, and carrying on of the functions of nutri- tion and excretion by these germs; the products of fermentation being the volatile salts set free by the organ- isms, which, multiplying rapidly by forming seeds or eggs, rendered the fluid in which they developed thick, turbid, and foul. This theory, admirable as it was, and accurate as it has since been proved to be, could not then be based on any very extensive or detailed observation, and we find that some of the most prominent and brilliant men of the ‘ period did not feel justified in accepting the explanation that Plenciz had offered as to the causes of disease and fermenta- tion processes; and it was not until the years following 1831 that any real advance could be made in our knowledge of the presence of a “ Contagium vivum,” or living contagium element in the production of disease and fermentation. Previous to this, however, there was being gradually accumulated a large mass of facts bearing on these wonder- fully interesting minute living organisms, and numerous isolated observations were constantly being made by various workers, none of whom, however, were sufficiently master of their subject to enable them to make any systematic attempt at classification of their accumulated facts, and the scientific results were consequently comparatively small, standing in no proportion to the amount of work expended and the number of observations made. The first attempt to reduce this chaos to something like order was made by Otto Friederich Miller, of Copenhagen. ‘He thoroughly appreciated the work he was taking in hand. 54 BACTERIA. With a well-defined plan he set himself to systematize and arrange the various organisms that had been described by previous observers—commencing with Leeuwenhoek and ending with Spallanzani. When the nature of the optical apparatus Miiller had at his disposal is taken into consideration, it must be acknowledged that he succeeded in a most marvellous manner in classifying, on the Linnzean system, the minute organisms with which he had to deal. Under the head of Infusoria he divided them into two classes—those that could be seen with the naked eye, and those that were invisible except with the assistance of a microscope. The latter class he again divided into Membranacea, or those forming thin surface membranes, and Crassuiscula, or those forming thick membranes ; these latter, including Monas, Proteus, Volvox, Enchelys, and Vibrio, representing, he maintained, the lowest forms of animal life. Of Monas he described no fewer than ten species, and of Vibrio he was able to distinguish, by utilizing the characters of form, motion, nidus or cultivation medium, and other biological features, thirty- one species. Relying, however, principally on the form of the organism, he described rounded and slightly oval forms, shorter and longer rods, rounded and truncated cork-screw-shaped and snake-like organisms, ee but not spiral in their movements, and also long threads or acilli. Although he did not fully recognize the importance of his observation he described in certain organisms little shining points, arranged in series at regular intervals, especially in the rod-shaped forms, points which we must now conclude were spores. It is certainly not remarkable that he should never have understood the full significance of these spores, as even ninety years later, with all the additional light that had then been thrown on the subject, these bodies were still not properly understood. Later observers laid stress on the rapidity of movement of the vibriones or lineole, which were gradually separated from the other forms of lower organisms., The vibriones then described, including lineola, rugula, bacillus, and spirillum, correspond more or less per- fectly with our bacteria of the present day. Many advances were made after Miller’s work was com- pleted as regarcs the morphology of these organisms, but the question eas to whence these minute forms came still remained unanswered. Whether they were the result of spontaneous generation, or were the progeny of pre- existing forms, was the question which for over a century occupied the minds of those engaged in scientific research and speculation. Some observers, prominent amongst whom THE HISTORY OF BACTERIOLOGY. 55 were Hartsoeker, Reaumur, and Joblot, considered, though they had no great amount of evidence to adduce in support of their theory, that bacteria were the progeny of minute organisms which were present in myriads in the air, from which they were deposited on fruits, plants, and other matter, whence they made their way into the various infusions prepared from them. In this country a prophet arose in the person of Dr. Needham, who was really the first to suggest an attempted solution of the question by a theory of abioge- nesis, or spontaneous generation. Needham at first thought that these vibriones, or “plant animals,” as he called them, arose from plants by special vegetative power, and that from the plant-animals, by a process of evolutionary accretions, other organisms again arose. He tried to prove, by boiling a beef infusion and keeping it and allowing it to putrefy in a well-stoppered bottle (a most scientific method), that these zoophytes could not owe their origin to germs which outside insects or organisms had brought into the infusion, as he eonsidered that the boiling should have destroyed the germs originally in the fluid, and as no new germs could, he thought, make their way into the closely-stoppered vessel, the resulting organisms must be the result of the action of a special vegetative force.. This apparently logical and fascinating theory was accepted by many whose names had great weight in the scientific world. Needham’s observations were repeated time after time with the same results, and his theory met with wide acceptance. To very critical minds it appeared, however, that these experiments of Needham’s left loopholes for the inser- tion of other explanations than those which he gave, and Bonnet, of Geneva, suggested that the vessels used by Needham were not hermetically sealed, that an almost invisible opening would be quite sufficient to serve as a means of entrance to organisms so minute as those with which he was dealing, and that on the other hand there was a possibility that the germs were so far resistant to increase of temperature that they might live through a short period (afew minutes only) of treatment with boiling water, Abbot Spallanzani followed up, by his wonderful experiments, the theoretical criticism of Bonnet. After convincing himself that organisms did actually develop in unboiled infusions even when the outer air was rigorously excluded, he argued 56 BACTERIA. that the germs of micro-organisms, or eggs, as he termed them, might exist on the walls of the vessel, on the material of which the infusion was made, or suspended in the air within the vessel. To get rid of these germs from the vessels he heated the latter on the fire, then filling them rapidly with his infusions he allowed them to cool, and sealed them hermetically, but he still found that after a few days a number of organisms made their appearance. Could the organisms have got in along with the air during the process of cooling ? Toset this question at rest he made a number of infusions in hermetically-sealed flasks, and boiled them for a whole hour, with the result that in flasks so treated no organisms made their appearance : if, however, the sealing was in any way interfered with, organisms soon made their appearance, and he concluded that living germs were’ necessary for the development of putrefactive organisms. This fact once established, the whole question was much simplified, and the principle on which it rested was soon utilized in Paris agd else- where in the methods adopted for insuring the preservation of various food stuffs—methods which, with few modifications, have been handed down to the present day. It was of course objected that Spallanzani had shut out air from his vessels, or, that he had so altered the constitution of the air which still remained, that it was not possible for these minute organisms to develop in it. This objection was met in 1836 by F. Schulze, who put the question to himself, “If the access of atmosphere, light, and heat, to substances in flasks included of itself all the conditions for the primary formation of - animal or vegetable organisms.” To prove that this was not the case, Spallanzani’s conditions of absolute freedom from germs capable of development in the infusion must be obtained, and, secondly, air must be admitted to this infusion in considerable quantities ; but the air so admitted must be perfectly free from germs. Schulze proceeded as follows : he filled a flask half full of distilled water, to which he added various animal and vegetable substances. He gives the following description of the further methods of procedure :—‘‘I then closed it with a good cork, through which I passed two glass tubes bent at right angles, the whole being air-tight ; it was next placed in a sand bath and heated until the water boiled, and thus all parts fad reached the temperature of 212° F, While the watery vapour was escaping by the glass tubes I fastened, at each end, an apparatus which chemists employ for collecting THE HISTORY OF BACTERIOLOGY. S7 carbonic acid gas ; that on the left was filled with concentrated sulphuric acid, and the other with solution of potash. By means of the boiling heat every living organism and all germs in the flask, or in the tubes, were destroyed, and all access was cut off by the suphuric acid on the one side and by the potash on the other. I placed this easily-moved apparatus before my window, where it was exposed to the action of light, and also, as I performed my experiments during the summer, to that of heat. At the same time I placed near it an open vessel, with the same substances that had been introduced into the flask, having also subjected them to the boiling temperature. In order now to renew constantly the air within the flask I sucked with my mouth, several times a day, the open end of the apparatus filled with a solution of potash, by which process the air entered my mouth from the flask through the caustic liquid, and the atmosphere entered the flask from without through the sulphuric acid. The air was of course not at all altered in its composition by passing through the sulphuric acid in the flask, but if sufficient time was allowed for the passage the portions of living matter, or matter capable of becoming animated, were taken up by the acid and destroyed. From May 28th till the beginning of August I continued uninterruptedly the renewal of the air in the flask without being able, without the aid of a microscope, to perceive any living animal or vegetable substance, although, during the whole of the time, I made my observations almost daily on the edge of the liquid, and when at last I separated the different parts of the apparatus I could not find in the whole liquid the slightest trace of infusoria confervee or of moulds; but all the three presented themselves in great abundance a few days after I had left the flask standing open. The vessel which I placed near the apparatus contained on the following day vibriones and monads, to which were soon added larger polygastria, infusoria, and afterwards rotatoria.” Schulze was thus able to prove that the sterility was not dependent upon any alteration in the air within the flask, or to the small quantity of air contained in it, and that it was not due to any alteration brought about in the liquid by the heat- ing process, as on the one hand a large quantity of air was passing through the flask, whilst on the other the fluid that had been boiled, but which was left exposed, rapidly underwent decomposition, a decomposition that was accom- panied by the development of micro-organisms in very large numbers. The objection that some particles of sul- phuric acid drawn in with the air might affect the growth of organisms was met by Schulze by further experiments ; and Schwann, who, instead of using sulphuric acid, used heat as a means of destroying any particles that might be present in the air that was drawn into the flask, corroborated Schulze’s statements. Now came further objections from the supporters of abiogenesis, who stated, most definitely and categorically, that these workers were not dealing with germs at all, but simply with particles of 58 BACTERIA. albuminoid matter floating in the atmosphere, as a result of the vegetative power of which, organisms of various kinds, according to the conditions by which these particles find themselves surrounded, were caused to be developed. In 1854 Schroeder and Von Dusch made a great advance ; they proved that simple filtration through a layer of cotton wool was sufficient to deprive the air of its organisms, and so to render it unfit to produce decomposition in infusions from which germs had already been eliminated by heat ; and no longer than thirty years ago Hoffmann, Chevreul and Pasteur demonstrated that it was quite sufficient to draw out, and bend downwards, the neck of a bottle in which the germ- free infusion was contained, in order to ensure the continuance of a. non-putrefactive condition, and the perfect freedom of the fluid contained within the flask from germs: they argued that germs obey the law of gravitation, like all other solid particles, when not blown about by currents, and must settle down upon an upper surface, so that when the tube was bent downwards the organisms could not fall into the mouth. Tyndall gave demonstrative proof of this in his exquisite experiment of removing all particles from a glass chamber, first proving their entire absence by passing through the chamber a ray of light which could only be seen so long as particles remained suspended in the atmosphere. As soon as the ray of light disappeared from view he placed vegetable infusions which had been sterilized by heat within the chamber, with the result that they remained free from any trace of organic life for several weeks together. Schwann had already pointed out that blood, taken with certain precautions and introduced into a flask in which the air was kept germ free, might be preserved for a considerable length of time, without the ‘development of micro-organisms, and later (1857) Van der Broek showed that the juice of grapes, and urine as’ well as blood, might be kept free from decomposition and the presence of organisms if the apparatus into which they were received was first thoroughly sterilized by means of heat, and if the substances were not allowed to come in contact with the outside air. Numerous observers, especially Burdon Sanderson, Roberts, Lister, Chiene and Ewart, and Watson Cheyne in this country, and Rindfleisch, Klebs, Cazeneuve and Livon, Leube, Hauser and Marchand abroad, con- THE HISTORY OF BACTERIOLOGY. 59 firmed these observations and made many new ones in connection with the behaviour of milk, egg albumen, vegetable substances kept under certain conditions, and pieces of organs from freshly-killed animals to which organisms from the external air were not allowed to gain access. It seemed as though the adherents of abiogenesis had not a leg left on which to stand; but owing to the fact that certain organisms, especially when contained in such media as milk and cheese, withstand the action of very considerable heat, they still contested every inch of ground, though their foothold was being gradually but surely cut away from beneath them. Milk, which was one of the strongholds of - the abiogenists, was first sterilized, with absolute certainty, by Schroeder, who attained his end by subjecting the fluid for a considerable time to a temperature of 100° C., and then by Pasteur, who heated it to 110° C., for a short time only. Cheese still remained to them, and as late as 1872 Bastian placed a small piece of this substance in an infusion of white turnip which had been filtered and carefully sterilized. This was then boiled in a flask for ten minutes, and whilst still boiling was hermetically sealed ; at the end of three days countless living organisms were produced, as Bastian held, from non-living albuminoid material ; but Cohn, repeating the experiment, explained that the resting spores or resistant germs were enclosed in the substance of the cheese, and that they were thus able to resist the high temperature to which the outer surface of the cheese, but not its centre, was exposed. Duclaux’s later experiments with the Tyrothrix of cheese, which resists the action of a very high temperature for a considerable time, also helps to explain Bastian’s results. The matter has now been set at rest, and it is an accepted belief that bacteria or microbes, as these lowly organized forms are now called, may be destroyed by heat and by certain chemical reagents, and that when once destroyed in any medium, no other organisms can rise from their ashes, the medium remaining perfectly free from putrefactive changes until fresh germs are introduced from without. Harvey's famous dictum, omne vivum ex ovo, has thus come to have a far wider meaning than that which he originally attached to it. The triumphs of surgery, of preventive inoculation, of hygiene in relation to specific infective diseases, of preservation of food, have had their origin in the knowledge gained during the battle which waged round 60 BACTERIA. the question of Spontaneous Generation or generatio @guivoca ; and to the disciples of that school every acknow- ledgment.must be made and due credit assigned for the attitude of scepticism and free ingenious and honest criticism which they passed concerning half-formed and inadequately- supported theories and imperfectly-conducted experiments, for to their efforts is certainly due the fact that the experiments of their opponents became more and more. perfect, and if to-day we have perfect methods of sterilization and of making pure cultivations, it is because nothing was taken for granted, and because able men on both sides of the controversy were ranged against one another to fight the matter to the death. Whilst this battle over the origin and development of these micro- organisms was going on, intermittent attempts were made to improve on the classification that had been drawn up by Miiller, but it was only as the optician supplied observers with better microscopic apparatus that any further advances could be made. Ehrenberg, however, took up the question and divided the Monad family into rounded and rod-shaped forms ; these latter—Vibriones—he described as undergoing transverse division, as they increased in length. These he sub-divided into Bacteria, or short, straight, inflexible organisms; Vibriones, longer and more flexible; Spirilla, or inflexible spiral forms ; and the Spirochzetee, or the flexible spiral organisms. If to this classification we add the Cocci or rounded forms, we have prac- tically a rude model of that adopted by authors at the present time. In his vibriones he had six varieties, of which lineola, rugula, and bacillus, had already been described by Miiller, but subtilis, tremulans, and prolifer, were new. In consequence, however, of the want of marks of characteri- zation, Ehrenberg himself was very doubtful as to the propriety of his system of division or nomenclature. His spirillum comprises three forms, the old vibrio undula of Miiller, the ordinary spirillum, and a new kind of spirillum (Tenue). These three differed from one another only as regards length and thickness, the Vibrio undula having only from one to one and a half spiral windings, the others being merely longer or thicker. His genus, Spirocheeta, contained a single form only, the Spirochzta plicatilis, an organism of great length, but of very small diameter. In consequence of the active snake-like and rotary move- ment of these organisms, Ehrenberg was fully convinced that he had to deal with animals, and this opinion was uni- versally accepted down to the time when Davaine gave his opinion that bacteria must be classified as really belonging to the vegetable kingdom. In 1840 colour characteristics were brought into play as a means of distinguishing certain organisms, and we find that Fuchs and Ehrenberg describe Vibrio cyanogenus and Bacil- THE HISTORY OF BACTERIOLOGY. 61 lus xanthogenus, as giving rise during their growth in milk to characteristic blue and orange colorations. He described the exciting cause as small chain-like organisms, and considered that they were the cause of both the colour formation and the acid fermentive changes. As early as 1819 we find the first description of bleeding bread, the organismal cause of which, Ehrenberg, in later years, was able to cultivate on Photo-micrograph of Bacillus Prodigiosus. Xx 1000.—Organism forms beautiful red colour of bleeding bread, bloody sweat, &c. various food media, boiled potatoes, Swiss cheese, and white bread. He describes the organisms of which the coloured mass was made up as being exceedingly minute, and as having a characteristic movement quite distinct from the so-called Brownian or molecular movement. Some idea of the size of these organisms may be obtained from Ehren- berg’s calculation that a cubic inch would contain from 46,656,000,000,000 to 884,736,000,000,000 plants ; this organism he called Monas prodigiosa, 62 BACTERIA, but it is difficult to associate the characters that he gave with any of the red pigment forming organisms now known. Felix Dujardin added very little to the actual classifications given by Miiller and Ehrenberg, but he brought out several most important facts. In certain large vibrios he was able to make out distinct bifurcation, the two new limbs becoming segmented transversely ; he was also able to recognize an outer membrane or resistant covering on these organisms, and within this a gelatinous or protoplasmic material. This led him to doubt whether, after all, he was dealing with an animal form, and several of the forms described he relegated to the plant kingdom as Algae— Vibrio subtilis of the Oscillaria. i As Léffler points out, although Dujardin was not able to make any further advances on the classifications of Miller and Ehrenberg, his observations on the chemistry of bacteria were most important. He describes as specially suitable for the development of bacteria, fluids containing such substances as phosphate of soda, hyponitrous acid and oxalate of am- monia and carbonate of soda, and he points out that the nitrogen from the oxalate of ammonia is gradually used up in the presence of organic substances. With the exception of Dujardin, as we have seen, all observers up to 1852 had looked upon bacteria as belonging to the animal kingdom, but in this year Perty announced that of these minute organisms, some belong to the animal and some to the vegetable kingdom, whilst a certain number appeared to him to stand on the borderland between the two. These vibriones, he says, are colourless or sometimes blue, yellow, or reddish, never green, organisms, with scarcely a trace of any differentia- tion of their substance ; they have a spontaneous or automatic movement ; they increase in number by transverse division, partial or complete ; when this is incomplete, chains or threads are formed. He divides them into Spirilla or spiral threads, Bacteria or winding or straight threads, and he thinks that the bacteria have not only an active animal life, but that they also pass through a stage during which they must be looked upon as vege- table in character: a double existence which he assigned to other forms. In 1854 Cohn insisted even more strongly on the plant nature of these micro-organisms. He, too, described zooglcea masses, and he summed up his researches as follows :—(1) All vibriones seem to belong to the vegetable kingdom, and they exhibit a very close relationship to the larger alge. (2) In respect of their want of chlorophyll, and of their occurrence in putrefying infusions, the vibriones belong to the group of water fungi (mycophycez). (3) Bacterium termo is the active, moving form of a closely-related species of palmella and tetraspora zoogloea. (4) Spirochzte plicatilis belongs to the species spirulina, which we can at once indicate as a THE HISTORY OF BACTERIOLOGY. 63 particular species. (5) The long non-wavy vibrios (vibrio, bacillus, &c.), are related to the more delicate beggiatoa (oscillaria). (6) The shorter vibrios and spirilla, which correspond in both form and motion to the oscillarie and spirulinz, he was unable to localize in his system of classifi- cation. In 1857 Naegelicollected all the forms then known, which had certain characteristic physiological features in common, into a group which he termed Schizomycetes or fission fungi, a group which is now fully recognized by botanical mor- phologists and physiologists. He included all those lower forms of plant-life in which chlorophyll was absent, and which contained carbon, oxygen, hydrogen, and nitrogen in definite proportions, which elements they were not able to assimilate and utilize in building up their own substance from inorganic materials. They, like the other fungi and animals, can utilize as food only such material as is presented to them in the form of living or dead organic matter held in solution, or combined with a,considerable quantity of water. The processes going on within their protoplasm are so intimately connected with oxidization that they usually set free no uncombined oxygen, and this characteristic feature along with their want of chlorophyll colouring matter Naegeli looked upon as the special feature by which they might be dis- tinguished from ordinary fungi. Amongst his fission fungi he placed the forms bacterium, vibrio, spirillum, sarcina, the mother of vinegar, the yeast fungus, the organism associated with the silkworm disease—a small colourless oval organism, somewhat resembling a yeast, which he named Nosema bombycis—most of which were characterized by the features which are now recognized as belonging to the fission fungus group. The relation of these to disease and fermentation Naegeli declined to discuss. Although Leeuwenhoek had described certain micro- organisms in the tartar of the teeth and in various secre- tions and excretions so accurately and minutely, it was not until 1837 that any definite attempt was made to associate them with the products of a disease; in that year, how- ever, Donné described an Infusorian, which he likened the vibrio lineola of Miller, as occurring in pus in syphilitic diseases. This he thought at first was simply a vibrio associated with the putrefaction of the pus, 64 BACTERIA. but as he was afterwards unable to find this same infu- sorian in the pus taken from other abscesses, and in putre- fying material that had been exposed to air, he was led to inquire whether it was really characteristic of syphilitic contagion, and whether it played any part in the trans- mission of syphilitic infection. Although he afterwards retired from his first position he carried out a series of most careful and ingenious experiments, through which he was Jed to believe that very characteristic vibriones were associated with the causation and transmission of syphilis, and he thus opened up a series of most interesting questions, the answers to which, though differing somewhat from those given by Donné himself, were nevertheless destined ulti- mately to be very much on the same lines as those that he had laid down. It is interesting to notice that the monad forms which at the present day are again coming prominently forward in connection with the produc- tion of certain diseased conditions, were early recognized and described by Donné, and that Rudolph Wagner also described a species of monad as occurring in cancer of the lip. As was to be expected, however, the connection of micro- organisms with fermentation was proved long before a similar association was made out between micro-organisms and disease, and in the same year that Donné published his results, Cagniard-Latour and Schwann, who had been work- ing independently, announced that the yeast cells (torula cerevisiz), originally described by Leeuwenhoek, and which were found to grow in grape juice and malt wort were to be associated with fermentation—that they were indeed the cause of this process. For long, although the intimate connection between the process of fermentation and specific infective diseases was widely recognized, the efforts of most scientific observers were directed towards the elucidation of the causes of fer- mentation and putrefaction. It was, in fact, suggested that cholera might be due to the action of some ferment-causing organism, which might become lodged in and multiply in the intestine. In 1837, moreover, Bassi described a kind of yeast fungus, which he thought must be the cause of a miasmatic con- tagious disease in silkworms. He found extremely minute spores, on and within the bodies of the silkworms affected, THE HISTORY OF BACTERIOLOGY. 65 and although parasites had long been accepted as the cause of certain diseases in plants, this was really the first fully- described and well-authenticated instance of a fungus para- site giving rise to disease in any members of the animal kingdom. As the direct outcome of these researches, Henle, in 1840, was led to believe that the cause of miasmatic, infective and contagious diseases must be looked for in living fungi or other minute living organisms, and although he was unable, experimentally, to satisfy himself of the accuracy of his position, he was fully convinced that he was working in the right direction. It would indeed have been difficult, at that period, to satisfy every condition that he required to be fulfilled; the methods now in use were then unknown and have only been perfected by workers as it has been found necessary, from time to time, to comply in the most minute detail with Henle’s conditions, and as, one point being carried, it has been found necessary to advance on others. The first of these was that a specific organism should always be associated with the disease under consideration. As such presence, however, might be accidental, these organisms were to be found not only in pus but actually in the living body. As they might be, even then, merely parasitic, aud not associated directly with the causation of the disease, it would be necessary to isolate the germs, the contagium organisms and the contagium fluids, and to experiment with these separately with special reference to their power of producing similar disease in other animals. We now know that it has only been by strict compliance with all these conditions, again postulated by Koch, that the most brilliant scientific observers and experimentalists in England, France, and Germany have been able to determine the causal connection between micro-organisms and disease. After Henle’s work appeared a regular fungus fever set in; many skin diseases were proved to be the result of the action of fungi, and numerous internal diseases were, on very imperfect evidence, said to be due to parasitic agency ; and a large number of diseases which we now consider to be caused by the action of certain specific organisms in the system, were deemed, on very imperfect data however, to be due to these fungi. Cholera and typhoid fever became subjects of great interest, the stools from patients suffering 6 66 BACTERIA. from these diseases were carefully examined for organisms, and bacteria and monads were carefully described, but in no case could any definite proof be obtained that any of _these stood in any causal relation to either of these diseases. It was at this stage that Pasteur took up the work initiated by Cagniard-Latour and Schwann, who had first noted the connection between the growth of the yeast organism and fermentation. He applied to other processes of fermentation, such as those of lactic acid, butyric acid, and acetic acid, the same process of experiment and reasoning which they had fol- lowed, and he was able eventually to prove that the organic ferment in each case had specific characteristics, not only as regards its physiological action in setting up acertain definite form of fermentation, but also as regards the special mor- phology and mode of growth of the organisms that were found during and at the end of the process. As these experiments of Pasteur’s are now classical it may be well briefly to indicate the lines on which he worked. He first carefully observed the nature of the organic material in which certain fermentations took place, studying, both synthetically and analytically, the best medium for his purpose ; he then, by careful microscopical study, determined what organisms developed most rapidly during the special fermentation process. After making an artificial solution of the substance to be fermented, he added a small quantity of albuminoid material, and a trace of the ash of the special yeast that he wished to grow, in order that there might be sufficient of the necessary salts for the nutrition of the organism. This fluid was carefully sterilized by being boiled in flasks, to which only filtered air afterwards had access. To the germ-free solution he added a small quantity of his special yeast, and if he then obtained a characteristic fermentation with the production of the natural special fermentation products, accompanied by rapid growth and multiplication of the organism that he had introduced, he came to the conclusion that this organism was the cause of the special fermentation. In 1857 Pasteur described a new yeast (the cells of which were much smaller than the ordinary beer yeast), which gave rise to the formation of lactic acid from sugar, and he pointed out that the nitrogenous material which was necessary for the production of lactic acid was really needed for the THE HISTORY OF BACTERIOLOGY. 67 nutrition of the growing yeast, but that otherwise it did not exert any influence in transforming the sugar into lactic acid, as had hitherto been maintained by Liebig. Some idea of the delicacy of Pasteur’s experiments may be gathered from his observations on the conversion of racemic or paratartaric acid by a living ferment into right-tartaric acid (which in turn was capable of undergoing further fermentation) and into left-tartaric acid, which remained unaltered. He had already noted that the material in which fermentative changes took place, determined, in a very marked degree, the nature of the fermentation process. For instance, on adding dust, which of course con- tained a considerable number of different organisms, to sterilized urine, an ammoniacal or putrefactive fermentation took place; whilst on adding the same dust to sterilized milk an acid fermentation, as evidenced by the curdling of the milk, ensued, whilst in each case there seemed to be a special development of one particular organism. He had of course to contend with the difficulties involved in obtaining isolated organisms or pure cultivations, and for this reason he was for some time heavily handi- capped. All the forms of ferment-producing organisms which Pasteur had studied up to this point he spoke of as vegetable or yeast forms, and it was long before he was able to induce butyric acid fermentation. He at length found, however, a form which he distinguished as an infusiorian, in contradistinction to the vegetable or yeast form. This he describes as occurring in the form of small, straight, cylindrical rods, somewhat rounded at the ends, occurring either singly or in jointed chains of three, four, or more, about 24 broad and from two to ten times as long as broad. The organism has a slight gliding motion, and its reproduction takes place apparently by vegetative growth and transverse division. The physiological or bio- logical peculiarity of this organism is that it can exist apparently without a trace of organic nitrogen ; whilst, like the vegetable ferments with which Pasteur had been working, it can also live without oxygen, and although in form it is like a vibrio, it differs from the vibrios in this respect and also in that it is able to bring about fermentation. In 1863 he found a second anzrobic “ vibrio,” which he succeeded in cultivating. He prepared a solution contain- ing tartarate of lime, ammonia, potassium, and yeast ash, rendered it sterile or germ-free by boiling, and covered it with a thick layer of oil. To the fluid thus prepared he added a minute quantity of the organic deposit resulting from spontaneous fermentation of tartarate of lime ; the 68 . BACTERIA. was followed by a typical fermentation in the fluid, from which air was excluded by the film of oil spread over the surface. On microscopical examination of both the artificial and the spontaneous fermentations, he found “ vibriones” Ip thick and sop long. It was objected that in the spontaneous fermentation a certain quantity of air must necessarily remain in the fluid in which the organism was growing, so that the presence of oxygen could not be inimical to the growth of this “anzrobic” organism (a name that Pasteur gave to those organisms that are not dependent upon the oxygen of the air for their growth and development). He met this objection with proof that, grown along with other organisms, such as bacterium termo, which were dependent upon free oxygen for their existence, the latter developed and grew for a short time in the fluid and so used up what oxygen there was present in solution, at which point they were unable to develop further, so that other resulting changes must be due to the growth of the special anerobic organism. Passing from the butyric acid fermentation, and taking it as an analogy, Pasteur continued his researches on putrefactive processes in which nitrogenous substances and acrid and offensive smelling materials are formed, and he eventually came to the conclusion, which had already been expressed by Mit- scherlich in 1843, that as yeasts gave rise to fermentation so “vibriones” must be the cause of putrefaction ; and, going further, he assumed, what has since been proved to be erroneous, that the whole of the vibriones of putrefaction were anzrobic, that is, they could give rise to their specific products only when they were removed from the influence of the action of the oxygen of the air. In connection with the subject of anzerobiosis, Pasteur pointed out that the mycoderms known as “ flower of wine,” ‘flower of vinegar,” &c., were able to produce different forms of fermentation according to the presence or absence of oxygen. Mycoderma aceti, for instance, bringing about the splitting up of sugar into alcohol, z.e., setting up an alcoholic fermentation when there is too little oxygen present, but in presence of abundance of oxygen giving rise to the formation of acetic acid, then setting up what is known as the acetic fermentation. These researches eventually led Pasteur to the conclusion well stated by Duclaux, that ‘‘ whenever and wherever there is decomposition of organic matter, whether it be the case THE HISTORY OF BACTERIOLOGY. 69 of a herb or an oak, of a worm or a whale, the work is exclusively done by infinitely small organisms.“ They are the important, almost the only, agents of universal hygiene ; they clear away more quickly than the dogs of Constantinople or the wild beasts of the desert, the remains of all that has had life ; they protect the living against the dead; they do more : if there are still living beings, if, since the hundreds of centuries the world has been inhabited, life continues, it is to them we owe it.’ Without them the surface of the earth would be covered with dead organic matter, the remains of plant and animal bodies, which, retaining the elements necessary for the building up of new plant-life and animal bodies, would soon cut off the food supply of new plants and animals; life would be impossible because the work of death would be incomplete, or, as Pasteur puts it, “because the return to the atmosphere and to the mineral kingdom of all that which has ceased to live would be totally suspended.” From his experiments on fermenta- tion and putrefaction Pasteur, by a very natural transition, turned his attention to the diseases of wine, and then to those of the silkworm—diseases that specially affected two important French industries. After most careful research he found that the acetic fermentation, viscosity, bitterness and turning flat of wines, were all due to the action of certain organized ferments, most of which he was able to isolate and study. ; The acid fermentation, he found, was produced by the mycoderma aceti, which consists of short rod-like forms, about double as long as broad, slightly constricted in the middle; these individual elements, being joined together in long chains, which, as they grow on the surface of the wine, interlace with one another and form a regular film or skin. The bitterness, of wine he ascribed to the presence and action df branched tortuous filaments of about 1.54 to 4 in diameter, these chains‘ having a peculiar knotted appearance. The turning flat of wine was due to the presence of delicate unbranched filaments about Ip in diameter, which under certain conditions are broken up into short segments which some- what resemble the bacilli met with in a lactic acid fermentation. The cause of the viscosity of wine was an organism made up of cocci about 1.24 in diameter, often arranged in chains of considerable length. He found, indeed, that the relations of cause and effect were invariable ; wher- ever certain forms were present in addition to the yeasts, or were intro- duced after the yeasts, the result was a special fermentation superadded to the wine fermentation. ; The genius who had shed such a flood of light on the 70 BACTERIA. causation and prevention of wine disease now turned his attention to the ravages of the “ spot ” disease or “ pebrine” amongst silkworms, a disease that at one time threatened to destroy the flourishing silk industries of France. The organisms found in this disease had already been described by Naegeli as Vosema Bombyczs, and by Latour as Panhis- tophyton, small, glistening, oval corpuscles which appeared to -be endowed with life and to lead a parasitic existence in the tissue of the silkworm caterpillars. Pasteur was able to demonstrate their presence not only in the butterflies which develop from these worms, but also in the eggs they laid, and he found that where any one of the three forms was affected, the corpuscles were passed on to the next stage. He was able to show that they increased in the body, that they were the cause of the disease, and that by careful ex- amination and destruction of the affected eggs and the preservation of the healthy ones, the disease could gradually be eliminated. He found that another disease, the lethargy of silkworms, was also probably caused by the presence of micrococci, arranged in the form of chains, in the intestinal canal of the worms. Pasteur had thus succeeded in showing the relationship between certain micro-organisms and certain wine diseases, but he had also been able to demonstrate a causal relation between certain lowly-organized, parasitic organisms and a special disease in animals or insects. He had, in fact, demonstrated that certain specific organisms, endowed with definite morphological and physiological characters, gave rise by their presence to specific and characteristic diseases ; he had by observation and experi- ment extricated the theory of a living contagium from a condition of chaos, and he had assigned to definite organ- isms, each a special réle in the production of certain forms of fermentation, of putrefaction, and of disease ; and although much was still left, and still remains to be done, in the identification and classification of those organisms, he had separated a few distinct forms, and instead of assigning to these a general and common action in the production of the above processes, he had allotted to each one its own part. This he was able to do with such clearness and to place his experiments so lucidly before the scientific world, that there could be no doubt as to his meaning ; the consequence being that he soon secured a large following of enthusiastic THE HISTORY OF BACTERIOLOGY. 71 workers from amongst his contemporaries. On the other hand, however, he brought forward those who were by their researches led in opposite directions, or who, with less perfect methods, could not make their facts fit in with his theory, or who could not repeat or confirm his experiments. The most important point that he wished to demonstrate was that which related to the specific character of the various ferments in fruit juices. He was attacked most vigorously ‘on this subject by Lemaire, Bechamp, Hoffmann, and others, each of whom pointed out that in any fermentation experi- ments that he had made he had never seen a single organism only. (He had, in fact, never been working with pure cultiva- tions). This was undoubtedly a very forcible objection, and one which had to be met, but one which was easily enough over- come as methods of separation and isolation became perfected. Bechamp, who had found what he termed granu/es in the cells of living plants and animals, and even in fossil remains, held that these microzymas, as he called them, remained alive ; that they set up various forms of fermentation ; that under different conditions of food, separation from the cell, and external influences generally, they ran together, became altered in shape, and underwent various changes, so giving rise to the various forms which Pasteur had described ; all these processes going on concurrently or subsequently to the various changes that occurred in fermentative and pathogenic processes. He would, however, have nothing to do with any specific organism ; he considered that all organisms were merely the result of a new grouping and alteration of these microzymas separated from the cells, and that they were specifically affected by the various altered conditions in which they found themselves when removed from the cell in which they naturally occur. Both Pasteur’s positions were thus attacked. His first contention was that germs were the cause of fermentation in disease, and secondly, that each fermentation was due to the specific action of a definite organism. In his first contention his position was materi- ally strengthened by the observations of Lemaire who, after proving that the presence of carbolic acid was inimical to the life of the higher animals and plants, carried his re- searches a step further, and proved that the lower organ- isms were similarly affected by the same material, and he found that the addition of a small quantity of carbolic acid q2 BACTERIA. to fluids, in which putrefaction and fermentation would ordinarily take place, prevented the incidence of these processes. He found at the same time that the fermen- tations set up by chemical ferments, such as diastase and synaptase, remained entirely unaffected by the action of carbolic acid, and the result of his earlier experiments led him to believe that the process of fermentation was due to the action of living organized creatures which, like the higher plants and animals, could be killed by the carbolic acid. When they were allowed to develop freely they brought about fermentation ; when their growth was stopped, or they were killed, fermentation could not go on. The same reasoning, he thought, might be applied to infection and miasma, and he concluded that disease pro- cesses were the result of fermentations or decompositions going on within the tissues, and brought about by the above or similar organisms. Pus formation was the result of the action of germs falling from the surrounding air into a wound. By the application of his germicidal re- agents to wounds, vaccine vesicles, and suppurating surfaces, he attempted to destroy these organisms out- side the body whilst they were actually attacking the weak points. This was really the first step in the direction of an anti- septic treatment of wounds. Whilst treating by his method wounds in the human subject and in the dog he saw “ that pus remained entirely absent, or was reduced to a minimum, putrid alterations were absent,” and the wound healed rapidly. All these results were due, he maintained, to the destruction of the microzoa or infusoria by his carbolic acid lotions. The paramount importance of this theory was only afterwards fully appreciated and worked out by Lister, who saw that, owing to the difficulty of killing germs after they had once made their way into the tissues, it was absolutely necessary that such organisms should be prevented from gaining access to the wounds at all, and it is upon the attainment of this end that his well-known antiseptic treat- ment depends for its success. Accepting the truth of the statement that germs were the cause of fermentation, Lister also came to the conclusion, independently, that germs entering the wounds from outside might be the cause of suppuration, and since germs were THE HISTORY OF BACTERIOLOGY. 73 floating in the air, were suspended in water, and were attached to the instruments and bandages that were used in the treatment of wounds, he determined that it was necessary, by using some germicidal reagent, to kill all such suspended and adherent organisms before the various materials mentioned were allowed to come in contact with the wounded tissues. With a combination of experimental resource, patience, and brilliancy almost unparalleled in the history of surgical science, he, step by step, built up a theory and practice of antiseptic surgery, a theory and practice which rapidly revolutionized the treatment of wounds and the routine of ward management. He thus introduced a system which has affected the practice not only of those who believe in its accuracy, but of those who cannot bring them- selves to accept all its details, but who have nevertheless accepted its principles, sometimes even unwittingly. LITERATURE. Authors already mentioned. Duclaux, Davaine, Dujardin, Ehrenberg,* Hueppe,* Loeffler, Naegeli and Roberts. Anpry, Nicotas.—De la génération des vers dans le corps de 'homme. Amsterdam, 1701. Bonnet.—Considération sur les corps organisés, 2 t. Amsterdam, 1762. Bastian.—Beginnings of Life, London, 1872 ; Proc. of the Roy. Soc., No. 145, 1873; ature, 1873 ; Centralbl. f. d. Med. Wissensch, p. 521, 1876 ; Linnean Soc. Journ., Vol. 14, 1877 ; On Fermentation and the Appearance of Bacilli, Micrococci and Torulae in boiled fluids. Lond., 1877. Basst.—Del mal del segno, calcinaccio o mocardino. Milan, 1837. CHIENE AND Ewart.—Journ. Anat. and Phys. p. 448. April, 1878. *CHEYNE, WATSON.—Antiseptic Surgery. London, 1882. CAZENEUVE AND Livon.—Revue Mensuelle, p. 733, 1877. Coun.—Beitr. zur Biol. der Pflanzen. Bd. 1, heft 3, 1875. CAGNIARD-LATOUR.—Comptes rendus de l’Acad. des. Sc., t. 4, p. 905, 1873, sete HarTSOEKER.—Essai de dioptrique. Paris, p. 226, 1694. Horrmann.—Botanische Zeit., 5 and 6, 1860. Henie.—Handbuch der rationnellen Pathologié. 1853. 74 BACTERIA. Kiess.—Arch. f. Exp. Path. u. Pharm. Bd. 1, p. 206, 1874. LEEUWENHOEK.—Omnia opera sive arcana nature ope micro- scopiorum Exactissimorum detecta, 1722. ListER.—Trans. of Roy. Soc. Edin., 1875 ; Mzcro. Journ., 1878. Lruse.—Zeitsch fur. Klin. Med. Bd. m1., 1881. Mo ier. — Animalcula infusoria fluviatilia et marina, _. Hauniae (1786). NEEDHAM.—Observations upon the generation, composition, and decomposition of animals and vegetable substances, London, 1749 ; Notes sur les Nouvelles découvertes de Spallanzani, Paris, 1768. Pasteur.—Etudes sur la biére, 1876 ; Comptes rendus, t. L., p. 306, 1860; Comptes rendus, t. LVL, p. 734, 1863 ; Annal de Chimie et de Phys., 1862-1864; Etudes sur la Maladie des vers a soie, &c., Paris, 1870. Louis Pasteur.—His Life and Labours. By his son-in- law. Paris, 1883. Trans. Lady Claud Hamilton. London, 1885. RINDFLEIScH.—Virch. Arch. Bd. tiv., p. 397. 1872. SPALLANZANI.—Phys. u. Math. Abhand. Leipzig, 1769. ScuuLze.—Gilberts Ann. de Phys. u. Chemie. Bd. xxxix,, p. 836, 1836. Scuwann.—Gilberts Ann. de Phys. u. Chemie. Bd. u. 1837. SCHROEDER AND von Duscu.—Ann. der Chemie und Pharm., Bd. uxxxix., 1854; Journ. f. pract. Chemie, t. -xr., 1854. SANDERSON, BuRDON.— Quarterly Journ. Micr. Sctence, vol. XL, p. $23, 1871. TyYNDALL.—Phil. Trans., 1876-77 ; Haze and Dust, ature, Jan. 27, 1870 ; Essays on the floating matter of the air = sean to Putrefaction and Infection, London. 1881. VAN DER Brocx.—Annalen. der Chemie und Pharm., Bd. CXv., 1860. Wacner.—Arch. der Heilkunde. Bd. xv., p. 1, 1874. Those marked * give full lists of references. CHAPTER IV. BACTERIA AS THE CAusES OF DISEASE. Anthrax—Pollender--Davaine—Rayer—Laplat and Jaillard’s Observations controverted by Davaine—Pyzmia and Septiceemia—Salisbury on Bacteria as the Cause of certain Fevers—Johanna Liiders’ and Hallier’s observations on Pleomorphism or Polymorphism—Burdon Sanderson, Hoffman, and others, state that there is no Connection between Bacteria and the Higher Fungi—Demonstration of Infective Element in Anthrax Blood—Bacteria found in Organs in certain Diseases—Sarcina in Stomach—Specific Organisms—Specific Activities. * THE bacterium which, in its relation to the cause of a specific infective disease in man and the higher animals, has been most thoroughly studied is the Bacillus anthracis, an organism which, not only on account of its size but also because of its powers of adapting itself to conditions both outside and within the body, has been recognized comparatively easily, and cultivated in artificial fluids and on other nutrient media in which it grows luxuriantly, so that it has been possible to study more or less carefully its morphological and physio- logical characters. This bacillus was observed by Pollender as early as 1849 in the blood from the enlarged and pulpy spleens of cows that had succumbed to anthrax or splenic fever. He recognizéd that the bacilli were not fragments of broken-down vessels or coagulated fibrin, as had been sug- gested, but that they were probably small vegetable organ- isms similar to those described by Dujardin as “vibrio bacillus,” and he suggested that it was quite possible that these organisms were in some way or other associated with the appearance of anthrax disease. In the following year, and before Pollender’s description had been published (his full description was published in 185s), Davaine and Rayer described motionless thread-like organisms and rods in the blood taken from animals affected with splenic fever. These observations were confirmed by other observers, but it was not until 1863 that the micro-organ- 76 BACTERIA. ism was supposed to have any definite relation to the fever. Davaine, at that time stimulated by Pasteur’s investigations on the relation between micro-organisms and the butyric fermentation, was led to suggest that these rods were, in all probability, the actual and specific cause of the disease, which suggestion, along with his observations on certain cases of malignant pustule that were published in the following year, may be looked upon as the first real attempt to demonstrate the connection between the Bacillus anthracis and the diseases known as malignant pustule and splenic fever. Although he did not furnish rigorous proof of the connection he was so far successful that, except for the” cultivation of organisms outside the body and the pro- duction of the disease by means of the inoculation of pure cultivations, work which Koch afterwards completed, Davaine left no proof wanting. is In the meantime, however, Delafond had demonstrated the constant presence of the rodlets in the blood of animals affected with splenic fever, and had also suggested their plant-like nature. Davaine found that he was able to transmit the disease to healthy animals by means of the-inoculation of blood that contained these rods ; whilst where the rods were absent, although the blood might be exactly the same in other respects so far as he was able to observe, he could not produce the disease. He found that a single drop of diseased blood contained from eight to ten millions of these forms, and that by diluting the blood a million times, he was still able to produce the disease by inoculation. Subsequent observers, especially Leplat and Jaillard, objecting to his conclusions, said that similar rods had been found in other diseases. They pointed out that other elements in the blood—an extremely complex fluid—might be the cause of the infective disease, and that the rodlets themselves might be only an accidental factor. Leaving this safe ground, however, their further criticisms were based on the supposition that all the rod-shaped bodies were identical in structure and life history, and that conse- quently they should have the same effect when introduced into the body, and they concluded that because similar organisms taken from vegetable infusions did not produce anthrax, that therefore the rodlets in the blood could not be the specific infective agent. Davaine’s answer was that their contradiction of his statements was due entirely to misconcep- BACTERIA AS THE CAUSES OF DISEASE. 77 tion. He pointed out the different physiological characters of organisms taken from different media, and showed that different conditions were essential to the existence of different organisms, and, in connection with a number of inoculation experiments that they had carried on (with material sent by him, which they allowed to undergo decomposition, and by means of which rabbits were killed but no rods were afterwards found in their blood), that they had produced no anthrax because the incubation period was too short, because the splenic enlargement was absent, because the animals underwent much more rapid putrefaction, and because the disease was capable of being transmitted to birds—a class which up to that time had not been found to be susceptible to this disease. During this controversy Davaine pointed out that Pyeemia and Septiceemia could not be produced by the inoculation of true anthrax virus. He described the bacteridia of anthrax as totally devoid of movement in the blood, and carefully distinguished between them and putrefactive bacteria ; he demonstrated how these latter could, by their presence and activity, diminish the activity of the anthrax bacteria, and could in turn produce a septic condition, which was in all essential respects absolutely different from splenic fever. He demonstrated that these vibriones were vegetable and not animal, and recognized the most important fact that the environment, mode of nutrition, and products of excre- tions of the anthrax micro-organism had a most marked influence in modifying its activity and virulence. He did not believe in the possibility of infecting the organism of the foetus in utero, basing his belief on Brauell’s and his own experiments, and advanced the theory that the immunity enjoyed by the foetus was due to the filtering action of the placenta which, he contended, did not allow of the passage of solid particles either from the maternal to the fcetal circulation or in the opposite direction. He concluded also that certain species of animals were much more susceptible to the disease than others. He was convinced at that time that malignant pustule, malignant cedema and anthrax were all due to the presence of the same organisms in animals or in man, and in fact he initiated the whole theory of contagion from animals to man and vce versa, and opened up the immense and fertile field of the comparative pathology of infective diseases. In 1868 he 78 BACTERIA. almost completed his proof of the causal relation of the organism to the disease by the ingenious method of mixing a drop of virulent blood with a large quantity of water, and using the bacteria which fell as sediment as the medium with which to inoculate susceptible animals. With this sediment he was always successful in producing anthrax, whilst inoculation with the water taken from near the surface invariably gave negative results. It was not a perfect proof, however, and it was left for Pasteur with his filtration process, and for Koch by his pure cultivation process on solid media, to complete the proof that Davaine so ardently desired and worked to obtain. In view of our latter-day knowledge of bacteria, it is interesting to note that as late as 1870, or only twenty years ago, these bacilli of anthrax were declared to be albuminoid crystals ; whilst within the last ten years they have been described as being built up from the dédrzs of fibrinous filaments. About this time a great impetus was given to the theory of a living contagion—an impetus which unfortunately im- pelled numerous workers and theorizers to see the cause of disease in every germ that they found. First, Pasteur had formulated his germ theory of fermentation and putrefac- tion ; Davaine, in his descriptive and controversial papers, had insisted upon the connection between his anthrax rods and splenic fever, and in the animal parasitic world the etiological relation between trichina spiralis (a small round worm) and an acute fever, met with especially in pigs, had been fully demon- strated. Salisbury, in this country, thought that he had found the organisms that were the cause of intermittent and remit- tent fevers, of malaria, and of certain other forms of specific disease, but he was quite unable to give proof in any single instance. In Germany, Hallier took up the subject with eagerness. He was led to investigate the subject of Poly- morphism of the bacteria or fission fungi—a theory that had been advanced by Tulasne in 1851, and had been later worked out by Tulasne and De Bary. It was held, indeed, that yeast was simply a form or stage in the develop- ment of certain mould fungi, such as the Penicillia or Aspergilli. Pasteur had already pointed out the physiological likeness between the yeasts and the bacteria in their power of pro- BACTERIA AS THE CAUSES OF DISEASE. 79 ducing fermentation processes, and Hallier, who was well acquainted with these researches, concluded that if the yeast cells were part of a developmental cycle, the bacteria might also be taken to represent only short resting stages of the same or similar cycles. This was an especially seductive theory, as up to this time the origin of these minute forms had, as we have seen, been enshrouded in mystery, and had provided matter for the keenest controversy. A lady, Johanna Liders, was firmly convinced that the lower bacteria and yeasts developed in some way or other from the individual parts of the mycelium of certain fungi, or from their spores. Her observations were repeated, and there was a general concurrence of opinion that the bacteria were derived in some way from fungi and from other higher plant forms. Hallier, with his isolation apparatus, which consisted really of Schwann’s apparatus, to which an air-pump and a cotton wadding filter were added, and his cultivation ap- paratus, which corresponds practically to the potato-jar of to-day, with water to take the place of bichloride of mercury solution, came to the conclusion that the cause of almost every infective disease was to be looked for in bacteria, monads, and cocci, which in their turn were nothing but forms produced during the developmental cycle of one or other of the fungi aspergillus, penicillium, mucor, &c. Léffler says, in summing up the results of Hallier’s re- searches, “ He put forward the hypothesis that all contagia and miasmata are the products of fungi or algz which alone, on account of their small size, are able to pass through the fine capillary vessels, and that it was only necessary, in order to determine the nature of the original cause, first to find out the micfococcus and then to trace it back to the fungus to which it owed its existence.” By such new ideas, propounded with such an air of conviction and authority, Hallier made a most rofound impression on both the lay and scientific world. he whole system was so simple and clear and every part contributed so easily and naturally to form one harmonious whole ; every assertion was so definitely supported by micro- scopic observation and cultivation experiments that no doubt as to the correctness of the demonstrations seemed to be possible. : It was a somewhat noteworthy fact, however, that the pent- 80 BACTERIA. cillium occurred so frequently in his cultivations, and, as Brefeld and others pointed out, however complete Hallier’s isolation apparatus might be in itself, he did not take sufficient care to prevent the entrance of the spores of these various fungi when he introduced the micrococci and bacteria which he wished specially to study, so that, although nothing fresh might be added after he had introduced the seed material, his seed material itself might be a mixture of various kinds, and along with it he could not be sure that the spores of the larger fungi had not entered. It was a case, said Brefeld, of covering with a waterproof a man already drenched with rain. So faulty, indeed, were these experiments considered to be by Burdon Sanderson in this country, by Hoffmann, Rindfleisch, Manassein, and Ferdinand Cohn abroad, that these observers undertook various experiments to prove that not only was there no connection between bacteria and the higher fungi, but that there were actually cases in which the micrococci did not develop into the longer rod-shaped bacteria. Moulds could only be developed in artificially pre- pared food solutions when the seeds or spores of moulds were sown; whilst bacteria seeds or germs, when obtained free from the germs of moulds, would in similar solution give rise to the development of bacteria only. There was soon a reaction against the whole of Hallier’s teaching, and it was now pointed out that he had seldom or never been able to reproduce any disease by inoculating cultiva- tions of the organisms that he grew, and the theory of living contagion fell into discredit, though the fact must not be ignored that Salisbury’s and Hallier’s work led to further consideration of many points associated with the relation of bacteria to disease, and that eventually it exerted a marked influence on the germ theory of disease. Hallier undoubtedly laid great stress on the fact that a micro- coccus was the cause of certain diseases, and he pointed out that its extreme minuteness was in favour of its being able to enter readily and retain firmly its position in the body. In 1868-9 Davaine and Chauveau succeeded in demon- strating that in all probability the infectious element in anthrax blood (Davaine), and in glanders pus and vaccine lymph (Chauveau), and in vaccine lymph (Burdon Sanderson) was not merely a soluble poison, but some solid material, such as a leucocyte, and probably a BACTERIA AS THE CAUSES OF DISEASE. 8i tnicro-organism. The blood or pus was diluted many times with water, the sediment was washed again and again, each time being allowed to settle at the bottom, after which the supernatent fluid was found to have no effect in producing any disease, whilst the sediment which contained pus organ- isms and “fine granulations” almost invariably set up the disease process. The virus must accordingly, these observers thought, be a solid poison, and must be looked upon as a particulate body. These observations thus confirmed, to a certain extent, Hallier’s suggestion that a micrococcus or a bacterium was the cause of most specific infective diseases. From this time onwards a large number of observations were made on various infectious diseases and micro-organisms. Micrococci were found in diphtheria, in scarlet fever, in rinderpest, septicaemia, and in other specific infective conditions, though Traube, in 1864, had made what might be con- sidered the first practical application of what had been discovered to be an important pathological condition when he demonstrated the fact that, if bacteria found their way into the bladder by means of a dirty catheter, a severe attack of inflammation of the bladder followed—an observation which was supplemented by Klebs, who demonstrated the connection between small abscesses in the kidney and the introduction of micro-organisms into the bladder. But with all these records there was very little of definite value to demonstrate the causal relationship between bacteria and disease, and even when fragments of diphtheritic membrane and of the wall of abscesses were introduced under the skin of an animal, and gave rise to both local and constitutional symptoms, there was no proof Prthooming that these were due to the micro-organism, and not to such special chemical products as had already been separated from putrid and diseased materials. Panum had demonstrated, in 1856, that it was possible to obtain from decay- ing flesh infusions an extremely poisonous substance, and his results were confirmed by numerous observers, some of whom succeeded in combining with acids the ‘‘ basic” substance that Panum had separated ; the sul- phate of this base when injected into frogs proved fatal, and eventually Ziilzer and Sonnenschein prepared what they described as a septic alkaloid which was stable in character, and in its reactions resembled most remark- ably the vegetable alkaloids, atropin and hyoscyamin. It was natural that as ihese materials could be separated by chemical means from diseased and putrefying materials, they should be looked upon as the primary and real etiological factors inthe transmission of disease. In 1871, however, Reckling- hausen, turning his attention to bacteria, was able to show that in the organs of patients affected with various infective diseases (such as blood-poisoning, and puerperal fever, typhoid fever, acute articular rheumatism, gangrene of the lung) small accumulations of micrococci were present, and that these were probably the cause or the agent by which deposits of the abscesses or gangrenous patches occurred in different parts of the body and in different organs. These micrococci were described as having an exceedingly sharp outline, as being extremely resistant to strong acids and alkalies, and, in fact, as being in most other respects like those that had been described as 7 82 BACTERIA. occurring in diphtheria and in abscesses of the kidney. . These results, with some modifications and additions, were almost immediately confirmed by Waldeyer and Weigert; Recklinghausen and Weigert concluding that these micrococci were all in the lymphatic vessels, Waldeyer, on the other hand, holding that some, at any rate, were contained in the blood-vessels. In 1872 E. Klebs found in pus, organisms which he de- scribes most graphically as rod-like bodies, the so-called bacteria, motionless, frequently grouped in short chains, or in longer threads. He also found numerous micro-spores, very minute refractile organisms, whose diameter might be at most .5z, some lying isolated and free, and having oscillating movements, others linked together in rosary-like threads. Having found them in the discharges he examined granulation tissue (raw or “ proud” flesh), in lymph canals, in spaces in the septaor partitions between muscles, in inflamed marrow of bones, and in the ulcerating cartilage of diseased and injured joints, in the walls of blood-vessels and in thrombi or clots formed in the vessels and attached to their walls. These organisms were in all cases situated near the primary wound (the researches were carried on during the Franco- Prussian war), but having found the micro-organisms in this position he next traced them to the abscesses that formed in distant internal organs in cases of pyemia. In fact, wherever there were points of secondary disease or suppura- tion, there he was able to demonstrate the presence of these organisms ; whilst in very severe cases of blood-poisoning he was actually able to demonstrate their presence in the circu- lating blood. Inconsequence of this constant presence of the bacteria and micrococci in these various’ disease areas, he felt justified in concluding that the organisms he had observed and described were the cause of the pyemic and septiceemic conditions, and that also to their action was due the forma- tion of pus, of abscesses, and even of ulcerative inflamma- tions of the, vessel walls. There can be little doubt that the ingenious experiments devised and carried out by him and his pupils formed a groundwork on which succeeding investigators found it possible to build up the present mag- nificent structure. He conceived the idea of separating the micro-organism from the poison which it produced by means of baked clay cylinders, and found that fluid so treated, although it gave rise to constitutional disturbance when in- jected into the blood or under the skin, did not induce BACTERIA AS THE CAUSES OF DISEASE. 83 suppuration, nor did it cause death : but if to this a quantity of the micro-organisms were added, and the fluid was then injected into dogs, these animals succumbed to a true pyemia, accompanied by the formation of abscesses, especially near the points of inoculation. He showed that these organisms were not present in the normal healthy blood; he also, by means of ingenious apparatus, which he contrived or modified, was able to observe the actual multiplication of the micrococci under the microscope. He introduced the method of fractional cultivation, but not in the complete form in which it was afterwards used by Lister and Pasteur, as he relied on the more vigorous growth of a certain organism that was placed in a special fluid and under special con- ditions, rather than on the dilution and isolation of individual organisms in small drops of fluid. Klebs was, however, able, by his biological method, to obtain comparatively pure culti- vations of several bacteria and micrococci, and he was the first to distinguish the division going on in various planes. Klebs’ anatomical investigations were confirmed by numerous observers, Birch-Hirschfeld, Heiberg, Orth, and Hueter, the last of whom tried to build up a system of pathology on the basis of the researches that had already been carried out ; these, however, afforded very incomplete data on which to work. Pysemia was, he considered, the result of the invasion of the tissues by micrococci, with the formation of little plugs in the vessels, around which abscesses were developed ; septicemia was a general poison- ing by absorption from a localized formation of bacteria in the body ; anda third form, which he looked upon as putrid poisoning, was the result of the absorption of poisonous matter formed by vibriones existing outside the body. The only difficulty in the matter was that the same organism appeared to produce very different conditions, such as pyzmia, septi- ceemia, puerperal fever, pyelo-nephritis, typhoid fever, phthisis, small- pox, diphtheria, cholera, rinderpest, whilst even in the healthy ‘body organisms were sometimes found, especially in certain positions, a fact which at that time was not reconcilable with the various theories that had been advanced. Another objection stated was that as the bodies of patients who had died of pysemia and septicemia putrefied rapidly, they formed an especially good medium in which innocuous organisms might grow, and it was maintained that organisms were found in such bodies after death, because of the special suitability of the soil for their growth, and that they were therefore probably rather to be looked upon as accidental concomitants and consequences than as essential factors in the production of disease. There now also appeared great activity in the French school: Pasteur had shown that most of the processes of putrefaction were due to the presence of motile, anzrobic vibriones, whilst Davaine had observed only non-motile rod- 84 BACTERIA. lets in the blood of animals that died from anthrax. The latter was further able to prove to Jaillard and Laplat that these non-motile organisms of virulent anthrax were actually rendered inert by the growth amongst them, or alongside of them, of motile putrefactive organisms, and that the action of the anthrax organism was also considerably modified by the growth of septic organisms, which, in turn, when introduced into an animal by inoculation, were capable of producing a disease which might perfectly easily be distin- guished from anthrax. The knowledge that the septic con- dition was not due to ordinary putrefactive organisms was further augmented by Birch-Hirschfeld, who demonstrated that pus which when fresh was infective lost much or all of its specific activity when putrefactive organisms were allowed to grow in it; so that these putrefactive bacteria at any rate could not be looked upon as playing any part in the production of wound infection. Then specific forms began to be associated with specific disease conditions, or kinds of putrefaction and fermentation ; the Goodsirs described sarcina in the acid con- tents of dilated stomachs; Trecul found his Bacillus amylobacter, with its characteristic tadpole shape, which gave rise to no wound infection, and numerous others were observed, none of which could be proved to have produced. septic conditions. An organism which produced the color- ation met with in blue milk was described by Fuchs, and another perfectly distinct micro-organism was described by the same observer as giving rise to a yellow colour in milk. That the milk was not absolutely necessary for the nutrition of these organisms he proved by making artificial cultivations on other media, and he gave very fully the conditions under which they could continue to exist ; they were destroyed by a temperature of 50° R. to 55° R.; freez- ing did not interfere with their power of propagation when again thawed, and after being dried and again moistened they were able to develop in milk and to give rise to the characteristic coloration. The way was thus being prepared for the discovery that specific organisms had, under certain conditions, specific actions and activities. If a peculiar organism was found to be associated with the production of a special kind of colouring matter, and if special fermentation and putre- faction processes were induced by individual organisms, was BACTERA AS THE CAUSES OF DISEASE. 8 5 it not probable, in the light of Davaine’s work, that special diseases would ultimately be found to be associated, not all with the same organism, but each with a special form ? LITERATURE. Authors already mentioned. Cohn, Chauveau, Dujardin, Hoffmann, Klebs,* Léffler, Schwann. BrRAvVELL.—Virchow’s Arch. Bd. 11, p. 132, 1857 ; Bd. xiv P- 432, 1858. BREFELD.—Bot. Unters ii Schimmelpilz. Leipzig, 1074. BircH-HirscHFELp.—Archiv. der Heilkunde. Bd. xm, p. 389, 1872, and Bd. xiv., p. 193, 1873. Davaine.—Revue Scientifique. Ser. 1, t. v., p. 78. Comp- tes rendus, pp. 320, 351, 386. 1865. Drtaronp.—Recueil de méd. vét. 1860. ; pE Bary.—Untersuch. i d. Brandpilze. Berlin, 1853. Bot. Zeitung, 1854. Fucus.—Gurlt. u. Hertwig’s Magazin f. d. gesammte Thier- heilkunde, Bd. vi. Goopsir.—Fdin. Med. Journ., vol. tv. 1842. HerserG.—Die puerperalen u. pydamischen Processe. Leip- zig, 1873. HvetTer.—Die allgemeine Chirurgie. Leipzig, 1873. JAILLARD AND LapLtatT.—Comptes rendus, t. LIx., p. 748, 1864 ; and t. LxI., pp. 368, 523 ; 1865. Kocu.—Cohn’s Beitr. z. Biol. d. Pflanzen, Bd. u., p. 277, 1876. Livers, JoHANNA.—Schultze’s Archiv. fur Mikr. Anat., Bd. 1., p. 317, 1867. ManassEin.—Wiessener’s Mikr. Unters. Stuttgart. 1872. OrtTH.—Virch. Arch. Bd. Lvut., p. 437, 1873. PasTeuR.—Comtes rendus, t. Lu. 1861. Panum.—Bibliotek for Laegar, 1856. PoLLeNDER.—Casper’s Vierteljahrschrift, Bd. vim, p. 432, 1855. RINDFLEIsCH.—Virch. Arch. Bd. Liv., p. 397, 1872. Sauispury.—Amer. Journ. of Med. Sctence, Jan., 1866, and Jan., 1868. Sanperson, Burpon.—/Vature, vol. vit, p. 478. Bret. Med. Journ., p. 119, 1878. Quart. Journ. Micro. Se. 1871. 86 BACTERIA. TRECUL.—Comtes rendus, t. LXI., p. 156, 1865 ; and t. LXV., p- 927, 1867. Puteswes—Comtes rendus. t. XXXII, p. 427-470. March, 1851. Von RECKLINGHAUSEN.—Virch. Arch. Bd. xxx., p. 366, 1864. WEIGERT.—Centralbl. f. d. Med. Wiss. No. 39. 1871. ZUELZER UND SONNENSCHEIN.—Berl. Klin. Wochenschr. No. 12, p. 121, 1869 and 1872. * Full list of references given. CHAPTER V. FERMENTATION. Fermentation ; a Key to the Whole Position—Chemical Fermentations— Illustrations—Organic Fermentations—Alcoholic Fermentation—Re- sult of Activity of Living Protoplasm—Lactic and Butyric Fermenta- tions—Cagniard Latour—Schwann—Reess—Hansen—Liebig’s Theory of Fermentation— High Beer—Low Beer—Method of obtaining Pure Yeasts—Hansen’s Classification of the Saccharomyces—Spore Forma- tion—Film Formation—Characters of Species of Saccharomyces— Metschnikoff’s Monospora—Torule. From what has preceded it will be evident that if any light is to be thrown on the subject of the production of organic poisons in the course of disease a careful study of the subject of fermentation is a necessary preliminary ; for, taken in its widest sense, fermentation includes all those processes in which there is the formation of special ferments and of special products as the result of the life-history of certain vegetable organisms. It is quite as rational to speak of a putrefactive as of an alcoholic fermentation, and we might even go beyond this and speak of a colour fermentation or a disease fermentation, as the organisms by which one or the other is started, and the conditions under which they are carried on, have many features in common, and, in fact, do not differ more than do the causes and conditions that are already known as associated with true alcoholic fermenta- tions, Fermentation consists, essentially, in the breaking up of chemical compounds, the molecules of which they are com- posed being separated from one another for a brief period, and then allowed to combine and form simpler and more stable compounds. Owing to the setting free of such energy as has been stored up in the highly complex fermentable substance which is no longer required to maintain the- - high level of combination, a certain proportion of this energy is released in the form of heat, the temperature of 88 BACTERIA, a fermenting fluid being found to rise without the addition of any external heat. An object lesson may be used to illustrate the processes of breaking down and re-combination that go on in fermentation. Let us suppose that we have a tall tower built up of two kinds of blocks, placed one on the top of another ; first a large block and then three small ones, then another large block and then three more small ones ; let us consider that there are pulling on these blocks elastic bands of different strengths, one very strong one between the two large blocks, another very strong one between each pair of the small blocks, and two sets of three weaker bands run- ning from the larger blocks to the smaller ones; these sets of blocks are so arranged that if left perfectly undisturbed they will remain piled up one on another forming a tower of considerable height. The higher the tower the more easily, as a rule, will it be upset, though this depends on the way in which the larger and smaller blocks are distributed in the structure. If, now, some disturbing element comes in, and if one of the blocks is withdrawn or is even slightly shaken, the whole structure may collapse, even the movement of a small top block may bring this about by altering the tension on one of the elastic bands, so disturbing the equilibrium of tension on the whole tower, that the two large blocks, with their strong elastic band, are set free and fall to the ground ; the pairs of smaller blocks, with their strong uniting bands, also fall to the ground, and the other thin bands of which we have before spoken are now so far stretched or broken that their influence in holding the different sets of blocks together may now be left altogether out of account. But in falling from the top of the tower to the bottom energy of position has been lost, and in falling and striking the ground a block may do a certain amount of work ; if it were in connection with a series of. pulleys it might be made to lift a certain weight ; if it were to strike the ground with sufficient force it might produce a flash of light, or a certain amount of heat. We are, perhaps, not justified in stating that this is exactly analogous to what takes place in fermentation, but it is sufficiently so to explain the nature of the process, if we consider that the kinds of blocks that are built into our tower are multiplied. In the most characteristic and best form (for our purposes) of fermentation that can be taken as an example, we have FERMENTATION, 89 grape sugar, consisting of three kinds of blocks, six of car- bon, twelve of hydrogen, and six of oxygen, all built into a high tower of twenty-four blocks (sugar, C,H,,O,).- In the process of fermentation the equilibrium is disturbed, and we have the tall tower replaced by four smaller ones, two con- sisting of three blocks each (2CO,, carbonic acid), and two consisting of nine blocks each (2C,H,O, alcohol), and the heat that is evolved in the process may be represented by the sum of the figures representing the distances which these various blocks have had to fall from the high tower into the lower ones, multiplied by the figures representing the mass of the volumes. Other features have to be taken into account, but the above example will serve to explain part of our meaning. But this is not all. The children’s line of “soldiers” may be used to explain another feature: a child arranges its wooden “bricks” in a long line, placing _ them on end ; if they are all of equal length the distances be- tween them are also made equal ; if they are of unequal length, they are so arranged that one block falling will just touch the one next to it. If the end block be now so disturbed that it loses its uustable equilibrium, and so falls as to acquire a stable equilibrium, it not only loses its own unstable equili- brium, but it upsets that of the brick next to it, which in turn acts on its neighbour, and so on till the whole line is brought to the ground. If, instead of upsetting the first brick at once, you simply give it a slight tap and only just move it, instead of falling over, it sways backwards and forwards fora little but then settles into its original position ; you give it another tap, it sways a little further, but still returns to its erect position ; whilst if now you give it a good strong push, or if two of you tap it at the same time, over goes the brick; bringing along with it the whole line to a condition of stable equilibrium. The towers or the rows of erect bricks are fermentable substances, the disturbing forces are those agents that bring about fermentation, and the smaller towers or the bricks laid down in rows instead of on end are the products of fermentation. The larger number of smaller towers contain exactly the same elements as did the smaller number of larger ones. They had energy of position, which is converted into kinetic energy, exactly in proportion to the distance that the elements have to fall; some of the energy appearing in the form of heat, other parts being used up in 90 BACTERIA. the rearrangement that takes place when the smaller towers are formed. It may be asked, How does the tilting over of the bricks bear on’all this? The first tap that you give them may be said to represent the action of some ferment ; the tap that your friend gives at the same time may be held to represent one of.the conditions essential for the production of a ferment, say the presence of a certain amount of light ; whilst the tap that a second friend gives may be looked upon as representing a certain degree of temperature ; it may be a forcible tap, representing 40° C., or it may be a slighter one, representing a temperature of 20°C. only, in which case you do not get sufficient energy out of the ferment to start the process. One or two only of your taps may upset certain towers or certain lines of bricks, but other towers that have a somewhat broader basis or a’ more definite arrangement require the whole three forces to be applied, and these have to be applied in certain definite proportions if the same results are to be obtained in every case. Thus, for example, by graduating the power of the disturbing force, the equili- brium of your tower may be simply upset and the elastic bands drawing in certain directions cause a rearrangement of the blocks according to the strength with which these bands pull; if, however, instead of simply just distributing the equilibrium to allow of the new arrangements taking place, your tower is struck with a sledge hammer, or over- turned by a pistol shot, or by the explosion of a charge of dynamite near it, the conditions are so altered that you cannot rely upon any plan of rearrangement being adhered to. Asaresult you simply obtain a series of disconnected blocks or of much smaller and irregular towers. It will be said that this regular breaking up is exactly what takes place in the breaking up of chemical combinations. This is perfectly true, for that is exactly what fermentation is. It is the upset- ting of the equilibrium of unstable compounds by most deli+ cately adjusted forces which are so accurate, so constant, and so delicate, that the stronger affinities of certain elements for one another are allowed to act to their full extent, and regular stable combinations are formed always in a definite manner. Let us take first such a substance as nitrogen trichloride or nitrogen teriodide. If a sharp blow be given toa small quantity of either of these materials, or if either be heated to a certain temperature, it will immediately break down into nitrogen FERMENTATION. g!I and chlorine, or into nitrogen and iodine. Thus a tower made of 2NCI1,, or of two large and six small blocks, is trans- formed into two towers, one composed of two large blocks and one of six small ones, or rather into four towers, one composed of the two large blocks and three each composed of two small ones (2NCl,=N,+3 Cl,), and along with the falling of these molecules into their lower positions there is a setting free of energy which manifests itself in the form of heat and light. Consider now that in your tower or in your row of bricks you have continual oscillation going on ; the elastic bands are being continually pulled upon by certain forces which we may say are light and heat ; there is continual motion in every part of the tower, or the bricks standing on end are continually oscillating backwards and forwards, but never sufficiently far to disturb the equilibrium completely, when suddenly a third element of disturbance sets in, a small organism comes near and wishes to take out one of the blocks from the tower for its own use; it seizes the time when the oscillation is greatest, and giving a little extra pull it removes the block, seizes on it firmly and immediately, and the rest of the tower collapses ; or in the case of the swaying bricks, although it has no power alone to upset the first brick in the row, by striking it just when its oscillation is at one extreme phase it assists light or heat, pushes this first sway- ing brick a little further and causes the collapse of the whole line. Bunge gives a series of examples of the breaking down of such chemical substances into simpler materials, and shows how certain ordinary chemical substances along with heat, or even heat alone, may act as fermentation exciters. Thus he points out how a blow can initiate the breaking up of nitro- glycerine into carbonic acid, water, nitrogen, and oxygen. Nitro-glycerine is highly unstable, not so much from the elements which it contains as from the method of arrange- ment of the atoms of the elements. Some oxygen has been induced to unite with nitrogen,*a substance for-which under ordinary circumstances it has little affinity, it having at the same time a much stronger affinity for both carbon and hydrogen than these have for one another ; rapid and exten- sive oscillations are constantly going on, the slightest increase of which must be followed by a new arrangement of mole- cules.’ A sharp tap so increases these oscillations that the equilibrium of the tower is disturbed, the weak bands between 92 BACTERIA. the oxygenand the nitrogen are severed, and the free oxygen is immediately pounced upon by the carbon and the hydrogen, which are set free from one another, each of these elements taking up a certain quantity of the freed oxygen; the atoms of nitrogen having, of course, a strong affinity for one another, combine, and a small portion of oxygen is set free. The amount of energy released here is very great indeed, and it is the more readily observed, and even measured, from the fact that the process goes on rapidly and violently, as it usually does where the resolution is that of a very complex body, into extremely simple substances. Other examples given by Bunge are the resolution of nitrogen trichloride into its constituent elements by “contact with various substances, such as phosphorus, phosphorus com- pounds free from oxygen, selenium, arsenic, some resins (other kinds being inert), non-volatile oils, &c.;°? and he instances chlorate of potash, which splits up into chloride of potash and oxygen at a certain temperature, and, in the presence of binoxide of manganese, ferric oxide or oxide of copper at a much lower temperature ; he says: “The presence of this substance probably so modifies the heat-wave, that the atoms of the chlorate of potash are more easily thrown into responsive vibrations, and thus decom- posed. In the same way peroxide of hydrogen decomposes on being brought into contact with platinum, gold, silver, binoxide of manganese, &c. In these cases it is called an effect of contact, or a catalytic effect. We can form the following hypothesis of the process which goes on here, as in the cases above cited: The substance which acts ‘cata- lytically’ exercises an attraction on one of the atoms in the unstable molecule. It does not necessarily always unite with the atom, but the unstable arrangement of the atoms in the molecule is invariably altered to a stable one.” . Now let us see what actually takes place when grape sugar, of which we have already spoken, is being split up into alcohol and carbonic acid. We have seen that there is a rise of temperature quite distinct from any heat that is applied to bring about the fermentation. It has been proved that in some way or other the presence of the yeast-plant has a very definite effect in starting a process of fermenta- tion, and there are theories as to the rdé/e that this yeast- plant plays in starting the fermentation, In the first place, FERMENTATION. 93 it can only bring about the detachment of the molecules or bricks of a substance when the motion of the molecules of that substance is started or extended by the action of a certain degree of heat ; thus fermentation will not take place unless the material to be fermented is kept at a temperature of from 10° to 40°. This increased temperature acts, pro- bably, in two ways: first, by increasing the motion of the molecules as above stated, and secondly, by enabling the protoplasm to act more energetically, by increasing the rate and extent of molecular motion within the organism itself. The determining motion, according to Bunge, “ might pro- ceed from the vital functions of the cell. But it is likewise conceivable that certain substances occur in the cell, and that these substances act in a similar manner to the catalytic bodies in the examples adduced above.” Heat and moisture are both necessary factors in all processes of fermentation, but neither of these alone can give rise to it. Pasteur, who was really the first to understand this sub- ‘ject so far as to be able to throw light upon it for others, looked upon “ fermentation, properly so called, as a chemical phenomenon, co-relative with physiological actions of a pecu- liar nature,” the elements in which the peculiar physiological actions were manifested being spoken of as ferments which were not dead albuminoid matter, as held by Liebig and his school, but actually living organisms “ of a peculiar nature in this sense, that they have the property of exercising all the functions of their life, not excepting ‘ vegetative multi- plication,’ without necessarily employing the oxygen of the atmospheric air ;’’ and he thus generalizes his results : “Guided by all these facts, I have been gradually led to look upon fermentation as a necessary consequence or mani- festation of life when that life takes place without the direct combustion due to free oxygen.” This opened up exceed- ingly wide and important questions: Was it possible that all living plant-cells might have the power of inducing fermentation in a more or less marked degree? and did yeasts differ from other living cells only in the fact that they had more marked powers of acting on certain carbo- hydrates, and of exciting alcoholic fermentation? Experi- ments on fruit, on barley, on leaves, all went to prove that the elementary cells of plants possessed within themselves this power of inducing fermentation of sugar that was already 94. BACTERIA. ! present or of sugar that was artificially introduced. By a natural transition from the observation of vegetable cells to a study of animal cells, we are led to consider how impor- tant may be the part played by these latter in the digestive tract and in the tissues of the body, especially when they are called upon to act in conjunction with vegetable ferments, either in’ normal or in abnormal positions. Fermentation, then, may be looked upon as an ordinary chemical trans- formation of certain substances taking place as the result of the action of “ living ” cells, the nature of the fermentation and of the substances ultimately resulting being due, firstly, to the nature of the fermented body ; secondly, to the nature of the organism which induces the fermentation ; and thirdly, to the physical conditions under which the fermentation takes place. ‘Thus the results may be extremely complicated if a . mixture of ferments, say an alcoholic, a lactic, and a butyric, be sowed in a single nutrient material ; but if we sow only one, say the alcoholic, the sugar will split into alcohol and carbon dioxide ; if we sow the butyric fermentation, butyric acid will be formed as a result of the splitting up of the sugar ; and soon. As will later be seen, the special name of any fermentation serves to indicate merely that some special product predominates. In the alcoholic fermentation, alcohol is the chief product, but there are also formed as bye pro- ducts, glycerine, succinic acid and a number of other substances, the amount and nature of these bye products: varying, first, with the yeast, and second, with the condi- tions under which it is allowed to grow. The character and aroma of beer and wine indeed depend essentially on the formation of such bye products—compound ethers. It is of course possible, nay, even probable, that what bacteriologically may be termed impurities, may effect the same result, and that special aroma and flavour may depend upon the presence of small quantities of other organisms than the special yeast " used. Further, the activity of the process is dependent in a very marked degree upon the nature of the fermenting substance, and a medium which may afford ample material for the carrying on of one kind of fermentation is absolutely valueless as a medium for other fermentations. The only real difference that exists between a pure alcoholic or butyric fermentation and the complicated fermentations which take place in the animal body or in putrefactive processes is, FERMENTATION, 95 that in the one we have a single ferment only, playing its part, acting on comparatively simple and non-complicated media, whilst in the other we have a complex substratum for the growth of the organisms and a considerable variety of organized ferment cells. . Fermentation may be considered from two points of view— first, as merely a chemical process which is started by the products of micro-organisms or yeasts, in which it resembles many other chemical reactions which are initiated by light, heat, a blow, or some other molecular activity unassociated directly with organic life ; whilst, under the second heading, it may be looked upon as due to the action of living proto- plasm or cells, special fermentations being induced by special organic forms. The.soluble products of these organisms, however, appear to play a secondary part in the process of fermentation, some accelerating, others interfering with it. The process appears to be associated with the necessity which there is for the organized ferments to obtain certain elements for their growth and development—elements which can only be obtained under special conditions, and which, if obtained otherwise, do not lead to the breaking down of the substance which should be fermented in the usual fashion. As we have already seen, the early history of bacteriology was almost entirely associated with the work that was done in connection with fermentation, and it was not till Cagniard- Latour demonstrated that his yeast was made up of small cells which appeared to be capable of reproducing themselves by budding, that the inevitable conclusion was drawn that these globules of yeast were really composed of vegetable proto- plasm, and that it was in consequence of their growth and proliferation that sugar solutions underwent the process of fermentation with the evolution of carbonic acid gas and the production of alcohol. Schwann and Kiitzing, indepen- dently, arrived at the came conclusions after obtaining the same results, and other observers? soon corroborated the observations made by these pioneers, although the whoie facts were not discovered at once, and the knowledge ot the structure and life-history of the yeast-cell that we now * Kieser, 1814 (Schweigger’s Journal, No. 12, p. 229) described spheri- cal corpuscles, all of nearly the same size, which were transparent and motionless, and Desmaziéres depicted yeast globules in 1826 (‘‘ Ann. des Sciences Naturelles,” p. 4). 96 BACTERIA. possess has only gradually been accumulated. These cells are composed of a granular protoplasm surrounded by a definite envelope. When these vesicles or cells are watched during their development, growth, and multiplication, there may be seen, at or near one or other extremity of each, small protoplasmic bodies, which are projected beyond the general outline of the cell, and which gradually but surely increase in size. Ultimately there is a constriction, more or less marked, between the original cell and the bud, and the bud grows to the size of the parent cell; the same process is repeated time after time, until there is formed a chain or series of ellipsoidal or rounded yeast-cells. At one time it was supposed that there was no development either of spores or of mycelial chains, but, thanks to the researches of Reess, by whom the presence of spores within the cells of certain forms of yeast was demonstrated, and to those of Hansen, who was able to confirm their observations as regards spore formation, and also to demonstrate the presence of typical chain mycelia as well as of the budding form of mycelium, these organisms have been put into a separate family by botanists, who have given them the name of Saccharomyces, sugar fungi, or yeasts. These saccharo- myces are indeed to be looked upon as fungi, for although they are closely related to the alge in many respects they contain no chlorophyll. Many of the later observers made very definite statements, founded apparently on very accurate data, that the process of alcoholic fermentation is closely bound up in the question of organized ferments ; nevertheless Liebig continued to defend his doctrine of unorganized* ferments with great ingenuity and vigour. His theory was that fermentation was the result of “internal molecular motion which a body in the course of decomposition communicates to other matter in which the elements are connected by a very feeble affinity (Schitzen- berger, on Fermentation, p. 40). ‘Yeasts, and in general all animal and vegetable matter in a state of putrefaction, will communicate to other bodies the condition of decom- position in which they are themselves placed. The motion which is given to their own elements by the disturbance of equilibrium is also communicated to the elements of * The term unorganized is not here used in its modern signification, which will be mentioned in the next chapter. FERMENTATION. 97 bodies which come into contact with them.” This was nothing more than an extension of Willis’ and Stahl's view of fermentation ; they held that a ferment is a body which has a peculiar internal motion which is capable of -being transmitted from the ferment to a fermentable matter. So fascinating and plausible a theory, of course, received wide recognition, and until Pasteur’s admirable demonstrations of his theory of fermentation were made, had received very general acceptance, especially amongst German chemists and biologists. ‘The mechanical theory and the theory of catalytic forces as used in the old sense have now been laid aside, and the vitalist theory—expressed in the following words by Turpin : “ Fermentation as effect, and vegetation as cause, are two things inseparable in an act of decom- position of sugar "—has taken the field against all opponents. This theory is that living organisms build up structures and develop energy from the materials in which they live, and break up by their vital activity, either directly or through a soluble ferment, the sugar in which they grow. In this theory albuminoid material is considered to be necessary for the process of fermentation or decom- position only in so far as it is required for the nutrition of the micro-organism, it being denied that nitrogeneous elements play any such part, as that ascribed to them by Liebig, of producing the molecular motion, which brings about the splitting up of the sugar, by undergoing a spon- taneous decomposition. Albuminoid material, in fact, is merely an accompaniment of the process of fermentation— a necessary one, no doubt, but one not in any way playing the part of causal factor. ; What takes place in brewing, a process which, though until recently incompletely understood, has long been carried on on an enormous scale in most northern countries ? Malt is barley in which-a certain proportion of the starch of the grain has. been converted into sugar by the process known as “malting.” This consists essentially in moistening the grain several times, keeping it at a temperature high enough to promote its sprouting, during which a substance called diastase is developed as the result of the vital activity of the cells in the germinating grain which acting on the starch converts it into sugar. As soon as this takes place the sprouting is stopped by ee the temperature and then by 98 BACTERIA. drying the grain to kill the young plant and so prevent further sprouting. To obtain a fermentable liquid, a solution of the sugar and of the other soluble constituents of the malt is made in hot water; this is allowed to cool to a temperature of about 16°C. A certain quantity of “ high” yeast is then added to the solution, and during the pro- cess of fermentation the temperature may run up to 18° or 20°C, After a time little bubbles of carbonic acid gas are seen to rise, the yeast increases in quantity and gradually ‘rises to the surface, whence it is skimmed off, and may be again used to set up fermentation, if still pure. The fluid becomes bright, clear, and sparkling (from the presence of carbonic acid), and contains a certain proportion of alcohol ; whilst the sugar, if the fermentation has been properly carried on, has almost entirely disappeared. This is what is known as “high yeast” fermentation. It goes on most readily at a comparatively high temperature, and the yeast rises to the surface as it is formed, bringing up with it a certain proportion of the impurities contained in the liquid, the heavier particles falling to the bottom. The process goes on rapidly, but unless great care is taken it is said that there is a danger that impurities may get in, and that secondary fermentations may be set up, though this is a position now scarcely tenable in these days of India Pale Ales. The “low” fermentation is brought about by a ferment which acts more slowly, at a much lower temperature, and through the agency of yeast-cells that sink to the bottom as they areformed. This fermentation of beer must be allowed to go on at a temperature of from 4° to 5° C., and the fluid is not completely ripened until the end of about fourteen days. This low temperature is maintained in the small breweries by inverted cones of metal, containing ice, which are allowed to float in the fermenting liquid ; they are kept constantly supplied with ice, and the number used is regulated according to the temperature of the external air. In the larger breweries the same results are obtained by passing currents of purified cool air over the surface of the fermenting tanks, which, as a rule, are underground, so as to allow of the temperature being maintained at an extremely equable level. Formerly all beer was made by the high fermenta- tion process, a system that still prevails in this country, but FERMENTATION. 99. in Germany, Austria, and Scandanavia, and also in France, the low fermentation has now almost entirely ousted the high form. There can be little doubt that this is due, in part at any rate, to the impetus that Pasteur’s studies gave to the subject of the careful examination of yeast ; he pointed out that at the higher temperature there was greater danger of contamination by organisms which produce other fermenta- tions than the alcoholic, and that these micro-organisms unable to flourish at the lower temperature, would at the higher temperature grow most luxuriantly. This of course is undoubtedly true, but it should be remembered that where the brewing is in the hands of men ina smail way of business the conditions are very different from those where all the apparatus and skill that capital can command are at the disposal of the brewer. If it were once known which was the best kind of yeast for high beer fermentation it would be possible to obtain pure cultivations, and with it so to conduct the beer fermentation by attending most carefully to the cleanliness of the vessels in which beer is made and stored as to obtain a very pure beer, having excluded all organisms that by their fermentation might render itunsound. ‘The low beer may be brewed in smaller quantities and under less favourable conditions as regards the possibility of keeping the yeast pure than at higher temperatures. The yeast then used causes the fermentation to go on at such a low temperature, that a large number of the organisms which usually giverise to impurity in the beer cannot multiply. Such beer, however, can only be stored when the temperature is kept very low, for as soon as this begins to rise the dormant spores of other organisms begin to develop, set up various acid fermentations and spoil the beer. The different methods of brewing are also deter- mined in part at any rate by the climates of the different countries the cool, light beer apparently being more palatable in warm continental countries; the heavier, with its own peculiar flavour, being usually more sought after in this country. Given pure yeasts, thorough cleanliness, and means of keeping out other organisms, both beers may be kept sound even though the temperature be comparatively high. To -obtain such pure yeast it is necessary first to take a single cell, and from this to grow a series of buds and chains in a sterilized wort, to break this up into separate portions of seed 100 BACTERIA, material, to produce other crops from this, and so on until a sufficient quantity of pure yeast is obtained to bring about the special fermentation in the large mass of wort. This method has another very great advantage—it enables an investigator to take a single yeast-cell and to follow it in its life-history ; in doing this Hansen completed the work that Pasteur had commenced. As the process of obtaining pure cultures is of great interest not only to scientific investigators but also to practical men, we may here give it in brief,* In a Pasteur flask containing wort, a cultivation of the yeast to be experi- mented on is started and carried on as vigorously as possible. A quantity of water, that has previously been sterilized by boiling, is added to the growth ; the yeast-cells in a drop of given size are then counted under the microscope. Let us suppose that ten cells are found ; a drop of similar size is then transferred to a flask containing a2 known volume, say 20 c.c. of sterilized water, so that we have one yeast-cell for each 2 c.c. of water. The flask containing the 20 c.c. of water with the added 10 yeast-cells, is now thoroughly shaken and then divided equally, 1 c.c. being placed in each of twenty flasks containing sterilized wort.? If the separation has been complete ten out of twenty flasks should contain one organism each ; but this of course cannot be absolutely depended upon. After carrying the process to this stage Hansen shakes the flasks very vigorously, by which he separates the cells as far as possible from one another, puts them in an incubator, and allows them to remain perfectly still, so that the cells may sink to the bottom or become attached to the walls of the flask. If there are more cells than one in any flask, they should have been completely separated by the final shaking, and each will probably take up a separate position on the wall or bottom of the flask, and at the end of several days “the flasks are carefully lifted and examined, and it is noted whether one or more white specks have been formed on the walls of the glass ; if only one such speck is found we have thena pure culture.” This method is especially useful in the case where the yeast-plants are at all weakly, as all yeasts grow much more luxuriantly and strongly in a fluid medium than they do on gelatine plates, even when wort is added to the gelatine in order to render it more suitable for the nutrition of the yeast. Where the species are mixed, but where the individuals that we desire to separate are vigorous, the gelatine method may be used with advantage, especially as the individual colonies can then be examined from time to time, or continuously observed under the microscope as they pass through their various phases of development. The characteristic appearances that were said at one time to belong to the yeast-plants have been proved by Hansen to be really non-existent, except in a very limited sense. He maintains that the shape and relative size of the cells and the appearance of the protoplasm of a yeast-plant are + For the method of carrying on the process on a large scale see Jérgen- sen’s work. : . oe 2 1 c.c. or one cubic centimetre = about 16 minims. FERMENTATION. 10I not really specific characteristics. He finds that within certain limits the same species, under different external conditions, may exhibit very different appearances, but he also holds, and brings forward very strong proof in support of his position, that there are limits to the influence which can be exerted on the cells of a species, and that different species exhibit very different characteristics when placed under similar conditions ; thus, for example, Saccharomyces 7] Thotomicrograph of Saccharomyces Cerevisiz. x 500. Mother cells with small buds coming off. Yeast from an Edinburgh Brewery. cerevisiz, when developed in the ordinary manner and then grown vigorously for twenty-four hours at a temperature of 27° C., exhibits the ordinary appearance of rounded or ovoid cells with a formation of septa in some cases, and with more or less well-developed cells in others; whilst this same organism, when cultivated at 7.5° C., occurs in the form of dense colonies with beautiful mycelium-like branchings. 102 BACTERIA. As Hansen’s work is the mostrecent and most complete that has yet been carried out, we may give a short account of the species that he describes in his classification as quoted by Zopf. He divides the yeasts into three groups : Saccharomyces cerevisiz, 11-5# most frequently 8-6 in diameter, which contains a single species only ; Sacch. Pastorianus, 17-2.5p average diameter 8—7p, in which he describes three species, all of them oblong or sausage-shaped ; whilst the third group, Sacch. Ellipsoideus, 13-2.5# in diameter, the most usual size being about 8-74, in which most of the cells are oval or rounded. These forms so overlap one another that by mere microscopic examination it is impossible to determine whether we are dealing with absolutely pure cultures or not. Hansen’s researches were carried on practically, and on a most extensive scale, in connection with a large brewing industry at Old Carlsberg in Copenhagen, and he very naturally turned his attention to the classification of beer yeasts for the purpose of obtaining pure and healthy yeasts for the production of beer. Taking up the subject where Pasteur nad left off he, however, again went over some of the old ground, and studied the-various yeasts with the greatest care ; cultivated them in all kinds of media, noted their behaviour under different conditions as regards temperature, moisture, presence or absence of oxygen and the like; and as the result of prolonged and ingenious experimental work he was able to dispel much of the inaccuracy that had grown up around the subject, and to give detailed accounts of the life histories of the saccharomyces. He found that the older observers had not understood the conditions under which spore formation occurs ; that it was not possible, as Reess had stated, to classify the saccharomyce by a mere microscopic examination, in which the characters relied upon were necessarily merely the form and size of the cells and spores. He also pointed out that even Reess’ observations on the conditions under which spores were formed were not to be relied upon. That they were not the result of the yeast being attacked by mould fungi or bacteria, as Reess and Van Tieghem had, maintained, both of whom believed that spores were evidence of disease‘ in the yeast-cells, he was soon convinced. Nor could he believe that Wiesner and Brefeld, who made very careful study of yeasts, without, however, enjoying the advantages of FERMENTATION. 103 methods for obtaining pure cultivations, were right. These observers had ascribed to certain forms of yeasts the power of forming spores ; whilst in others the same power was denied, Wiesner holding that spores could not develop from pressed yeast, although they could from beer yeast, and Brefeld maintaining that cultivated yeast had lost its power of forming spores, whilst the wild yeast still retained this faculty. “To evolve some kind of order from this confusion was the task that Hansen set himself ; he wished to determine the conditions under which ascospores could be formed. For this purpose he used the method of complete aeration that is obtained by the use of Engel’s gypsum blocks. To well-baked plaster of Paris add distilled water until the plaster is nearly liquid; pour this on to a sterilized - glass plate, on which rests a small mould of thin metal or paper, made rather less than the vessel in which the experiment is to be carried on. These blocks are first thoroughly sterilized by means of heat, after which a small particle of yeast is placed on the upper surface of one of them, an air chamber is sterilized, and in this a small vessel containing water, in which the block rests, is kept. The whole vessel may be placed in an incubator if any special temperature is required, or it may be left at the ordinary temperature of the room. He found that the following conditions were necessary for the perfect formation of spores: a plentiful supply of air (oxygen) and moisture; a certain temperature—the most suitable for the six forms that he examined being about 25° C.; a young condition of the protoplasm of the yeast-cells, the older cells with their thickened walls appearing seldom, if ever, to give rise to spores. As regards temperature, he found that the extremes, at which the individuals of the different species grow, vary somewhat, the lowest tempera- ture at which they are developed being from .5° to 3° C. ; whilst at the other extreme he found that they could still grow at a temperature of 37.5° C. He found that spores became visible as irregular bodies formed from the cell contents in a period of about thirty hours from the com- mencement of the sowing of the yeast-cells when the temperature was kept near the higher extreme, or as low down as 25° C.; but working with lower temperatures, differences occur in the different species, though in none of ‘them does the development of spores take place so rapidly as at the higher temperatures ; for example, he found that at 11.5° C. the Saccharomyces cerevisize does not form spores until a period of ten days has elapsed ; whilst, on the other 104 BACTERIA. hand, Saccharomyces Pastorianus II., kept under exactly the same conditions, gives evidence of the commencement of spore formation after seventy-seven hours ; z.e., supposing that the previous conditions have been the same for the species experimented upon. , This single observation proved to be of enormous impor- tance to brewers, for Hansen’s pupils, Holm and Poulsen, determined that it was possible by Hansen’s method to show that pure cultivated yeasts produced spores at a much later date—under similar conditions—than did those saccha- romyces that were capable of producing certain diseases in beer when present in proportions of jth of the yeasts intro- duced ; and as it was found possible to determine by this method the presence of wild yeasts when they occurred in the proportion of 44th of the whole of the yeast used, it was an easy matter to determine within a very short time whether a yeast was fit to be used for brewing purposes or not; in the case in point, forty hours at the temperature of 25° C. was sufficient for the purpose. It was found, indeed, that all ‘the bottom yeasts might be separated from one another by ae cultivations at a temperature of 15° C. and at 25°C, Having obtained these pure cultivations, and being so successful with the study of the spores, Hansen turned his attention to the other characters, by the use of which he was able to separate in a still more definite manner the various species of saccharomyces one from another. He subjected to a most careful examination the films which appeared on the surface of liquids that were undergoing fermentation. It had been supposed that the Sacch. mycoderma was the result of a yeast fermentation; but Hansen found that the saccharomyces film was something quite different from the film formed by the Sacch. mycoderma, and he came to the conclusion that film formation must be looked upon as a phenomenon that may be met with under certain conditions wherever micro-organisms are or have been growing. Here again the presence of a plentiful supply of air is an absolute necessity ; another essential condition is a state of perfect rest of the surface of the fluid in which the process of yeast growth and fermentation is going on. It is interesting to note that when carbonic acid gas is passed through the fluid and is allowed to accumulate on the surface these films arenot formed, FERMENTATION. 105 so that in fermenting tanks in which, of course, large quantities of carbonic acid gas accumulate the films are not usually met with. From this one may gather how laboratory experiments may go wrong, or give untranslatable results, when the exact conditions met with in nature, or on a large scale, are not adhered to; it also explains the different results that have been obtained by various workers, the conditions in their experiments not always being the same. For instance, in Chamberland or ordinary flasks covered with filter paper the film forms will develop rapidly as soon as the primary fermentation is completed ; whilst in the closed Pasteur flasks, in which the air cannot be changed rapidly, the films grow comparatively slowly. To produce these films Hansen proceeded as follows: Having obtained his pure cultivations, drop cultures were made into carefully sterilized four-ounce flasks, half filled with sterilized wort and protected from falling particles by being covered with sterilized filter paper. As soon as the films be- came apparent to the naked eye they were examined. They appear, according to Hansen, as small opaque points, which gradually increase in size and then run together, forming irregular patches floating on the upper surface, with a con- vexity on the side resting on the fluid. The film at length overspreads the whole surface and becomes adherent to the wall of the flask at the margins; on shaking the flasks much of the film can be made to sink, but a new one forms to fill up the gaps that are left. These young films he divided into two groups, the first consisting of Sacch. cerevisiz, Sacch. Pastorianus II., Sacch. ellipsoideus IT., in none of which could he find mycelium-like colonies (at one time it seemed that Hansen’s researches had demonstrated this fact) ; whilst in the second group, which included Sacch. Pastorianus I. and III. and Sacch. ellipsoideus I., such mycelial colonies were early developed. Later researches have tended to show that this division is very much a matter of time, and that it cannot now be looked upon as having any real scientific value. As regards the temperature at which these films are developed Sacch. cerevisie and Sacch. ellipsoideus I. are developed between 38° and 6° C., the three Pastorianus varieties between 34° and 3° C.; Sacch. ellipsoideus II., 38° to 40° C., and 3° C. The film of this latter appears to be the 106 BACTERIA. most vigorous at all temperatures, at 13° C. it develops so quickly and vigorously that it out-distances all the others, giving at 23° C. a film covering the whole of the surface in from six to twelve days ; whilst the others at the same tempera- ture only gave a much more delicate film in from three to five weeks. The next to this as regards rapidity of the forma- tion of the film is Sacch. Pastorianus JII., which at the temperature of a warm room forms a film much more rapidly than any except Sacch. ellipsoideus IT. It may be noted, in connection with the limits of tempera- ture at which this film formation takes place in the three Pastorian varieties, that they cease to develop and form spores at a temperature of 36° to 38° C., if this be con- tinued for ten or eleven days ; whilst the other three species all continue to grow for a much longer period. In addition to the forms studied by Hansen others may be mentioned. Below is appended a short classification of the more important forms of yeasts, with some of their more characteristic features. The Saccharomycetes or yeast fungi are a family of ascomycetes, divided into two very unequal groups—the Saccharomyces of Reess and the Monaspores of Metschnikoff ; the mode of spore formation is somewhat similar in the two, the only difference being that in the saccharomyces the spores are rounded and are sometimes multiple in the same sporangium, whilst in monaspora there is in each cell a single needle-shaped spore developed. In the first genus are included the following forms : 1. Saccharomyces cerevist#, may be looked upon as a typical English high yeast, some of the characteristics of which have already been described. It grows as rounded or slightly ellipsoidal cells, which give off small cells by budding ; in the earlier stages of film formation there are formed delicate mycelial-like threads, but as the film becomes older longer and more regular threads are formed. In the yeast-cells nuclei are frequently to be made out, especially on being stained with osmic acid or hematoxyline. These nuclei are very distinctly seen in old cultivations. The development of ascospores takes place most rapidly (after twenty hours) at 30°C., most slowly (after ten days) at from 11° to 12” C., and stops alto- gether below this. The spores may be very distinctly seen, as they are highly refractile, and their walls are well defined. They are usually from 2.5 to 6 FERMENTATION. 107 in diameter. Film formation takes place most rapidly (seven to ten days) at a temperature of from 20° to 22°, most slowly (two to three months) at 6° to 7°C., and ceases altogether at 38° C., and at 5°C., at the other extreme. Between 20° and 30° C., the cells are frequently sausage-like and irregular ; from 6° to 15° C. the cells are usually like the parent cells in younger cule Ds but in older cultivations the forms are like those already escribed. This species, like all those investigated by Hansen, secretes a peculiar substance, which, acting on saccharose or crude cane sugar, inverts it to ‘invert sugar ; the yeast then brings about the fermentation of this latter substance, and also of dextrose and maltose, giving rise to the formation of alcohol and carbonic acid gas, with an evolution of heat and great multiplication of the yeast-cells. It does not seem to exert any action on lactose or milk sugar. In this respect these ferments resemble mucor racemosus, which first brings about the inversion of cane-sugar ; it also secretes invertase, which causes inversion of saccharose, the products of which it ferments ; it also sets up a weak fermentation in beer wort of the maltose and dextrose present in that liquor. Reess’ genus of Saccharomyces ellipsotdeus Hansen divides into two—l. and II. 2. Saccharomyces ellipsotdeus I. is really a ‘‘ wild” species of wine ferment ; in beer wort it grows as a low yeast. It is usually rounded or ellipsoidal in shape, though it sometimes assumes the sausage form. The spores, of which two to four are usually found in a single ascus, are from 2 to 4p in diameter. These spores develop most rapidly (in twenty-one hours) at 25° C., most slowly (eleven days) at 7.5° C., and are not formed at all at 32.5°C. at the one extreme, and at 4° C. at the other. Grown on the surface of beer wort gelatine, its colonies form a peculiar net-work along the line of the inocula- tion streak. The surface membrane is formed rapidly (eight to twelve days) at a temperature of from 33° to 34° C., most slowly (sixty to ninety days) at 6° to 7°C. ; it is always a delicate membrane, and cannot be grown at 5° C. on the one hand and at 38° C. on the other. The most characteristic growth takes place at from 13° to 15° C., when it occurs as a complicated branching mass, with elongated cells, or threads, arranged in rows, with several lateral processes coming off at the points of junction. Secondary branches are formed at the constrictions of the primary branches. : It appears to exert as powerful and rapid a fermenta- 108 BACTERIA. tion process as does the Saccharomyces cerevisize on the various carbo-hydrates on which that ferment acts. . 3. Saccharomyces ellipsoideus II. is also a “ wild” or wine fermentation yeast which gives rise to the muddiness of beer. It is essentially a low yeast, the film that forms is exceedingly delicate ; it makes its appearance in from three to four days at a tempera- ture of 33° to 34° C., but not for five or six months at 3°to 5°C. At 2° and at 40° C. no film forms. Young cultivations at 15° C. are usually somewhat rounded or egg-shaped, whilst the older cultures show longer mycelial rods, with forked transverse shoots given off at the joints. Asci containing from two to four spores may be egg-shaped, slightly irregular or elongated. The spores measure from 2 to 5m in diameter; they are developed most rapidly at 29° C., most slowly at 8° C., and are not formed at above 35° or below 4° C. Hansen does not look upon Saccharomyces Pastortanus as a pure species ; he divides it into three. 4. The first of these, Sacch. Pastordanus I. (Hansen), is a wild yeast, spores of which frequently occur in the atmosphere of breweries. It gives an unpleasant bitter taste to beer ; it, also, is a bottom ferment, occurring as elongated ellipsoidal or pear-shaped cells, from which small. apical or lateral branches may be given off. The asci are usually elongated or rounded; they may contain two spores or multiples of two, up to eight or even more. The spores vary very much in size from 1.5 up to 54; are developed most readily (twenty-four hours) at a temperature of 27.5° C.; most slowly (fourteen days) at a temperature of 3° to 4°C. The spore formation ceases at .5° C., and at 31.5° C. The films are usually very delicate, are developed most readily (seven to ten days) at from 26° to 28° C., most slowly (five to six months) at from 3° to 5° C., development ceases altogether at 34° and 2° C. At from 3° C, to 15° C. mycelial-like threads are developed pretty freely in this film and most irregular forms make their appearance ; many irregular club and skittle-shaped and other forms are formed in the older films, but fewer in the younger ones ; in these films the cells are usually smaller. 5. Saccharomyces Pastortanus II. (Hansen) was also separated from the air of the brewery. In gelatine made with yeast water it grows along the line of the inoculation streak (at 15°C. at the end of sixteen days) in the form of colonies with smooth edges. It is a feeble top fermentation yeast when grown in beer wort. The sedimentary cells of this yeast are mostly elongated, but they may be slightly rounded, varying considerably in size. " The cells found in the film are rounded, egg-shaped, or somewhat elongated. The spores are from 2 to 5u in diameter; the asci are usually elongated and the spores occur in multiples of two. They are formed most rapidly (twenty-seven hours) FERMENTATION, 109 at a temperature of 23° C., most slowly (seventeen days) at 3° to 4°C., and cease to be formed at 29° C. and at .5° C. This yeast gives rise to neither cloudiness nor to any unpleasant bitter taste. It secretes an invertase and causés fermentation of all the carbo-hydrates that are fermented by the other yeasts of this group. In old cultures of the films the cells are small, very irregular in shape, and thread-like, like the preceding. 6. Saccharomyces Pastortanus III. (Hansen) is, according to Hansen, one of the causes of turbidity in beer. Grown on yeast water gelatine at a temperature of 15° C., at the end of sixteen days the colonies present peculiarly fringed edges ; grown in wort it gives rise to a top fermentation, and causes considerable turbidity with a production of alcohol and carbonic acid gas. The spore formation is very much like that in the preceding species: it takes place most rapidly (twenty-eight hours) at 25° C., most slowly (nine days) at 8.5° C., and ceases at 29°andat 4° C. The film appears in the form of small flakes most rapidly (seven to ten days) at 26° to 28° C., most slowly (five to six months) at 3° to 5°, and ceases altogether at 34° and 2°. Here again the elongated or sausage form predominates, but large and small rounded and oval cells are also present in the sedimentary forms in the films at from 20° to 28° C. The cells are of much the same shape as are those of the sedimentary yeast, but at a temperature of from 15° down to 3° C. there are elongated mycelial-like threads which in old cultures become still more characteristic. These mycelial-like threads are developed at the above temperature, which is much lower than in the case of the threads in Saccharomyces Pastorianus I., where they are most characteristic at a temperature of 13° to 15° C. At the same temperature, 15° to 3° C., the cells in Saccharomyces Pastorianus II. are oval and rounded. Hansen describes in less detail a number of other ferments which produce alcohol from sugar. 7. Saccharomyces Ludwzgiz, though found in the sap of oaks, grows freely in yeast water, when it appears as a peculiar caseous mass or as fungus-like specks which float in the fluid. One great peculiarity of this form is that it may be so modified by cultivating it in beer wort through several generations at a temperature of 25° C. that it does not form spores, or that it forms them but slowly. The spores, when formed, are usually from one to four in number, but there may be more; the cells of the film are usually con- siderably elongated. The film formation goes on most rapidly at about-25° C.; at the ordinary temperature of IIo BACTERIA, the room it goes on very slowly, taking a whole month to form a comparatively delicate membrane. In very old: - cultures well-marked mycelium formation may be met with in which the cells are ellipsoidal, elongated, or sausage- shaped, or somewhat club-shaped. It is capable of acting on grape sugar ; it inverts cane sugar and ferments it ; but ‘has no action on maltose, lactose, or dextrine in yeast water, nor does it attack starch. 8. Saccharomyces Marxianus, named after its describer, was first found in wine. Hansen studied it most closely. He found that in beer it develops as small ellipsoidal and egg-shaped cells, with here and there sausage-shaped cells, which are often combined into colonies. There are developed on a quiescent fluid small viscid masses, some of which remain on the surface whilst others ‘sink to the bottom. The film develops exceedingly slowly, but in it are found cells which resemble very closely those of the films of the first six species of Saccharomyces described. The true film contains oval and short sausage-shaped cells. The spores are not freely developed. When it is grown on solid nutrient media spores are more frequently formed, in which case they are usually oval or kidney-shaped. In beer wort this yeast is not very active, nor is it able to ferment mal- tose, but it acts vigorously on saccharose, inverting it and then fermenting it with great activity ; it also acts upon dextrose. 9. Saccharomyces exiguus, found by Hansen in German yeast, differs from the preceding saccharomyces in the fact that it forms no mycelial threads on beer wort, or on solid nutrient media. It forms spores, but sparsely, and the film is exceedingly delicate, the cells of which this is made up being short rod-shaped or ovoid. It acts on the sugars exactly as does the Saccharomyces Marxianus. 10, A somewhat peculiar saccharomyces belonging to this group is the Saccharomyces membranefactens, which forms on beer wort a bright yellow tough scum, composed of long and sausage-like cells, which may be closely packed together or may occur singly. It forms spores rapidly, liquefies nutrient gelatine, and is peculiar from the fact that it does not cause fermentation of any of the ordinary carbo-hydrates, nor has it any effect in inverting cane sugar. 11. Saccharomyces minor (Engel), who describes it as spherical cells 6# in diameter, arranged in chains of 6~9 FERMENTATION. Ill elements. The spore-forming cells are larger 7-8.5y, and contain from 2-4 spores 3.5# in diameter. He ascribes to this ferment the action of fermentation in bread, a notion that has since been scouted and again accepted. 12. Saccharomyces conglomeratus (Reess), Hansen thinks is simply a form that may be met with in old films of all the six species that he specially investigated, and in recent works this species has been dropped. 13. Saccharomyces apiculatus, described by Reess, can scarcely be said to be a true saccharomyces, although it is included amongst them by Zopf as a doubtful member of the group; it has not yet been ascertained to have any spore formation, and is therefore retained by Hansen in this group only provisionally. It occurs in fer- mented wine and spontaneously fermented beer, and, in the hot seasons, on sweet succulent fruits, such as cherries, goose- berries, plums, or grapes ; whilst in the winter it is found in the soil beneath the trees that bear these summer fruits. It occurs in cultivation fluids as lemon-shaped cells—hence the name—though under certain conditions it assumes elongated, crescent-shaped, and rod-shaped forms. It gives off buds of two kinds : one oval, the other lemon-shaped. It isa bottom yeast giving rise to a feeble alcoholic fermentation ; it does not invert cane sugar, but acts on dextrose in yeast water, but does not ferment it completely. Mixed with Saccharo- myces cerivisize it retards the action of the latter. An organism that was long classified with the true yeasts is the Rosahefe or Pink yeast of the Germans. Accord- ing to Hansen, however, no spores are formed during any phase of its development and for the present he excludes it from the Saccharomyces or true yeasts. It belongs rather to the Torule. Genus II. (1) Monospora (Metschnikoff). The single mem- ber of this group, Monosfora cuspidata, is of interest princi- pally because of the elaborate researches that have been made by Metschnikoff on its relation to a peculiar disease of the Daphnia, a small fresh-water crustacean. It occurs as a bud- ding mycelial thread made up of elongated cells which before spore formation become elongated ; there then appears a long, thin needle-like body situated in the centre of the cell in its long axis. This spore is taken into the alimentary canal of the Daphnia, whence it is driven through the walls by the Ilz2 BACTERIA. peristaltic action of the muscles of the intestine. It thus passes into the body cavity or into other tissues, where it is immediately attacked by the white blood corpuscles, or by the connective tissue corpuscles ; or it may first become developed into the rod-shaped vegetative form. When it has once commenced to divide it is no longer attacked by the above cells (which Metschnikoff speaks of as the phagocytes) of the insect. If the Daphnia is in moderately good health the Photomicrograph of Rosahefe. x 1000. Rose-coloured yeast (?) No spores have been found, and Hansen does not classify it with the yeasts. monospora is gradually overcome, but if the insect is feeble, or if the monospora is ingested in very large quantities, it multiplies so rapidly that the animal may eventually succumb to its attacks. This form is specially interesting from the fact that it afforded some of the strongest proofs of Metschni- koff’s phagocyte theory that he was able to obtain. We have already seen that the yeast-cell is usually observed in a fermenting liquid as a rounded or ovoid body, FERMENTATION. 113 that it gives rise to buds by sending out small processes from its wall, and that these latter then become detached from the mother cell. This cell consists of a distinct membrane and of protoplasm, the former of which may be thicker or thinner according to the age of the cell, whilst the latter may vary very considerably. Whilst the cell is merely growing actively, the plasma or cell contents are homogeneous and highly re- fractile, but when it is placed in beer wort or other highly nutritive media, it multiplies rapidly and gives rise to the fermentation of the sugar and maltose, and the protoplasm becomes differentiated and undergoes certain changes. Large clear spaces, which are supposed to contain the more fluid part of the protoplasm (vacuoles), are formed; cloudy granular change occurs in the other protoplasm ; and larger fat globules also make their appearance. As the cell gets older, and consequently less active, the finely granular proto- plasm accumulates as a thin layer inside the cell wall, whilst the centre is occupied by clear fluid, in which are floating a number of fatty granules and globules. It would appear that this last isa somewhat degenerated condition, and that it is due to imperfect nutrition, for if a few of these cells are transferred to a fresh rich nutrient medium, the protoplasm becomes again modified, the cloudy granules disappear, small shoots of the clear plasma pass into the large central cavity, small rounded vacuoles are formed in place of this large central cavity ; these in turn become subdivided, and eventually the whole cell is again occupied by clear protoplasm. By appropriate staining, especially of an older cell, a nucleus may be distinguished, whilst under certain conditions, to be afterwards mentioned, spores or ascospores are formed. Other organisms which in certain respects resemble the yeast fungi are the Torulz, which Pasteur described as being somewhat of the nature of yeasts, but different in the fact that they were unable to give rise to such marked alcoholic fermentation. Hansen, however, was able to show that this was not a sufficiently distinct characteristic, as some of the saccharomyces give rise to the formation of little or no alcohol, whilst, on the other hand, some of the Torule set up very marked alcoholic fermentation. The great point of distinction is, that none of the Torulz, so far as has yet been observed, are capable of producing endospores ; all 114 BACTERIA. of them multiplying by budding, some of them also giving rise to the formation of mycelia. It is of course quite possible that such a classification may not hold good, as it has been suggested that some of the torule could not be definitely brought within it, as far as non-sporulation is concerned. Hansen describes seven species varying in size from 1.5 to 84; some invert cane sugar; some give rise to scarcely a trace of alcohol, whilst others produce as much as 6.2 per cent. of alcohol. They occur as spherical or elongated cells, and cannot be distinguished by the microscope alone from the round cells of the different species of saccharomyces. As a group they have not much action on maltose, and only some of them affect dextrose. CHAPTER VI. FERMENTATION (continued). Soluble Constituents of Yeast—Action of these upon Sugar—Conversion of Glycogen inthe Liverand other tissues—Growth of Yeast-Cellsin Organic and Inorganic Fluids—Fermentation of Fruit Juice—Zrobic and Anz- robic Fermentations—Effect of Free Oxygen on Yeast-Cells—Fermen- tation not necessarily equal toGrowth of Yeast—Enzyme or Unorganized Ferment a Secondary Function—Function depends partly on Organism, partly on Medium in which it is Growing—Peptonizing Function usually requires presence of Oxygen—Various kinds of Fermentation : Lactic, Urea, Butyric, Ammoniacal, Acetic—Formation of Fatty Acids—Mycoderma Aceti—General Processes of Fermentation— Hoppe-Seyler’s Classification. On approaching the subject of fermentation by yeast we find at the very outset that Pasteur and others had noticed a very remarkable peculiarity of the water in which yeast had been mixed, and from which it had again been separated. It was observed that a certain substance, perfectly soluble in water but precipitable by alcohol, had the peculiar property of inverting saccharose into equal quantities of dextrose and levulose, and it was at once assumed that this invertin, invertase, or some similar substance, produced through the vital activity of the yeast-cells, was necessary to bring about conversion of starch into sugar (diastase), or of saccharose into invert sugar (invertin), before the cells could bring about a true fermentation, or splitting up and hydration into alcohol and carbonic acid gas, with certain other products to be mentioned immediately. It was noticed by Bertholot and Hoppe-Seyler, moreover, that, even if the living organisms were first killed by the addition of ether, the invertase or invertin still continued to act, and was able to invert a quantity of saccharose altogether out of proportion to the- amount of inverting material present. In consequence of the latter part of this observation, many physiologists and chemists maintain that the action of the invertin is essenti- ally due to the setting up of a certain rate and length of 116 BACTERIA. molecular vibration, such wave rate and iength being trans- mitted through the whole body of material to be inverted. In fact, that the addition of the invertin is simply the light- ing of the spark that fires the whole train. There is some- thing very fascinating in this theory, and it certainly explains many, otherwise, obscure chemico-physical questions con- nected with these two subjects—inversion and fermentation. We see at any rate that part of the process takes place entirely outside the yeast-cells, and may go on even when the organisms that produce the inverting material have been killed or completely removed. There are other similar examples of conversion by purely chemical means, as, for instance, where, on the addition of a dilute acid, such as sulphuric acid at a certain temperature, starch is converted into glucose, the heat and the acid setting up such molecular vibration amongst the molecules composing starch, that in the dilute acid there is a rearrangement of molecules, water is taken up, and by hydration of the starch glucose is produced. In this case the conversion takes place much more rapidly and completely at a temperature of 130° C. than at 100° C. In a similar manner glycogen may be converted into a sugar that will reduce Fehling’s solution. This process goes on in the liver and other organs and tissues of man and animals, in which, probably, the secretions of the cells take the place of the sulphuric acid and the high temperature, or of the protoplasm of yeast and other vegetable cells. Let us now, however, see what relation the yeast-cells themselves (as apart from their products) are supposed to bear to the real process of fermentation. If it were possible to obtain an absolutely pure solution of sugar, z.e., a solution containing no nitrogenous elements of any kind, and if we were to place in this a minute quantity of yeast, we should find that a very slight fermentation might take place—z.e., there would be an almost inappreciable diminution in the quantity of sugar present ; a small quantity of alcohol and carbonic acid gas would be developed, but very shortly the process would stop, and there would be no marked increase in the number of yeast-cells found in the whole solution. If now we were to add a smalt quantity of nitrogen in the form of an albuminoid substance and a certain quantity of ex- tractives and salts, such as are found in the ashes of burnt yeast, there would very quickly be observed a very different FERMENTATION. 117 state of matters ; first there would be very marked turbidity of the fluid. If we were using a high yeast this turbidity would rapidly become more and more marked, and there would rise to the surface a yellow scum ; bubbles of car- bonic acid gas would be seen rising in the liquid, and a spirituous or alcoholic taste would soon become pronounced. If from the first fluid, z.e., the fluid in which there was nothing but pure sugar, we were to examine the very minute quantity of yeast, we should find that, although there was apparently an attempt at budding in a few of the cells, in most cases there are a series of clear globules with- in the yeast-cells, that there is a very large proportion of thick walled granular cells (which have certain other peculiarities), that, in fact, throughout the whole we have evidence of very little proliferative activity, and that young, vigorous, healthy, yeast-cells, budding and giving rise to other cells by vegeta- tive activity, are conspicuous by their absence. A micro- scopic examination, in the case of the yeast growing in the sugar solution containing a small quantity of albuminoid material and extractives, reveals a very different state of matters ; here the yeast-cells are in a state of extraordinary activity, buds are being thrown off from the extremities, terminally or laterally,the protoplasm is evidently exceedingly active, vacuolation is conspicuous by its absence, except in certain cells, and we are at once struck by the extraordinarily large proportion of young vigorous cells—the more active the process the greater the number of the new cells, and the ‘larger the quantity of yeast formed. In one case the yeast-cells die of starvation, although large quantities of sugar are present; and as the yeast-cells have not been able to grow and reproduce by the exertion of their vege- tative activity, they have not been able to resolve the sugar into alcohol and carbonic acid ; badly nourished or dead yeast-cells, therefore, exert little or no influence in bringing about an alcoholic fermentation. In the other case the cells have supplied to them all the elements necessary for the nutrition of their protoplasm. The carbon, the hydrogen, and the water, may all be obtained from the sugar solution, albuminoid material of course supplies the requisite nitrogen, whilst the ash of yeast, which has been added, con- tains all the other elements necessary for the nutrition of the yeast-cells, 118 BACTERIA, Under these conditions, the cells, as we- have seen, may undergo rapid development, growth, and multiplication, and we have an increase in the amount of yeast, and at the same time in the amount of alcohol and carbonic acid gas gene- rated. These two experiments afford most exact evidence that Liebig’s theory was essentially incorrect, and that Pasteur's theory that true fermentation was the result of the action of the living protoplasm, gives us the key to the whole situation. It was, naturally enough, objected that as the fermen- tation process could not go on except in the presence of an albuminoid material, this might really be the cause of the whole process, and many most elaborate experiments were brought forward to prove that it was this organized but dead material that was the real and primary factor in the process. Pasteur, however,. equal to the occasion, was able to demonstrate in a most convincing manner, that the nitrogen might be supplied to the organism in the form of inorganic salts, instead of being presented as albuminoid material. He utilized for his purposes a mixture containing 150c.c. of a io per cent. solution of sugar candy, .5 grammes of the ash of yeast, .2 grammes of bitartaric of ammonia, and .2 grammes of sulphate of ammonia. He found, on introducing Saccharomyces Pastorianus into this solution, that a some- what slow but very complete transformation of the sugar took place, and that the nitrogen from the ammonia was used up by the growing. yeast-cells, which at the same time increased enormously in number. It was thus evident that these mineral salts could take the place, in fermentable liquids, of “ media of natural composition.” He found, however, that the process went on more slowly, that somewhat peculiar forms of yeast showed themselves, and that an essential factor for the success of the experiment was that no other organisms should be allowed to make their way into the fermenting solutions—‘e., the absolute purity of the various materials of which the nutrient solution was composed, and of the ferment itself, must be guaranteed, and any relaxation of the strict ‘conditions of extreme purity was invariably followed by an interference with the vital manifestations and physiological actions of the yeast organisms. It would appear, indeed, that although the fermentation FERMENTATION. 11g is, under such conditions, very complete if sufficient time is allowed for the process, the yeast-cells experience a certain difficulty in wresting the nitrogenous elements from the inorganic ammonia salts, and that all other conditions must be extremely favourable, in order to allow of their taking up, and utilizing for their own use, inorganic nitrogenous material. The presence of other organisms, for example, that have a stronger affinity for nitrogen in this form, z.e., organisms which are better adapted to exist under such conditions, and which can, by their vital activity, interfere with the growth of the yeast-cells, remove from the sphere of action of the yeast-cells material that is absolutely necessary for their rapid and perfect morphological and biological development, as a result of which fermentation and hydration of the sugar do not take place in the ordinary way ; other substances or bye products are formed, and the decomposition of the sugar is incomplete, or is irregularly carried out. What, then, are the conditions necessary for the growth of the fermenting organism in a fermentable fluid ? In the pro- cess of wine-making it isa well-known fact that the fermenta- tion is set up by some organism, which, though present either as young cells or as spores on the outside of the grape, cannot attack the juice so long as the skin remains unbroken—a fact brought out by Davaine at a very early stage of his researches. When, however, the grapes are plucked, the skins are bruised and the juice is set free, the fermenting organism,—the wine yeast or Saccharomyces ellipsoideus, or some similar variety—utilizing the grape juice, which contains not only sugar but also all the ele- mentary constituents necessary for its nutrition,—grows vigorously and sets up the vinous fermentation. It is a remarkable fact, but one well known to wine pressers and fermenters, that for the commencement of this vinous fer- mentation there must be an access to free oxygen, or oxygen mixed merely mechanically with nitrogen of the air to the fluid that has to be fermented, as without this the yeast spores and old yeast-cells are utterly unable to develop or to give rise to active and vigorous yeast-cells. Pasteur insists that we have evidence of the necessity for the presence of such free oxygen in the fact that the fermentation of grapes takes place much more rapidly and completely when the grapes are left attached to the stalks of the bunches, by 120 BACTERIA, which means there is a freer circulation of air allowed to take place through the fermenting mass than when they are plucked from the stalks and pressed and fermented. Of course, it might be objected that the air is useful merely in carrying the yeast cells into contact with the fermentable fluid, but numerous experiments have been carried on to prove that the presence of oxygen is absolutely necessary for the resuscitation of old and spore-bearing yeast-cells. This is in itself a most remarkable circumstance, and a very significant one when it is borne in mind how very different the conditions are under which the later stages of fermenta- tion are best carried on. Jt must be remembered, however, that the conditions most favourable for the multiplication and production of a sufficient number of active cells are not by any means the conditions most favourable for the decom- position by the cells of the largest amounts of sugar In our large breweries (as is sometimes brought home to us only too closely by the reports of the death from suffocation by carbonic acid gas, of men who go down into vats to clean them out) there is always, as the fermentation process goes on, a very great accumulation of carbonic acid gas on the surface of the fermenting liquid ; so dense and so deep is this layer that it is at once seen how impossible it is for much free oxygen from the atmosphere to obtain access to the yeast-cells. Any oxygen they utilize for the building up of their protoplasm must be derived either from air held in solution in the fer- menting liquid (which can only be a very small amount, as the boiling of the wort must have driven out a very large proportion of such air from the fluid), from the small amount of oxygen that can pass through the layer by diffusion, or it must be derived from the breaking down of those substances rich in oxygen that are contained in the malt solution. In the same way in the later stages of wine-making the fer- mentation is allowed to go on in large casks ; carbonic acid gas here also rises to the surface, fills the cask up to the bung-hole and gradually flows over, and, as the carbonic acid gas comes in minute bubbles to the surface from all parts of the fluid, it can scarcely be imagined that any large amount of free oxygen can be left in suspension in the fermenting grape juice ; and certainly very little can find its way from without, as the car- bonic acid gas is pouring out from the bung hole in such considerable quantities. The fermentation at this stage, then, FERMENTATION. 121 is going on very rapidly; there is a growth and multi- plication of the yeast-cells, a transformation of the sugar into alcohol and carbonic acid gas—all without the presence of free oxygen. Pasteur set himself to reconcile these two apparently contradictory facts, and as a result of his observations he made one of the most important of his discoveries, important in its relation to the life history of both fermentative and pathogenic organisms ; and specially important because of the parts that the zrobic and anzrobic conditions under which organisms exist play in determining the growth of these organisms and the spread of certain specific infective diseases. He found that old cells with thick membranes, even con- taining spores, were not able to multiply in media other- wise suitable unless oxygen was present ; and that the vigour of the rejuvenating process depended to a very great extent on the amount of free oxygen that was contained within the fermenting fluid. He found, in fact, that with these cells, as: in the case of fungi and of other animal and vegetable cells, a sudden and complete cutting off of the supply of free oxygen immediately proved fatal to them, so that the cells which had been exposed to the air for some time—that is, which had acquired an zrobic habit—were rendered inactive by the cutting off of the supply of oxygen. “Along with this he found that the yeast organisms could multiply in liquids containing nutrient materials for a considerable length of time before there was any appearance of alcohol in the fluid. After a time, however, the free oxygen being used up, it would naturally be expected that the growth of the yeast-cells would also come to an end, but such was not found to be the case. The yeast-cells continued to multiply, the oxygen in the fluid was used up and replaced by carbonic acid gas, and car- bonic-acid gas was found on the surface, cutting off any further oxygen supply from without. How was it that these cells went on multiplying? Pasteur answered the question as follows : During the gradual elimination of oxygen the yeast- cells, having once become vigorous from the presence of a good supply of oxygen, and of other nutrient requisites, had acquired an activity that they did not before possess ; they had at the same time become gradually acclimatized, and their protoplasm had become so far altered that, as the free oxygen was partially cut off, it was able to wrest from the sugar what oxygen it required for the building up of its own 122 BACTERIA. substance, and as the free supply was more and more cut off it became gradually more able to take what it required from the sugar solution—that is, it was gradually acclimatized. In reading this if instead of oxygen the word energy be used it would appear that we should have a more accurate physiological statement. It has already been stated that, in the presence of a free supply of oxygen, relatively less alcohol is formed than under conditions of anzrobiosis ; it appears as though the oxidation is too complete ; there is enough oxygen to supply not only the wants of the living organism but also those open bonds of combination in the various molecules that are unsatisfied when the yeast takes for its own use certain constituent atoms from these molecules, and as a conse- quence very perfect oxidation is brought about and alcohol is transformed into carbonic acid gas and.water. In the anzrobic condition that is brought about when the oxygen in the fluid is used up, and as the carbonic acid gas accumu- lates on the surface in the flask or in the vat, the vigorous yeast-cells are able to separate sufficient oxygen for their own use from the sugar, or.sugar and water, disturbing the com- positions of the fluid, and necessitating further rearrange- ment of the remaining molecules ; additional oxygen can- not be brought from without to satisfy the vacant bonds, and other very definite products are formed. These products are comparatively stable even when afterwards exposed to free oxygen but not to oxygen in a nascent condition, and we have as aresult the alcoholic fermentation. Here, in fact, are two essentially different metabolic processes at work in the proto- plasm of the yeast-cells. When oxygen is freely supplied the anabolic or building-up processes predominate, a fact evidenced by the rapid division of the yeast-cells under these conditions ; whilst, when oxygen is excluded, the catabolic or breaking-down processes are in the ascendant, as shown by the larger amount of the medium decomposed, accom- panied, however, by a slower multiplication of the yeast-cells. Summing up, Pasteur says :— ‘“ This being so, it is evident, we repeat, that to multiply in a fermentable medium, quite out of contact with oxygen, the cells of yeasts must be extremely young, full of life and health, and still under the influence of the vital activity which they owe to the free oxygen which has served to form them and which they have perhaps stored up for a time. When older they reproduce themselves with much difficulty when deprived of air, and FERMENTATION. 123 gradually become more languid, and if they do multiply it is in strange and monstrous forms. A little older still, they remain absolutely inert in a medium deprived of free oxygen. This is not because they are dead ; for in general they may be revived in a marvellous manner in the same liquid if it has first been aerated before they are sown. . . . At this point we must observe—for it is a matter of great importance—that in the operations of the brewer there is always a time when the yeasts are in this state of vigorous youth of which we have been speaking, acquired under the in- fluence of free oxygen, since all the worts and all the yeasts of commerce are necessarily manipulated in contact with air, and so impregnated more or less with oxygen. The yeast immediately seizes upon this gas and acquires a state of freshness and activity which permits it to live afterwards out of contact with air, and to act asa ferment. Thus, in ordinary brewery prac- tice, we-find the yeast already formed in abundance even before the earliest signs of external fermentation have made their appearance. In this first phase of its existence yeast lives chiefly like an ordinary fungus.” But as soon as the process of fermentation ends, and some- times even before the whole of the sugar has been converted, ‘the yeast if originally not sufficiently rejuvenated gradually loses its power of living by deriving its oxygen from its nutrient medium, and the cells revert to their original con- dition of senescence. They become dormant, and until again supplied with oxygen can bring about no further fermenta- tion. It is for this reason that to obtain the hest fermenting yeasts, cultivations must always be made in the presence of free oxygen, and although this was done before Pasteur explained the reasons for-its necessity, it is now carried on in a much more systematic manner. Brewers knew perfectly well that they had to clear out their vats from time to time, not only to get.rid of foreign organisms, but also that they might obtain oxidation or more perfect aeration in the early stages of the process of fermentation. It is very interesting to note that Pasteur, although laying such stress on the connection between vital processes in the cell and the process of fermentation, at the same time holdsa very definite opinion that the vegetative activities of the yeast- cells are independent of their characters as ferments; and he maintains that the presence of oxygen, although increas- ing the activity of the cells as regards their subsequent power of setting up a rapid fermentation, may, nevertheless during its presence, give rise to weakening of their fermenting action. He says :— “ Free oxygen imparts to the yeast an increasing vital activity, but at the same time gu@ oxygen, impairs rapidly its power as yeast inasmuch as under 124 BACTERIA. this condition yeast approaches the state in which it can carry on its vital process after the manner of an ordinary fungus; the mode of life, z.¢., in which the ratio between the weight of sugar decomposed, and the weight of the new cells produced, will be the same as holds generally among organ- isms which are not ferments. In short, varying the form of expression a little, it may be concluded, from the sum total of observed facts, that the yeast which lives in the presence of oxygen, and can assimilate as much of that gas as is necessary for its perfect nutrition, ceases to be a ferment at all. Nevertheless, yeast formed under these conditions, and subsequently brought into the presence of sugar, away front the influence of air, would decom- pose more zz @ given time than in any other series of conditions under which it could be placed. The reason is, that yeast which has formed in contact with air, having the maximum of free oxygen that it can assimilate, is fresher and possessed of greater vitality than that which has been formed without air or with an insufficiency of air.” In other words, when the ordinary respiratory power which it has in common with the fungi is reduced to its lowest point, after the protoplasm of the cell has been thoroughly rejuvenated, its fermentive power in the absence of oxygen reaches its maximum. It is thus evident that the processes which go on in fermen- tation are similar in kind to those chemical metamorphoses that are constantly being brought about in the animal and vegetable organisms ; the only difference being, first, in the nature of the material that is broken up; second, in the nature of the cells that bring about the metamorphoses ; and third, in the exact nature of the ultimate products of the various processes; the difference in all cases being not so much in kind as in degree. As to the exact nature of the process, there is much difference of opinion, some authorities holding that it is necessary for the whole of the sugar which is altered by the living cell of beer-yeast to penetrate or pass, by a process of endosmosis, through the membranous envelope of the cell and become an integral part of the cell protoplasm, and that, unless this takes place, the resolution of sugar into alcohol, glycerine, carbonic acid gas, succinic acid, &c., cannot take place. It is necessary, in fact, that the whole of the sugar should be, as it were, digested by the yeast-cells, and combined in great part into protoplasm before it can be converted into the various substances above mentioned, which are, on this assumption, merely the excretory products of the vegetable cells feeding on a definite kind of nutrient material. We have seen, however, that the cells of yeast secrete a definite FERMENTATION. 125 substance, invertin, which has the power of materially altering the carbo-hydrates to which it is added, and from our knowledge of the functions of other cells we should be led to expect that these cells may exert some influence on the fermenting fluid without the whole of the sugar actually be- coming part of the cell; or perhaps that, on the other hand, the protoplasm may set up such molecular motion in its immediate neighbourhood, especially at certain tempera- tures, that a certain area of the sugar present is so acted upon that some of its molecules are set free for the use of the yeast-cell, and that the others can only rearrange them- selves in a definite manner ; and that, as a result, we have hydration and the formation of alcohol, carbonic acid gas, and water, the most stable elements that can be formed under the existing conditions, and out of the molecules of oxygen, hydrogen, and carbon that are available. There may be slight modifications giving rise to the formation of succinic acid or other bye products, as a greater or less number of accidental or superfluous molecules are set free to become converted into the superfluous or additional substances. It is a well-known fact, that when yeast is placed under conditions of moisture and warmth suitable for its develop- ment, if there be sufficient nutriment present, but sugar be withheld, or even if nitrogenous elements be kept from from it, it becomes soft, and certain marked changes go on in the substance of the cell. Bechamp found under such conditions leucine and tyrosine, both of them products of protoplasmic metamorphoses, a soluble albuminous substance coagulable by heat, an enzyme, a peculiar gummy substance, phosphates and acetic acid, along with which there was of course the production of a certain amount of alcohol, some carbonic acid gas, and pure nitrogen. Schitzenberger, repeating the experiments, found other products, such as xanthine, hypoxanthine, carnine, and guaine, that pointed most distinctly to a process of meta- morphoses of the protoplasm, and by a series of most inge- nious experiments he found that yeast in distilled water lost in five days about 9 per cent. ofits protoplasm. That is, in yeast there are certain soluble elements that can be re- moved by washing with distilled water, leaving the insoluble 126 BACTERIA. protoplasm, which can be filtered, calcined, and weighed ; after the process of self-digestion, which goes on when the yeasts are deprived of saccharine fluids, a further quantity of 9 per cent. is transformed from insoluble protoplasm into the soluble elements above mentioned. he protoplasm has, in fact, been living on itself, and, showing how indis-. solubly these metabolic changes are associated with the process of fermentation, both alcohol and carbonic acid gas are formed in the process. Yeast, then, has the power of disassimilating its substance by a series of steps into simpler bodies, but under favourable conditions it may be said to exert its metabolic power only in breaking down in a very superficial way large quantities of the medium on which it is grown—sugar. It is specifically adapted to break down this sugar as far as the stage of alcohol formation, but most yeasts cannot carry disintegration further. Other organisms, how- ever, have the power of carrying on the process as far as the formation of acetic acid for example, at which point another organism may again intervene and take up the work. We have thus the yeast forming alcohol and then dying out as it were, then the Mycoderma aceti comes in and does its work, and later various putrefactive organisms may continue the breaking-down process. On the other hand it must be remembered that whilst yeast sets up the alcoholic fermenta- tion, Hueppe’s lactic acid bacillus gives rise to the production from sugar of lactic acid, whilst the Bacillus amylobacter or the bacillus of the butyric fermentation causes the sugar to be split up directly.into butyric acid. We have here an example of three specific actions or fermentations of the same substance by the intervention of three different sets of organisms, and it is quite possible that other organisms effect an even further transformation into H,O and CO, at one step, the organism being specially adapted at each stage for the work that it has to carry on, or perhaps it would be better to say that the conditions are adapted to the organism. As might be expected, such self-digesting yeast is materially weakened ; it is no longer in a position to absorb much oxygen, and, if the process be long enough prolonged, both. the power of absorbing oxygen and the power of inducing fermentation are lost. But if the water that has been used to wash yeast, z.e., water containing the soluble products necessary for the perfect nutrition of the yeast cells, be FERMENTATION. 127 added to the exhausted yeast, the conditions of nutrition are rendered so favourable that the yeast cells again acquire in a most marked degree their original and characteristic power of absorbing oxygen, of vegetative proliferation, and of setting free alcohol and carbonic acid gas. We may briefly give in his own words the position that Schiitzenberger holds, so that it may be compared with Pasteur’s position, previously stated. ‘* The cell ferment is not developed in the absence of free oxygen, even in its most favourable medium, the must of grapes. The ferment, in process of development, continues to increase in suitable media, even in the absence of all trace of oxygen, as M. Pasteur had already shown. The contrary assertions of Brefeld are erroneous. M. Pasteur’s theory, according to which yeast, in the absence of air, takes from the sugar the oxygen neces- sary for its development, is not well founded; in fact, this development stops long before the greater part of the sugar is decomposed. Is it from the albuminoid matter that the ferment takes its oxygen in the absence of air? Yeast sets up alcoholic fermentation in a solution of pure sugar in the absence of all trace of oxygen, but without developing. This is contrary to the affirmation of M. Pasteur that fermentation is bound up with the organization of the yeast, or is a phenomenon correlative to the vital activity of the cells.” . We find that in nature there is, in protoplasm, not only an extreme adaptability to surrounding circumstances, but an attempt to utilize, as far as possible, the whole of the energy set free from cells. We find that in this matter the protoplasm of yeast cells differs in no essential respect from other kinds of protoplasm, and we have already seen that in the process of fermentation of saccharose there is a preliminary change brought about by the soluble products of the yeast-cells (invertin), by which we obtain dextrose and levulose, both of which materials may, under certain con- ditions, be split up into alcohol and carbonic acid gas. It may now, further, be noticed that if the fermentation be stopped at a comparatively early stage, there is found in the fermenting solution a larger quantity of levulose than of dextrose ; z.¢., the dextrose is more readily converted into the characteristic products of fermentation than is the levu- lose. It has also been proved that if the yeast-cells be heated to a temperature of about 60° C., they are destroyed, as is evidenced by the fact that they are no longer able to multiply, and they never give further evidence of vitality. But there is left in the fluid a substance known as enzyme (é» Sun, leaven—yeast), which, added to the mixture of dextrose and levulose, does not affect the levulose in the slightest.. 128 BACTERIA. (This may also be done by using the Pasteur-Chamberland filter, which keeps back the yeast-cells, but allows the enzyme to pass.) It is only on the addition of living yeast, or that yeast in which there is active protoplasm capable of actually digesting and transforming food materials, that the levulose becomes transformed into dextrose. We have here three very significant statements, all of them attested by eminent authorities, which seem to explain many of the anomalies and disagreements in indi- vidual statements. The process of inversion is reduced to a purely chemical one, in which the chemical reagents bring- ing it about are manufactured by the living protoplasm ; apart from which, however, it is able to exist and act so that saccharose is converted into dextrose and levulose merely by the action upon it of the chemical products or enzymes contained in yeast water; whilst the conversion, and cer- tainly the fermentation of levulose, can only take place when this material comes into actual contact with the living pro- toplasm, from which it seems, necessarily, to be more closely associated with the actual process of digestion. This is a decided step in advance in assisting to explain the process of fermentation, of digestion in the higher animals, and of those changes that take place in putrefactive and pathogenic processes. It also appears to throw light on some of the points in dispute on fermentation.. For example, we can easily understand how different the results would be in cases where experimenters use yeast as the fermenting agent, and crude saccharose as the fermentable substance in one case, and yeast with dextrose in another. The process of inversion of the saccharose by the invertin into dextrose and levulose is carried out easily enough, whether oxygen be present or not, and this dextrose is easily broken up by the yeast. When we come to the levulose, however, it is a different matter, as this substance can apparently only be converted into dextrose when it is in direct contact with the protoplasm of the cell, and so long as food materials and oxygen can be obtained from other sources there is not the same tendency for the protoplasm to take up this levulose that there is when other sources of food supply are cut off. The active and rejuvenated cells are still able to utilize the levulose, whereas older cells, which, as we have seen, after self-digestion lose even their power of transforming FERMENTATION. 129° dextrose, lose their power of transforming levulose at a still earlier period. This enzyme function, although so intimately associated with the vital activity of protoplasm and so usually possessed . by cells, is considered by Hueppe to be a secondarily derived function of the protoplasm, and to be really a modification or development of the primary power of digestion possessed by all protoplasm independent of the action of enzymes, only to be met with when the conditions for the growth of the organism and the development of its functions are most per- fectly combined. It is, as Cartwright Wood says, a function developed in such a high degree, that it may actually be temporarily separated from the protoplasm which develops it. It must be looked upon as something separate, and as some- thing that can act, under favourable circumstances, apart from protoplasm, economizing the energy of the proto-: plasm, and utilizing the most suitable products for the nutri- tion of the organism and for the manifestation of its special functions. Under less favourable conditions, where the pro- toplasm has to struggle for existence, as it were, this enzyme function is withdrawn ; and although the protoplasm can still exert its characteristic powers of digestion and of forma- tion of special products, these processes take place within the cell, they are of a less specialized nature, and are, in fact, the primary and inherent functions of the cell protoplasm, which are capable of doing their work within the protoplasm, but are not so highly differentiated that they can act exter- nally to it. The primary function is, then, not separable from the protoplasm. This formation of enzymes is of special interest in relation to the processes of disease; for it is found that, by modifying the conditions under which an organism grows, or by varying its food, special ferments come into operation. For instance, Lauder Brunton and M’Fadyean, experimenting with a certain organism, found that if it were grown on peptonized beef jelly, it would give rise to an enzyme which was capable of liquefying the gelatine. If this organism were now introduced into a solution of starch, it would continue to grow, but it could give rise to no diastatic ferment—z.e., it was incapable of transforming, except within its own protoplasm, the starch material into sugar, or into material that it could utilize for its own nutrition. If, how- ever, successive generations of this same organism were cul- 10 130 BACTERIA. tivated on starch, there came a period at which it generated a diastatic ferment, which could be separated from the solu- tion, and which alone, and without any direct intervention of the protoplasm, could bring about a transformation of starch into sugar—ze., a secondary separable diastatic func- tion had been developed, in addition to the primary digestive function of the organism. When taken back to the gelatine from the starch, the organism had lost its power of liquefy- ing gelatine ; but this again it regained, after being again cultivated, through a series of generations, on gelatine. It is quite possible that this may be a somewhat too strong statement of Lauder Brunton’s and M’Fadyean’s position. It may be that they maintain only that the organism when grown on starch produces the diastatic ferment, whilst when on albumen it forms a peptonizing substance. It may be merely a temporary modification just as under analagous conditions, the cholera bacillus when grown on sugar produces butyric acid, whilst when grown on albumen it forms ptomaines. It is thus evident that special forms of fermentation require that, in addition to a special kind of protoplasm, there shall be a special nutrient material—a fact that explains some of the different results that have, from time to time, been recorded. The yeasts, as we have seen, are capable of fer- menting dextrose and of producing a substance, invertin, by which the saccharose, or cane sugar, is converted into dex- trose and levulose—both fermentable substances. Wood points out that only three yeasts are known that are able to ferment milk sugar directly, although many of -the bacteria are able to invert and bring about its fermentation. He says that— “* Of still greater interest is the varying manner in which the same organism condicts itself towards different albuminoids. . . . Asa general rule, those organisms which liquefy gelatine are able to coagulate milk, and then peptonize casein which has been separated; but some organisms which peptonized gelatine are without action on the milk, and some that are Inoperative on gelatine peptonize milk, though this is exceptional” ; and he further says that even the manner in which “ milk is peptonized is subject to considerable variations, and that, although the vast majority first coagulate the casein and then dissolve it, certain microbes seem to peptonize the casein directly. . . . Very striking is the way in which the same organism conducts itself to the different albuminoids, gelatine, fibrin, blood-serum, and egg albumen. One organism is unable to liquefy gelatine, but pep- tonizes fibrin ; another liquefies the gelatine, but cannot peptonize the FERMENTATION, 131 egg albumen. . . . They must, accordingly, be regarded as specific in their nature, depending on the specific nature of the protoplasm of which they are merely further differentiations.” Micro-organisms, when grown under such conditions that oxygen cannot gain access to them—under conditions of anzrobiosis—usually appear incapable of developing their separable peptonizing enzyme function, as they are no longer able to liquefy the gelatine in which they are growing. The fact that this separable enzyme function could be kept in temporary abeyance was utilized by Chantemesse and Widal, to prevent the liquefaction that takes place in plate cultiva- tions where a certain specific organism requires a considerable time for its complete development. By adding dilute carbolic acid to their nutrient medium they found that the peptonizing power of the organisms was interfered with ; whilst if the carbolic acid solution was sufficiently weak, even the more delicate organisms still retained sufficient vitality to enable them to grow on the surface of the carbolized gelatine. Having corroborated the above observations, Hueppe and Wood sought to obtain the same results while avoiding the dangers resulting from the use of such a substance as carbolic acid, by offering for the nutrition of the organism a certain preparation of a material which would enable it to develop the diastatic, instead of the peptonizing ferment ; and, by adding glycerine to the gelatine, they found, as they expected, that Lauder Brunton’s and M’Fadyean’s experiments were practically repeated. The organisms feeding on the glycerine did not in most cases attack the gelatine, and the peptonizing became exchanged for a diastatic ferment ; plate cultivations were not so rapidly liquefied, and bacilli of very slow growth could thus be separated from impure cultivations, in which organisms that ordinarily exert a liquetying or peptonizing function were present in considerable numbers. Our knowledge of most other fermentations, although being gradually increased, is still in a somewhat nebulous condition, for the reason that no one has as yet (with the exception of those who have worked at the matter from the industrial point of view) taken up the matter thoroughly since it became possible to obtain pure cultiva- tions of any single organism, the whole energy of bacterio- logists having been diverted to the consideration of the very important questions that have come. up to be answered in 132 BACTERIA. connection with the production of specific infective diseases in man and animals by micro-organisms. There has, indeed, been so much work to be done in separating out the causal bacterial agents in these diseases, that there has been little time or energy left to be devoted to anything more than a most cursory study of the chemical and biological processes of the non-pathogenic forms. We do know, however, something about the man- nitic fermentation of sugar, the lactic, the butyric, the ammoniacal, and the acetic fermentation ; and, speaking generally, these processes are essentially the same as those which take place in ordinary alcoholic fermentation, the differences being that the protoplasm cells of the ferment differ from those of yeast both in morphological and bio- logical characteristics, and that the composition of the end - products is also different. The food material acted upon by different protoplasm, and giving rise to different results, may be the same ; the fermentation is still brought about by a process of hydration or some other specific process, and is intimately associated with the vital activity of the special ferment. In the mannitic fermentation the sugar is converted by hydration into dextrose and levulose, after which comes the conversion of some of the sugar into gum, and of some into mannite, during which second part of the process there is also an evolution of carbon dioxide and the formation of water. Mannite is a sugar to which a molecule of hydrogen has been added; gum, a cane sugar from which one molecule of water has been sub- tracted. The viscous change that takes place as the fermen- tation proceeds is due to the formation of this gummy material in the fluid. Pasteur described the special man- nitic ferment as consisting of chains of small cocci, each coccus having a diameter of 1.2 to 1.5#@ of an inch. These, like the yeasts, require nitrogenous material, in addition to salts, extractives, and sugar, in order that they may develop. This form of fermentation is specially in- teresting, from the fact that it is to it that the ropiness of the white wines is due—a ropiness which, as Pasteur pointed out, might be prevented by heating the wine for a few minutes at a temperature of 60°C., or, as suggested by Frangois, by the addition of a certain proportion of tannin, a substance which appears to interfere most markedly with the development of the viscous fermentation organism. FERMENTATION. 133 In the lactic acid fermentation it does not appear to be necessary that any process of hydration should take place, as there is here merely an exact division of one molecule of sugar into two molecules of lactic acid, there being neither addition nor loss of either oxygen or hydrogen. It must, however, be borne in mind that the lactic acid fermentation is frequently accompanied by the formation of CO,, in which case, of course, the process is not nearly so simple. By fermenting lager beer at from 30° to 34° C., Chr. Hansen found that there was developed a form of bacterium made up of long chains, of dumb- bell or hour-glass shaped organisms, of bacteria as long curved or straight threads ; whilst at irregular intervals were formed spindle-shaped or club- shaped bacteria, even when the nutrient conditions still continue good at a late stage of the growth. ‘That these organisms, however, were not all alike, Hansen proved by the fact that some of them are stained yellow by iodine, whilst with others a blue reaction was obtained. The conditions necessary for the development of this organism, and the production of acetic acid, are a high temperature—from 30° to 34° C.—and a plentiful supply of oxygen. So necessary is this latter that a film forming on the surface is all that there is to denote the presence of an organism, the fluid beneath, from which free oxygen is cut off, remaining perfectly clear; and Hansen even gives this as a diagnostic feature to enable an observer to determine whether he is dealing with pure cultivations of these mycodermi or not, as, wherever other bacteria are present, roughly speaking, turbidity is set up. It is a curious fact that the pure lactic fermentation cannot go on when the medium is too acid, and it is only by remov- ing the lactic acid as it is formed that a complete trans- formation even of milk sugar into lactic acid can be obtained. This fact was observed long before the exact nature of the . process was understood; and in all the earlier methods devised for the preparation of this substance the daily neutralization of the fluid with chalk or carbonate of soda played'a most important part. That the process is essen- tially the same as the others, in so far as it is the result of the activity of micro-organisms, was proved by Pasteur, who found that by sowing the lactic acid ferment, which he described as composed of small globules or short joints, either isolated or in mass, in a fluid which he had found specially suitable for alcoholic fermentation, active lactic fermentation was coincidentally set up, and lactic acid was found present along with the resulting alcohol ; whilst, as we already know, if no lactic acid fer- ment be introduced, either accidentally or intentionally, no 134 BACTERIA, lactic acid fermentation ever takes place. Although this is true in a certain degree, it is found, as has already been stated, that there are actual differences in degree as to the readiness with which the various sugars undergo the alco- holic and lactic fermentations. For instance, although the glucoses are readily converted into lactic acid, the saccharoses, which are specially fermentable by the saccharomyces, are not very easily transformed into lactic acid; whilst, on the other hand, sugar of milk, which does not readily yield alcohol, may be comparatively easily converted into lactic acid. Muscle sugar and mannite, neither of which can be converted into alcohol, may both be broken up into lactic acid, as may all those substances that are specially affected by the butyric fermentation. It was for some time thought that the fermentation of urea, through which carbonate of ammonia is frequently met with in the urine, was the result of a single micrococcus, the micrococcus urez, a small organism about I in diameter, which may occur in pairs, in tetrads, or in longer chains, and which, in the presence of suitable nutrient materials, and of urea, either in artificial solution or in urine, determines a regular hydration of these substances and their conversion into carbonate of ammonium. Since the time of Pasteur’s researches, however, Leube has found an organism about double the size of the above and arranged in chains, which rapidly decomposes solutions of urea into carbonate of ammonia. It differs, too, from Pasteur’s organism in that it peptonizes or digests gelatine, causing it to liquefy, and it appears that, unlike some other bacilli and sarcine that can set up a feeble urea fermentation, it is quite as powerful an ammoniacal ferment as the small micrococcus ure described by Pasteur, as in both of them the fermentation can go on most vigorously, even in the medium that has become distinctly alkaline, ze., the carbonate of ammonia accumulates until about 13 per cent. of this substance has been formed ; these two microbes may be said to bear the same relation to the other urea-forming bacteria that the Saccharomyces cerivisie and the other more powerful alcohol-forming yeasts have to the weak saccharomyces and other sugar fermenting organisms. That this power is not confined to a single organism is not remarkable, since it is quite possible by boiling the urea in FERMENTATION. 135 water, or, better still, in slightly alkaline solution, to bring about direct hydration of the urea into ammonium car- bonate. When it is asked what value the study of fermentation has been to the cause of medicine, it is not necessary to go further for an example than this urea fermentation. In the operations of the older surgeons it was very frequently recorded that the introduction of instruments into the bladder (in which, of course, was a solution of urea) was followed by a regular fermentation, which too frequently led to the death of the patient ; and it having been determined that micro-organisms were the cause of this fermentation, it became an easy matter to prevent their introduction by careful sterilization of the instruments; and, as a result, such ammoniacal fermentation within the bladder is now a matter of very infrequent occur- rence, and is, in those cases in which it does occur, the result not so much of accident as of carelessness on the part either of the patient or of the surgeon. So marked, indeed, has this been, that even those who sneer at bacteriology and antiseptics have been compelled to accept new methods of procedure in regard to the cleanliness of their instruments, a cleansing which means simply the removal of these minute vegetable protoplasmic organisms, which, on being introduced into the suitable fermenting medium in the bladder (or elsewhere), give rise to the alkaline fermentive, putre- factive and other processes. In an interesting chapter on the butyric fermentation of putrefaction, Schiitzenberger points out that in the process of putrefaction or putrid fermentation, as it may be called, there is a formation of butyric acid, one only of a series of fatty acids formed under similar conditions ; accompanying this there occurs a transformation of glucose, starch, lactic acid, albuminoid substances, and the various fruit acids, into these fatty acids, into carbon dioxide, and into certain hydrogen compounds. From a very large number of these substances lactic acid with or without carbon dioxide is formed, whilst the lactic acid may be further split up into butyric acid, carbon dioxide, and hydrogen. From this chapter we may borrow some of the formulz to show how easy and how rational is the transformation from these substances into the fatty acids. Other substances may also be formed, these being further broken down or separated as the case may be. Glucose, as we have already seen, is, under the action of the lactic acid ferment, converted into lactic acid, and this under the action of the butyric. ferment, a small rod-shaped organism from 1.8 to 18 in length and 1.8#in breadth is converted into butyric acid, carbon dioxide, and hydrogen. These micro-organisms 136 BACTERIA. are straight, somewhat rounded at the extremities, single, or arranged in short chains of three or four elements ; if placed in a nutrient medium similar to that in which yeast grows, they are reproduced by a process of vegetative division, especially if this medium be neutral, or slightly alkaline, and if it be kept at a temperature of 40° C. The formula of the process of splitting up is given as_ first CoH1206 a 2C,H.O; 1 Molecule Glucose ~ 2 Molecules of Lactic Acid The two Molecules of Lactic acid are then transformed into C,H30, + 2CO, 1 Molecule of Butyric Acid 2 Molecules Carbon Dioxide 2M 2 Molecules Hydrogen In the same way two molecules of malic acid are broken up into two of lactic acid and two of carbon dioxide, and the lactic acid is again transformed as above, that is, 2C,H«O; = 2C;HsO; + 2CO, = C,HgO. +-4CO, + 2H2. This butyric acid formation is only one of a group, and we find that where lactic fermentation is going on in an impure condition propioni¢ acid, C,H.O., acetic acid, C2H,Oz, and valerianic acid, CsH1.O2, are produced, and glycerine fermented for some time with beer yeast, is split up into pro- pionic acid mixed with formic and acetic acids, giving a kind of compound of putrefaction products. A very ingenious formula is quoted by Schiitzenberger to show how the fatty acids are formed from sugar :— = (CéHi206) = [CnHenO2] (n—2)CH.O, Fatty Acid Formic Acid group, being immediately converted into carbon dioxide and hydrogen. The presence of lime or organic matter appears to modify this process some- what, and to give rise to the formation of secondary vegetable acids. For instance, two molecules of malic acid, 2C,H«O;, break up into succinic acid, C,H6O,, and tartaric acid, 2C,H6O., and from these are formed, from the tartaric acid the fatty acetic acid, C.H,O, + CO, + H., and from the succinic acid the fatty valerianic acid, carbon dioxide and hydrogen, C,H,.02 + 3CO. + H.. + Formic acid, the lowest of the fatty acid These transformations have this in common, that they are all brought about by the presence of minute organisms, that the conditions of the development of these organisms are similar to those met with in other fermentation pro- cesses, and that theirs are the principal products formed during the process of putrefactive fermentation, which also in all cases requires the presence of these minute organisms, the spores of which, as we have seen, seem to be everywhere FERMENTATION, 137 present. Here, as in the case of the yeasts, we find both zrobic and anzrobic organisms. The zrobic forms give rise to oxidation of certain of the products of decomposi- tion, one of these, nascent hydrogen, especially taking up oxygen, as a result of which water is formed; the an- zrobic organisms, which may be said to complete the process of putrefactive fermentation, only commence to grow when the zrobic organisms have used up all the readily available oxygen such as that dissolved in a liquid, on the surface of which there has formed a kind of film, by the intervention of which aeration from the external air is greatly interfered with. We have already noted this film growing on yeasts, when it appears to assist the carbon dioxide developed during the pro- cess of fermentation in keeping off the oxygen of the atmo- sphere, and to allow of these anzrobic organisms carrying on their growth without the further intervention of a free oxidiz- ing agent. The products of these anzrobic organisms, of course, differ from the products of the zrobic fermentations in so far that they contain less oxygen, the sulphuretted hydrogen, hydrogen, and nitrogen being released in a more or less free condition and uncombined with oxygen, the oxygen of the original fluid being used up in the formation of such substances as leucine, C,H,,(NH,)CO OH ; tyrosine CH, | ore (NH,)CO, OH) ; the volatile fatty acids ; some compound ammonias, and, of course, carbon dioxide ; the two latter of which, as already seen, may be formed from urea (CO(HN?),) by simple hydration, the more highly organized substances, such as leucine and tyrosine, being, during the process of fermentation, further converted inta ammonia and the fatty acids. From the variety of the products of these fermentations it is evident that we have to do, not with a simple conver- sion by means of any single organism into the simplest and ultimate elements of decomposition, but that we have a series of breakings down or stages of decomposition by which the highest (and most unstable) organic materials are gradually transformed into the lowest and most stable. It will be found, however, that the process is not quite so straightforward and simple as above stated, as some of the energy released during the formation of a number of lower 138 BACTERIA. and more stable compounds is utilized in the presence of animal and vegetable protoplasm in building up higher and more stable materials ; though at the later stages of decom- position these become fewer in number, and eventually become oxidized into the more stable forms—a free access of air to the products of anzrobic decomposition always leading to their oxidation. Hydration almost invariably occurs during each of these stages. Schiitzenberger says, ‘‘ Nothing resembles putrid fermentation, with reference to the derived products, more nearly than the change which takes place in the constituent parts of yeast, when left to itself without nourish- ment, deprived of sugar and oxygen. We see, in fact, the appearance of leucine, tyrosine, sarcine, &c. This is the first step; the action stops there, and goes no further; the yeast, or the special soluble ferment which it secretes, is unfit to attack these bodies again; but if we wait for the development of vibrios, we shall find the production of ammonia, carbon dioxide, and volatile fatty acids at the same time that the leucine partly disappears.” Fermentation by oxidation can only be set up by organisms in the presence of a free supply of oxygen, and the process in such cases appears to be due to special organisms which, attacking the substance to be fermented, remove some of its constituents, set free others, especially carbon and hydrogen, which, in their nascent condition, are seized upon by the oxygen of the air, and so water, or water and carbonic gas, are formed. ‘In the commonest of these fermentations by oxidation, the acetic fermentation of alcohol, the free access of air is as absolutely essential for the growth of the organism as it is for the special stage of the fermentation of the alcohol into acetic acid. If the mycoderma aceti be sowed in wine, freely exposed to the air at a temperature from 24° to 27° C., chemists hold that there is hydration of alcohol by the combination of an atom of free oxygen with two atoms of its hydrogen water, OH, and aldehyde C,H,O, being formed, the latter of which, by further oxidation, in turn becoming converted into, C,H,O,, acetic acid. At a certain stage this process of acidification stops, owing to the quantity of acid developed, but if this be removed and fresh alcohol be added, the acidification again commences, and so on, as long as the mycoderma remains active, can obtain sufficient oxygen, and sufficient alcohol FERMENTATION, 139 on which to act. If, however, the supply of either oxygen or alcohol be cut off, the ordinary mode of life of the mycoderma is at once interfered with. It uses up part of its own protoplasm by internal metabolism, continues to act on the acetic acid, and converts it into simple substances, water and carbon dioxide, at the same time retaining molecules of carbon, hydrogen, and oxygen for its own use. The mycoderma is, then, no longer so much an enzyme-former or an organism with a thick celled wall, that acts principally on the suitable nutrient medium by means of its chemical products, and gives rise to a definite chemical process ; but is now rather a mass of protoplasm endowed only with its primary digestive powers, by means of which it can convert organic matter into its very simplest forms or ele- ments. There is very complete oxidation, and instead of alcohol and acetic acid being formed as intermediate products that can be easily separated, the substances are at once converted into water and carbon dioxide, the ultimate products of perfect oxidation. It is sometimes said that the mycoderma in the true vinegar fermentation is in a weakly condition ; it is cer- tainly not in its most active condition, but the state appears to be one rather of quiescence or advanced development, in which the formation of a thick membrane and the produc- tion of a separable enzyme are characteristic features, rather than one that can be spoken of as a condition “ of incomplete or tardy development.” To show that the process was not merely a physical one, as was suggested even by Pasteur, Mayer showed that by simply heating the acidifying fluid to a temperature at which the mycoderma is killed all oxidation is arrested, and he also showed that the temperature at which oxidation of concentrated alcohol goes on in spongy platinum is at a temperature above 35° C., at which point it is completely ‘stopped in the presence of the mycoderma aceti, in addition to which “ physiological acidification” can only take place in a weak solution of alcohol. Pasteur’s observations and results led to the adoption of what is known as the Orleans process of making vine- gar, which, as given by Schiitzenberger, is as follows :— “The mycoderma aceti is made up of small, slightly elongated cells, with a transverse diameter of from 1.5 140 BACTERIA, to 3H, united in short chains, or curved rods. Con- striction and division take place between the segments, of the chain as the process of vegetation goes on. A small quantity of this mycoderma, which occurs as a wrinkled membrane on the surface of liquids that are undergoing acetic fermentation, is first sowed on the surface of an aqueous liquid containing two per cent. of alcohol, one per cent. of vinegar, and traces of alkaline and alkaline-earthy phosphates. When the surface is covered with the mem- brane the alcohol begins to acidify. This action being fully set up, some alcohol, wine, or beer mixed with alcohol, is each day added to the liquid, in small quantities ; this is continued till the oxidation becomes slower ; the acetification is then allowed to terminate, and the vinegar is drawn off. The membrane is collected, washed, and employed for a new operation. It is better always to give the plant sufficient alcohol, so that its activity may not be exerted on the acetic acid. Nor ought it to remain too long out of the liquid, or it would lose its active force ; finally, it is better to moderate its development, to prevent burning oxidation.” These last few lines are of special interest, as they show how markedly favourable conditions of nutrition, and the supply of special materials on which the organisms may act, modify the process set up by the acidifying organism ; just as certain organisms will select glycerine or sugar from a peptone gelatine medium before they begin to act on the gelatine itself, so the mycoderma aceti, under certain conditions, confines its attention entirely to the alcohol, converting it into acetic acid and then leaving it ; whilst, as soon as the glycerine in the one case and the alcohol in the other, are completely used up, or as soon as the activity of the protoplasm becomes so great that it cannot derive sufficient material from the one, it immediately attacks the other, and continues the oxidizing process. Not only so, but the various organisms appear to be specialized at the different stages of the resolution of organic matter; each, during the stage at which it can perform its share of the work, appearing not only to multiply much more rapidly than the other organisms with which it is found but actually holding them in check. In this respect. the vegetable protoplasm of the lower organisnis is only like the protoplasm of the cells of the higher animal organisms. In relation to pathogenic processes this is a FERMENTATION. I4l point of very considerable interest, for we find that under certain conditions there comes to be a contest between the animal cells arid the lower vegetable cells. As Wood well puts it, “the reaction of the cells upon each other may turn on the sum of the conditions of existence to which they are exposed happening to favour one more than the other, thus when two organisms are sown together in a culture fluid, which will overgrow the other may depend upon the relative quantity of the two primarily introduced, the nature and reaction of the media, and the temperature at which they are held.” Fermentation, then, is due to the action of highly specialized cells. That this is so is indicated by the usual presence of enzymes and also by the fact that they exert this specific action in their highest degree only under a certain set of specific conditions which vary in the case of each organism. Each organism has become adapted to a certain medium, and is so specialized as to have the power of splitting it up under certain conditions in a specific way. This is also indicated by the fact that as the conditions become unfavourable, less and less of the specific product (relatively to the bye products) is produced. Although we shall have more to say in connection with the subject of the specific infectivity of micro-organisms in disease, it may here be pointed out that fermentation is due, in great part, to the action of cells which have the power of developing a special enzyme function ; that these cells are usually more highly specialized during the stages when they bring about their specific action ; that this is associated to a certain extent with diminished activity of the protoplasm directly on the sub- stances to be fermented ; that most processes where there is- the formation of an enzyme, or a special poison, take place most actively when there is a complete or partial cutting off of the supply of oxygen ; that the cells in this condition usually develop a more or less perfect cell membrane which characterizes the formation of zooglcea masses ; that this in- complete oxidation also characterizes the formation of toxines ; that the same rules hold good in the formation of most of the products of the lower vegetable organisms and of the individual cells of animal tissues with complete oxidation and the formation of carbonic acid gas and water ; that when incomplete oxidation takes place various specific products make their appearance ; but that even in 142 BACTERIA. the presence of a full supply of oxygen, protoplasm under certain conditions cannot bring about complete oxidation, and as a result some of the intermediary products of fer- mentation and decomposition may be formed. lt is evident, then, that all ferments may be classed under two great heads: firstly, the organized ferments or the active unstable protoplasmic cells of yeasts, bacteria, of plants and animals. Secondly, the unorganized ferments or enzymes which we have spoken of as the separable functions of more highly developed cells, both of them being, however, as- sociated with the nutritive and other metabolic changes of protoplasm, be this protoplasm that of simple vegetable cells or of highly organized gland cells. Hoppe-Seyler classifies the whole of the fermentations as follows :—I. ferments which bring about hydration or cause hydrolysis, these being divided into (a) those that act like boiling mineral acids, all of which belong to the enzyme group ; and (4) ferments acting like caustic alkalies in which we have the process of decomposition (1) of fats into glycerine and fatty acids as in saponification, which results from the action both of the organisms directly and of the unorganized ferments, (2) decomposition of amido —or ammonia-nitrogen compounds which is also usuaily associated with hydrolysis. II. In the second group, Hoppe- Seyler places fermentations in which there is transference of oxygen from the hydrogen to the carbon atoms. As examples of this we have (a) lactic acid fermentation in which there is decomposition of certain carbohydrates into lactic acid ; this, as we have seen, being always associated with the presence and activity of a certain group of micro- organisms, (4) the alcoholic fermentation brought about in a similar manner by yeasts, (c) putrefactive decomposition changes, (1) of simple inorganic compounds such as combi- nations of the fatty acids with lime, or (2) of organic com- pounds such as fibrin and other proteids. Then we have also the acetic fermentation already referred to, nitrification or mineralization, chromogenic fermentation and that series of changes in which we have the production of ptomaines. Such a classification is specially important when we come to consider the relation of the ordinary fermentation pro- cesses to those that are set up during the course of disease in animals and in plants. FERMENTATION. 143 LITERATURE. Authors already mentioned.. Fligge, Davaine, Roberts, Tyndall, Fitz. — Becuamp.—Comptes rendus, t. LVI, pp. 969, 1086, 1231, 1863; t. LXL, pp. 374, 408, 1865; t. LXXV., p. 1036, 1872; t. XCIIL, p. 78, 1881. BoutTrovux.—Comptes rendus, t. LXXXVI., p. 1, 1878. Bunee.—Text Book of Phys. and Path. Chem. (Transl. : Wooldridge). London, 1890. ag ea pbaas Zeitung, 1875 ; Die Hefepilze, Leipzig, — 1883. es AND WinaL.—Gazette Hebdomadaire, p. 146, 1887. CaGniarD-Latour.—Ann. Chim. Phys., 2nd ser., t. xv., 1820. DesMazigRES.—Ann. des Sc. Naturelles, t. x., p. 4, 1826. Dusrunrant.—Comptes rendus, t. XLIL., p. 945, 1856. EnGet.—Les ferments alcooliques. 1872. HoprE-SEYLER.—Zeitschr. Physiolog. Chemie, Bd. x., p. 36, 1885. Huepre.—Mitth. a. d. Ges. Amt., Bd. 11, p. 309, 1884. Huepre anp Woop.—JZancet. Dec. 7, 1889. Hansen, Cur.—Meddel. fra Carlsberg Lab. Copenhagen, 1879, '81, '83, 86,88; Allgem. Zeitschr. f. Bierbrauerei u. Malzfabr. 1883-87 ; Zeitschr. f. d. ges. Brauwesen, 1884, and republished in Munich, 1888. Hoitm anp Poursen.—Meddel. fra Carlsberg Lab., Bd. 11, 1886. JORGENSEN.—Micro-Organisms of Fermentation (Trans.), London, 1889. , Kieser.—Schweiggers Journ., 1814, No. x11, p. 229. Leuse.—Virch. Arch., Bd. c., p. 540, 1885. Lirsic.—Ann. de Chimie et de Phys., 2nd ser., t. LXXL, p- 178, 1839. ; Lister.—Pharmac. Journ. and Trans., vol. vut., 3rd series, _ Pp. 555) 1878. . : Maver.—-Die Lehre von den chemischen Fermenten. Heidel- berg. 1882. PasteuR.—Ann. de Chimie et Physique, 3me Serie., t. Lvut., p- 323, 1860; Etudes sur le vin, 1866 ; Etudes sur le vinaigre, 1868; Etudes sur la biére, 1876; Comptes rendus, 1876, t. LXxxulL, p. 173 ; Lecture faite a l’Acad. 144 BACTERIA. de Méd., 1878, 26 Nov. ; Comptes rendus, 1861, t. LIL ; 1864, t. LVI. | Reess.—Botanische Untersuchungen itiber die Alkohol- gahrungs-pilz. 1870. ScHUTZENBERGER.—On Fermentation. London, 1886. ScHwann.—Poggendorf Annal., 1837, Bd. 41, p. 184. Trécut.—Comptes rendus, t. LXI., pp. 432, 553, 1865; t. LXV., Pp. 513, 927, 1867. Turpin.—Comptes rendus, t. vit, p. 369, 1838. vAN TIEGHEM.—Ann. d. Sc. nat. Bot. Série vi, t. 4, 1878. WIESNER.—Sitzungsber. der Wiener Akademie, Bd. Lix., 1869. WI.is.—De Fermentatione. 1659. Woon, Cartwricut.—Lab. Reports Roy. Coll. Phys., Edin., 1890, p. 253. CHAPTER VII. PARASITES AND SAPROPHYTES. Putrefactive Bacteria—Pigment-forming Bacteria—Enzyme-forming Bacteria —Non-pathogenic, Saprophytic, Parasitic, Pathogenic Bacteria— Sarcina Ventriculi—Leptothrix Buccalis—Facultative Saprophytes— Facultative Pathogenic Parasites — Anthrax Bacillus — Pathogenic, Parasitic, Saprophytic, merely Relative Terms. - In order that much of what occurs in the following chapters may be understood, it is necessary that some idea should be given of the way in which the terms saprophytic, parasitic, and pathogenic are used. The first of these is not so impor- tant; the term saprophytic as applied to bacteria is being gradually supplanted by the term non-pathogenic, especially in works dealing with the relation of micro-organisms to disease. Speaking generally, saprophytic bacteria are those bacteria that have the power of obtaining the nutriment they require for their building up, and for the carrying on of their vital functions, from dead vegetable or animal substances only. They are not able to invade the tissues of living animals and plants, but they play an important part, as we have already seen, in bringing about oxidation of various organic materials and of giving rise to certain definite com- pounds. To this class belong the putrefactive bacteria, and many of the pigment-forming bacteria ; the yeasts or fermen- tation fungi are also to be looked upon as purely saprophytic in character ; and such organisms as the Bacillus amylo- bacter, the lactic acid bacillus, and the like, are all probably to be classified in this group. The subdivision of the group has been attempted by many workers, and the saprophytic organisms have been arranged according to their power of action on the various carbo-hydrates and on proteid substances. Others have taken their products as a basis on which to classify them, and enzyme-forming, pigment-forming, or alcohol- II 146 BACTERIA. forming organisms have all been described ; but as their powers of decomposing different substances and of giving rise to pigment or to certain enzymes necessarily overlap one another, or are met with in the same individual under different conditions, it remains an exceedingly difficult matter to give any classification founded on the above mixed characteristics. Some of the organisms which, at present, are supposed to be saprophytic in character, may eventually be found to be parasitic under certain conditions, and it is for this reason that the term ou-pathogentc is gradually ousting the older botanical term of saprophytzc. Parasitic bacteria are those which are able to flourish on or within the substance of plants or animals. In animals they live in the various cavities of the body, or in certain cases in one or other of the nutrient fluids of the body, and they derive their food from these fluids, or from the nutrient materials that are taken in by the host. The ordinary terms of obligate parasites, facultative saprophytes, and facultative parasites, can scarcely be used in connection with these minute organisms, as, with very few exceptions, all the parasitic bacteria have now been cultivated outside the body and under such conditions that it is evident that some of those which were at one time looked upon as purely parasitic have a saprophytic stage of existence; so that nearly all, if not all, the parasitic bacteria must be looked upon as facul- tative parasites, or parasites that can develop almost as well saprophytically as they can parasitically, or facultative saprophytes, or saprophytes that can develop almost as well parasitically as they can saprophytically. We know of course that all parasitic bacteria are not pathogenic, and that we have along the alimentary tract, and even in the respiratory tract, a large number of para- sitic organisms. The Sarcina ventriculi, for example, first described by Goodsir, may be taken as a purely parasitic organism. It is found especially in dilated stomachs. A drop of fluid taken from the contents of such a stomach frequently contains a number of organisms which present a curious appearance under the microscope. They are very like the typical bale of wool or goods tied with a strong cord in three directions. Other sarcinz are saprophytic as well as parasitic, but the Sarcina ventriculi has not yet been PARASITES AND SAPROPHYTES, 147 satistactorily cultivated outside the body, or if it has, it has always become somewhat altered in appearance. Sarcine have also been found in the bladder, in the air passages, in the intestines, and even in the blood, but most of these can be cultivated on nutrient gelatine. None of these organisms are pathogenic so far as at present can be made out; they appear to be simply accidentally associated with certain conditions, as in the case of dilated stomachs, in which they are not always present, but in which they frequently occur, apparently because the acid fermentation that goes on in the accumulated contents renders them specially favourable media for the development of these sarcine, The non-pathogenic parasitic bacteria of the mouth were amongst the first organisms observed by Leeuwen- hoek ; they are found on the gums, on the sordes covering the teeth, and on the mucous membrane of the mouth. The forms of these we shall mention later. Although most of these are parasitic and non-pathogenic, one form, the Leptothrix buccalis (an old term embracing probably several species, and one not now very generally used), is also patho- genic in the sense that it invades the teeth, and gives rise to what is known as‘caries, or rotting of the teeth, but even this has to obtain help from some of the other non-patho- genic species found (some of the micrococci), which give rise to the formation of lactic acid from the sugar of old food, an acid that, combining with the lime salts of the teeth, softens them, and so allows of the pensiaiaan of the lepto- thrix form. Pasteur has suggested that other facultative saprophytes are those met with in the alimentary tract, where he thought they appeared to assist in carrying on the decomposition of the nutrient materials, assisting, under certain circumstances, the animal tissues of the body to carry on the process of digestion without interfering in any marked degree with the nutrition of the tissues of the host by absorbing any of the material that could be utilized by these tissues for their own nutrition. This, however, is now considered to be question- able. The organism of cholera finding its way into the alimentary canal, acts as a pathogenic parasite, but here only from the fact that by its growth and metabolic activity it gives rise to irritant toxic materials, which exert most injurious effects both locally and constitutionally. 148 BACTERIA, As another example of a facultative pathogenic parasite, the anthrax bacillus may be cited. It may develop actually in the tissues, as in woolsorters’ disease, where it occurs in the bronchial mucous membrane; in the lymphatic tissue spaces, and in the pleural cavities; or in the blood, as in cases of accidental inoculation. It may also occur in the blood in woolsorters’ disease, the bacillus readily making its way from the air vesicles and tubes into the pulmonary circulation. In this connection it may be mentioned that anthrax bacillus cannot undergo its whole developmental cycle within the body; the vegetative stages only having been found in parasitic anthrax; whilst growing on dead tissues at a favourable temperature spore formation is invariably noticed at some period or other during the life history of the anthrax rods, so that the bacillus must be looked upon as a facultative saprophyte. Another point to which attention may be drawn is that bacteria must be looked upon as essentially and primarily saprophytic organ- isms. By a process of long and gradual acclimatization or adaptation, certain species have become so altered, either temporarily or permanently, that they are able to exist as parasites. It will always be found, however, that the tendency to revert to the saprophytic or non-pathogenic condition is more marked than their tendency to become transformed into the parasitic and pathogenic condition. It has been found, for example, that anthrax bacillus passed through a series of cultivations in gelatine, or hog-cholera bacillus similarly treated, loses a great deal of its patho- genic power. This loss of pathogenic power may take place in two directions, either by the organism losing its power of development in living tissues, or by losing its virulent specific poison. The cholera organism cultivated outside the body usually loses much of its pathogenic power. By appropriate treatment, such as cultivating at the body temperature in specially prepared broth through a number of generations, this pathogenic activity may, however, be restored. We must therefore look upon pathogenicity, para- siticism, and saprophyticism as mere relative terms; the conditions necessary for the development of the saprophytic mode of life being more widely met with than are those in which the parasitic life may become developed, most organisms become more or less permanently adapted to PARASITES AND SAPROPHYTES. 149 them, so that it is only under special circumstances that parasitic and pathogenic organisms are developed. The importance of this fact will be evident when we consider the epidemiological importance of the presence and mode of life of micro-organisms both outside and within the body. CHAPTER VIII. CHOLERA. Cholera a Parasitic Disease—Early Observations on the Comma Bacillus —Characters—Methods of Staining—Methods of Isolation—Use of Plate Method in Practical Public Health Work—Tube Cultures— Motility of cholera bacilli—Potato, Blood-serum, and Milk Growths —Behaviour in Water and Sewage—Infection of Man through Agency of Bacillus — Early Inoculation Experiments — Koch — Nicati and Rietsch—Macleod—Difficulties—Cholera in Guinea-pigs—Position of Bacilli in Tissues—Presence of spores doubtful. On account of its general interest there are few examples that can more appropriately be taken to illustrate the rela- tions between a special bacillus and a special disease than that afforded by Koch’s discovery of the comma bacillus in cases of Asiatic cholera, and its relations to that disease. The study of the disease itself, since its appearance in this country in the great epidemic of 1832, has had a peculiar fascination for epidemiologists and skilled hygienists. Its development, behaviour, and whole general history appeared to be shrouded in mystery, and phenomena, the explana- tion of which seemed to be utterly beyond the powers of experts of all kinds to give, were at one period constantly being observed, recorded, and discussed. Now, however, through the laborious but brilliant researches initiated by Pettenkofer at the head of one school, and by Koch in a very different one, much of this air of mystery has been dis- pelled, and many of the doubts and difficulties that sur- rounded the subject have been gradually cleared away as workers under one or the other of these great leaders have gained fresh knowledge and elucidated new facts. Take, for example, the question of the spread of cholera from its home in Lower Bengal, in the delta of the Ganges, to surrounding districts and distant countries. At first such spread was thought to be most erratic and inexplicable. Now, however, although it appears from time to time to have CHOLERA, 151 made almost unaccountable leaps and divergencies, it has been found to follow a very definite line of advance in the course of the various epidemics, and although there may have occurred sporadic cases which could be traced to no definite source of contagion or infection, which have proved a stumbling-block to many conscientious workers and ob- servers, it is generally acknowledged that the evidence accumulated through the researches of Koch and his fol- lowers, both in Germany and in Great Britain, and of several enthusiastic workers in France, can leave little doubt in the minds of most of those who study the subject care- fully, that cholera is a parasitic disease, that it travels along the ordinary lines of commerce by railways, caravans and ships, from the regions in which it is endemic to those centres of trade and religion which, by their imperfect sanitary arrangements, by the want of cleanliness of their inhabitants, by meteorological conditions, and on account of bad water supply, are ready for its reception and propagation. In the European epidemics, of which up to 1885 there had been six exceedingly severe ones during the present century, the disease has, in every case, followed the lines of trade. Before the three last epidemics (1865, 1873, 1884) cholera usually came to Europe by what may be called the Con- tinental routes—the caravan routes through Persia, Asia Minor, and Russia; but in the three last it came by the Mediterranean or maritime route, first by land through Egypt, brought there by Mecca pilgrims, and thence to the seaports of France, Italy, and Spain, whence it gradually made its way northward and inland, spreading over the whole of Europe. At the mouth of the Yang-Tsze, as instanced by Macleod, of Shanghai, cholera breaks out regularly at certain seasons of the year, but he adduces evidence of great value to show that although it may be imported’ directly from India, between which and Shanghai there is at least weekly com- munication, just as there is between India and the Nile delta region, itis probably, in the strictest sense, an endemic disease of that region. It can be readily understood, after the fearful ravages which it made in places in which it was not actually endemic, and after it had decimated the population in certain parts of India, in Egypt, in the low-lying portions of Persia, and 152 BACTERIA, Asia Minor and in Europe, that many observers should be anxious to find out the ultimate cause of the disease, and as early as 1848 Virchow, and in 1849 Pouchet, Brittan, and Swaine found numbers of vibriones in the discharges of choloraic patients, without, however, being able to assign to them or prove for them any specific é/e in the causation of the disease. Following up these researches Philippe Pacini, setting himself to look for a causal agent, frequently found in cholera stools small micro-organisms which were charac- terized by active and peculiar movements. These observa- tions, however, as well as those of Klob, who looked upon the cause of cholera as an accumulation of fungi in the intestines, and those of Boehm and Hallier (all published in 1867), who believed that they had found the cause in a peculiar fungus which was found to grow in certain forms of grain imported from India, not only did not receive adequate proof, but were, like all preceding observations, entirely unreliable. Then followed the experiments by Hayem and Raynaud, carried on during the epidemic of 1873, which, however, again were equally futile and without definite results, and it was not until Koch, going out with the German Cholera Commission to Egypt and India, was able * to demonstrate a peculiar species of bacterium as the causal agent that any definite proof of the micro-organismal nature of the contagium of the disease could be obtained. Since that time, however, the amount of work published on the subject has been so great that cholera has now a special “ literature” of its own. The report of the German Commission was speedily followed by that of the French Government, who sent out MM. Straus, Roux, Nocard, and Thuillier, the last of whom fell a victim to his assiduity and zeal in carrying on the work of investigation in Egypt. In this country Klein and Heneage Gibbes, Cunningham and Lewis, Roy, Graham Brown and Sherrington, Watson Cheyne, and Macleod have, with very different voices, some . supporting Koch’s verdict, others opposing it, reported on the subject; with the general result that until further and more convincing opposing evidence is forthcoming, Koch’s comma bacillus, of which the following is a brief descrip- tion, must be looked upon as the causa causans of the disease. CHOLERA. 153 Th: ‘comma’ bacillus, which is now regarded as belong- ing .» the spirilla, usually occurs as a slightly curved rod, mei iring from I to 2p in length, with an average length of zbout 1.5m ; it is .5 to .6# in thickness, the average thickness being about one-third to one-fourth of the length. It is therefore from one-half to one-third the length of the tubercle bacillus, but somewhat thicker. In place of occurring as single rods these organisms may be grouped Photo-micrograph cholera bacilli. x 1000, Long spirilla, comma, S and ‘O shaped organisms. Some involution forms. in pairs, or in larger numbers, in which case the curve may be continuous or reversed, so giving rise to the formation either of half circles or of S-shaped curves. In cultivations in meat broth the bacilli may be so grouped that they form long wavy or spiral threads, each of which may be made up of 10, 20, or even 30 short turns. Such are the characters of the organisms which Koch found, 154 BACTERIA. almost invariably, in the stools of patients during the earlier stages of the disease, in the contents of the lower bowel and in the mucous membrane of the lower part of the small intestine. In fatal cases Drs. Straus and Roux thought that they could also see certain organisms in the blood of cholera patients, but they were unable to repeat their observations. Emmerich also described a bacillus which he said he found constantly in the blood, organs, and intestinal contents in cases of cholera. This organism was, however, according to Fligge, probably a common inhabitant of the intestine, the Bacterium Coli Communis, and it is now a generally ac- cepted fact that nowhere, except in the alimentary canal, have the comma bacilli been found in ordinary cases of cholera ; as the blood, the liver, the spleen, and other organs have all been carefully examined, and in no case, in which no fallacy crept in, could positive results be ob- tained. The best method yet described of demonstrating the cholera bacillus in the discharges is that recommended by Cornil and Babes, who spread out one of the small white mucous fragments on a microscope slide, and then allow it to dry partially ; asmall quantity of an exceedingly weak solution of methyl violet in distilled water is then flowed over it, and it is flattened out by pressing down on it a cover glass, over which is placed a fragment of filter paper, which absorbs’ any excess of fluid at the margin of the cover glass. Comma. bacilli so prepared and examined with an oil-immersion lens (xX 700 or x 800) may then be seen ; their characters are the more readily made out because of the slight stain they take up, and because they still retain their power of vigorous movement, which would be entirely lost if the specimen were dried, stained, and mounted in the ordinary fashion. The bacilli retain their curved shape and “rounded” extremities (which, however, may be either slightly pointed or thickened), but they seem to be somewhat larger when thus prepared than when completely dried. During the very early stages of the disease, and when the period of reaction is setting in, it is sometimes an exceedingly difficult matter to demonstrate the presence of the cholera organism in the dejecta, and several methods have been tried with greater or less success to obtain such a demonstration. Of these the plan described by Schottelius is found to be one of the most useful, especially in the later stages of the disease, where although the number of cholera bacilli may be com- paratively small, other bacteria are found, often in con- siderable numbers, in the intestinal contents. CHOLERA. 155 A small quantity of the material from the intestine is mixed with double its quantity of-iaintly alkaline meat broth, which is then allowed to stand at a temperature of 35° C. for about twelve hours; at the end of that time the comma bacilli have multiplied to such an extent, especially where they are in contact with the air, that if a small portion of the pellicle from the surface is stained and examined, as above, it is found to consist of an almost pure cultivation of comma bacilli which are, however, some what shorter than those usually met with. If the cultivation be left undisturbed for a longer period the bacilli generally grow larger, and eventually spirilla may be formed, but in the course of a few days the other bacteria grow so luxuriantly that the comma bacilli no longer predominate, and in many cases they are almost entirely ‘‘ overgrown.” From the earlier ‘‘ maass”’ cultivation so prepared, in which the relative proportion of cholera bacilli is enormously increased, ‘‘ plate” cultivations may now readily be made. The plate method as employed by Koch is the best, and the one least open to fallacy for obtaining pure cultivations. It is carried out as follows: On a sterilized platinum needle a drop of the above fluid, of the cholera discharge or of the contents of the small intestine is taken. This is introduced into a test tube one-third full of nutrient gelatine, liquefied at as low a tem- perature as possible by immersing the lower portion of the tube in warm water. The seeded gelatine is carefully shaken in order that the crganisms may be disposed widely and equally throughout its substance; three small drops of this are then taken on a looped platinum wire and added to a second tube, which is similarly shaken, and from this second tube five drops are transferred to a third tube. After the contents of each tube have been thoroughly mixed and the seed material taken for the next tube, the remainder is poured on toa glass plate previously sterilized ; each of the three plates is carefully labelled and placed under a bell jar in which the air has been thoroughly sterilized (see Appendix). The plate from tube No. 1 is found to contain a large number of organisms, that from tube No. 2 contains a much smaller number, and that from tube No. 3 a much smaller number still, so that as the organisms are developed (one colony from each organism or group of organisms) there is sure to be sufficient space on one or other of the plates between the different groups to allow of a careful study of the growths as they extend, and at the same lime there is a sufficient number of points to ensure the appearance of several growths of each kind of organism that was present in the original seed material. If plates made according to Koch’s method be kept in a chamber in which the temperature is. maintained at a little over 20° C. small greyish or white points are first seen; examined with a low power lens, each of these has a slight yellow tinge and a somewhat wavy margin; as the “colony” increases in size its colour becomes slightly deeper, the margins become more and more crenated, and the surface somewhat granular. Slow liquefaction is now found to be taking place, and small funnel-shaped depressions, any one of which seldom measures more than I in diameter, are seen in the gelatine; at the 156 BACTERIA. bottom of these depressions yellow pin-like masses are usually seen. In consequence of this liquefaction the colony has the appearance of a piece of ground glass with a finely notched margin surrounded by a clear zone. On the second or third day small air bubbles are seen, and on the fifth or sixth day the liquefaction is well marked ; at this stage the colony has sometimes a delicate pink tinge, a most charac- teristic feature when present. From these points or colonies growing, as we have seen, from individual organisms or small groups of the bacillus, pure cultivations may be made in test tubes containing gelatine, agar-agar, potatoes, or broth in or on.which the growth and special characters of the bacillus may be watched and noted. It is sometimes made a matter for reproach to bacteriologists that the methods employed in their work, useful enough though they may be in conducting scientific experiments, are found altogether wanting when they are required for actual prac- tical work. The following story, which went the round of the medical papers, may. help to remove the idea that bac- teriologists are mere viSionaries, and to prove that the science, some of the problems of which they are at- tempting to solve, is by no means so useless as many would have us believe. An Italian emigrant steamer, touching at New York, had on board a child suffering from a sus- picious form of diarrhoea, though it could not be said that all the symptoms of Asiatic cholera were present. In order to determine the true nature of the disease—whether it was cholera or not—gelatine plate cultivations of the dejecta were made by a doctor in port, and the vessel was detained for four days; during that period bacilli identical in ap- pearance with, and having all the characteristics of, Koch’s comma bacillus were developed, and it was at once con- cluded that this was a case of true Asiatic or Indian cholera. The diagnosis was subsequently confirmed, as a series of other cases occurred, in all of which unmistakable symp- toms of Asiatic cholera were developed. An equally in- teresting and instructive example of the.same thing was recorded by Pfeiffer and Gaffky when they were working in Koch’s laboratory. At a time when there was no general cholera epidemic, Gonsonheim and Finthen in Germany were suddenly ravaged by a most deadly form of diarrhoea which in many features resembled true Asiatic cholera. The CHOLERA. 157 recorders, in order to determine the exact nature of the disease, made plate cultivations from the dejecta of some of the patients, and found Koch’s comma bacillus; they were thus able to put the matter beyond all doubt. How the organism came to the district, and why the outbreak remained localized, are questions that still remain un- answered. During 1890 there was a similar, but less localized, outbreak of fatal diarrhoea in Spain. Koch's bacillus was here again separated, and all doubt as to the nature of the disease at once removed. In view of these facts the immense importance of bacteriological methods, as permitting of rapid and definite recognition of the disease, with the possi- bility of taking precautionary measures as early as possible, and so prevent- ing a wide dissemination of the disease germs, can scarcely be insisted upon too strongly. In a puncture culture in gelatine the growth takes place along the whole track of the needle, first as a delicate white cloud, then, as it gradually becomes more and more marked, it forms a delicate streak, around which there is usually a clear space, due to the liquefaction of the gelatine along the tract of the needle. Near the surface the liquefaction goes on more rapidly than deeper down, and at the end of forty-eight hours there is a distinct funnel-shaped area, in which a clear liquid portion usually sinks somewhat below the level of the gelatine, and it appears as though the top of the funnel was closed by a small clear glass globe or air bubble. This appearance is apparently due to the slow- ness of the liquefaction, time being given for the water to evaporate from the liquefied gelatine. This is proved by the fact that if 5 per cent. gelatine instead of 8 or 10 per cent. be used, the gelatine liquefying more rapidly does not allow time for evaporation to take place, and the bubble is never seen, About the fourth day of the growth this “funnel” is still more marked, but the upper part has become quite clear, the central thread having fallen to the lower and narrower part of the funnel, where it may be seen as a comparatively short spiral, the thread as it sinks being arranged in regular “ bights ” like those of acable. After a time the whole of the upper part, say two-thirds, of the gelatine is liquefied and perfectly clear, then comes a layer of “ white” cholera bacilli, which rests on the gelatine that has remained solid (this sediment has usually a yellowish tinge) ; and on the surface 158 BACTERIA, of the liquefied gelatine a greyish scum is found, in which “involution forms ” may be frequently observed. Cultivations made with a platinum needle on the surface of agar-agar, grow as elevated pale translucent streaks, the margins of which are well defined, and in the agar imme- diately below the streak there occurs a slight opalescence which is very characteristic of the bacillus. The agar, unlike the gelatine, is not liquefied. As the growth becomes older it spreads, though not very rapidly, the band increases in thickness and gradually assumes a brown colour especially in the opalescent substratum. When the organism grows in meat infusion it forms a delicate greyish pellicle on the surface, and in such a medium the movements of the bacillus are most characteristic. Ifa single drop taken from the surface of such a cultivation, or even a very small fraction of a drop be added to a drop of meat broth faintly tinged with a 1 in 2,000 solution of methyl violet and examined in the moist chamber (see Appendix), the bacilli are seen to have most rapid movements, especially if the temperature be slightly raised ; Koch says, ‘‘ when they accumulate in num- bers at the border of the droplet, and are swimming about actively, it seems quite as if they were a swarm of midges, between which, diving here and there, are long spiral-shaped threads, which are also moderately active.’ The shorter rods become more and more curved, and then stretch them- selves out, constantly moving, oscillating and twisting. From numerous experiments carried on by several observers it appears that this motility is dependent partly on tempera- ture, but chiefly on the desire for oxygen which these organisms exhibit. In the “ hanging drop” (see Appendix), for instance, it will be seen that the cholera bacillus separates itself from most other micro-organisms by its movements in search of a supply, of oxygen, which always bring it to the margin of the drop ; once there, however, it loses its motility and remains in its acquired position, where its wants are supplied without any effort on its part. The cholera bacillus also grows on potatoes, but here it should be noted the ‘growth does not go on at the ordinary temperature of the room, but only when the temperature is raised to 30° or 35° C. A fragment of blue litmus paper placed on the surface of a slightly moistened boiled potato indicates, by turning red, a slight acid reaction, which is due CHOLERA. 159 (according to Koch) to the presence of an organic (pyro- malic) acid. Here the acid reaction of the potato is the factor that interferes with the growth of the bacillus, and it is only when the temperature conditions become more favourable than usual that any growth can take place. The growth when started appears as a thin, moist “ light greyish brown” mass, very similar in character, as Koch points out, to the glanders bacillus as grown on potato, except that it has not the deep or chocolate brown colour so characteristic of that organism. A most interesting fact has been observed in this connection. Potato cultivations of bacilli from the various cholera epidemics vary somewhat in colour; there are also other slight modifications in the appearance of the different masses, by means of which a skilled observer, who has cultivated the various organisms from the different localities and epidemics, is enabled to distinguish the bacilli of each epidemic; and it is stated that in some of the German laboratories the directors can take up half a dozen potato cultivations and say, This is from the Naples epidemic, that from Egypt, and so on through the whole series. There are undoubtedly differences, and to the most inexperienced eye the colour of the Shanghai cholera growth may be seen to differ slightly from that of the Egyptian and that of the Naples growths. On blood serum and in milk these organisms grow most luxuriantly. They may cause slight liquefaction of blood serum, but in milk, which forms for them an exceedingly good nutrient medium, they give rise to no noticeable altera- tions ; it may therefore be readily understood how deadly the -cholera organism may become if it once finds a resting place in milk. Its presence cannot be recognized by any peculiar or characteristic appearance, by taste, or by smell, as it only gives rise to a faintly aromatic and sweetish smell, which can scarcely be distinguished, except by the most practised nose, from the slightly aromatic smell of the milk itself. Dr. Simpson in Zhe Indian Medical Gazette records a naturally prepared experiment, which shows at a glance what an important part these milk cultivations may play in the spread of cholera. On board the ship Ardenclutha, in port in Calcutta, ten men partook of the milk supplied by a native milk-seller who came to the ship daily ; of these ten men, four died of cholera, and five suffered from exceedingly severe diarrhoea, the tenth, who escaped, had taken only a small quantity of the milk. After careful investigation it was found that the milk had been watered with 25 per cent. 160 BACTERIA. of water, from a district in which it was known that several people were suffering from cholera. A case had been im- ported into this district on the second day of a certain month, the dejecta from this patient drained into a tank, near which the milkman’s house stood ; on the seventh day of the same month the first new case occurred among the milkman’s neighbours, and on the same day the first case of diarrhoea occurred on board the Ardenclutha, and two days later a case of undoubted cholera occurred in the ship. These facts are in themselves interesting and instructive, but they are rendered doubly so by the fact that of the other members of the crew, fourteen in number, who had taken none of the watered milk, not a single one was attacked by diarrhoea or cholera. Cholera bacilli are so little fastidious in their diet, that, within certain limits, they are satisfied with anything, but there are certain things at which even they draw the line— soup mazgre, for instance, they abhor, as also sour things ; acids are to them a deadly poison ; on the other hand, how- ever, they are somewhat exclusive in their habits, and the presence of other and more vulgar bacteria they cannot brook for long. For example, if intestinal contents or dejecta from a case of cholera are sprinkled on moist soil or damp linen kept at blood heat the comma bacilli increase at an enormous rate for the first twenty-four or thirty-six hours, and under these conditions, as already seen, there may be obtained almost pure cultivations of the specific organism, but on the third or fourth day it begins to die out, and other bacteria are found to be asserting themselves most strongly. Babes has found that at a temperature of 30° C. the bacillus will grow upon various kinds of meat, on eggs, on various vegetables, and on moistened bread. It also multiplies in the dejecta from healthy patients, on cheese, in coffee, chocolate, caz sucrée, and other kinds of fluid sugars ; but it can- not exist for twenty-four hours on acid fluids or vegetables, on mustard or onions, or in wine, beer, or distilled water. Distilled water and soup maigre are never sufficient for its nutriment, and if the strength of any of the usual cultivation media be reduced to about a fortieth of the original strength, there is gradual diminution in the number of organisms; even in water which contains an ordinary amount of organic and inorganic material in solution, multiplication of the comma bacillus does not take place. Where, however, there is a very large accumulation of organic matters, as at the margin of stagnant water, where there are large quantities of nutrient materials ‘from the presence of a variety of solid CHOLERA, 161 particles in suspension and of pieces of mud,” development occurs, “ especi- ally on the floating sohd fragments.” Koch’s experiments, however, have shown that cholera bacilli mixed with spring water were still alive after the lapse of a period of thirty days. In Berlin sewer water they lived only six to seven days, mixed with excrement only twenty-seven hours, and in cess- pool water they were not alive at the end of twenty-four hours. Babes’ experiments, as already stated, did not bear out these statistics in their entirety. Infection through the agency of milk has already been mentioned, and water has been referred to as a vehicle by which the bacillus may be carried. Macnamara gives an experience of infection through water, for the absolute accuracy of which, in the case of such a skilled observer, Koch contends there can be little doubt. In a region in which no cholera prevailed, the dejecta from a sporadic case of cholera became accidentally mixed with some water, which, after remaining exposed to the heat of the sun for a whole day, was partaken of by nineteen persons, of whom five were attacked with unmistakable cholera within thirty- six hours ; the evidence here given was so conclusive that little doubt can be entertained as to the relation of the dejecta to the water, and to the subsequent cases of cholera. It should here be mentioned, however, that Klein, who does not believe in the specific nature of Koch's cholera organism, in order to prove his position, drank a quantity of fluid which was said to contain cholera material without feel- ing any ill. effects; and that Bochfontaine swallowed cholera dejecta in pills without suffering any incovenience ; but in both cases there is ample evidence that the persons experimenting upon themselves were suffering from no gas- tric or intestinal derangement of any kind, and that the gastric juice was sufficiently active to prevent the multipli- cation of the cholera organisms, and it is probable that the fourteen men of the nineteen not attacked in Macnamara’s case were in a similar impregnable condition. As positive evidence to set against the above is a most striking case that occurred in connection with Koch’s cholera courses, carried on in the Hygienic Institute in Berlin. A doctor who had been for eight days engaged on work in the cholera course became affected with a slight disturbance of digestion, which was accompanied by diarrhcea ; he continued to get worse, and was at length so ill that he started for home, where he was almost immediately attacked with symptoms of true 12 162 BACTERIA. cholera, rice-water stools, great weakness, unquenchable thirst, diminished excretion of water, except by the bowel, and spasmodic contraction of the feet and toes. He was very anxious to have this rice-water material examined for the comma bacillus, and a quantity was despatched to Koch, who was able to demonstrate in and cultivate from it, true comma bacilli; the patient recovered. There wereno other cases of cholera in Germany at the time, but the doctor had been examining and making cultivations of the cholera bacillus shortly before he was attacked, and there can be little doubt that he had, through inattention to the rules of the laboratory, in some way or other, ingested some of the bacilli with which he had been working. ° The description of these cases of infection or non-infection of individuals by cholera material introduced into the alimentary canal, naturally opens up the way for an account of the results obtained experimentally. Thiersch was the first to make experiments on animals with cholera dejecta. He fed white mice with scraps of filter paper, impregnated with decomposing cholera dejecta, but he found that plain filter paper was equally efficacious in causing the illness of the white mice. He was quickly followed by Burdon Sanderson, who carried on a similar series of experiments, and obtained much the same results. Dr. Richards, of Goolando, also attempted to produce cholera in pigs by feeding them with large quantities of cholera material, but he found that the animals died in from fifteen minutes to two and a half hours after the administration of the poisonous material, and the contents of the intestine of the first pig that died, when given to another, produced absolutely no symptoms. It was argued from this that the poison had not multiplied, but had been destroyed or ab- sorbed, or rendered inactive in the stomach of the first host, so that none was left to act on the second pig ; death in the first case being produced by rapid intoxication through the introduction of a large quantity of some active poison, and not by a poison developed in the intestinal canal as in the case of true cholera. This poison, whatever its nature, had no effect on dogs. Following on these came a series of experiments by Koch, who attempted to produce symptoms of cholera by feed- ing and inoculating in various ways, mice, monkeys, cats, CHOLERA, 163 dogs, poultry and other animals with cholera dejecta and with comma bacilli; in’no case could he produce cholera, and on search being made for the bacilli in the stomach and intestine, they were invariably absent, apparently having been destroyed in the stomach, as in only a few instances did they reach the intestine at all, and then in very small numbers, To demonstrate the difference between these and certain other bacteria, a mouse was fed with a red micro- organism that had been isolated in a pure condition ; and after a time its intestinal contents were disseminated on potatoes, on which small red colonies of the same organism shortly made their appearance, showing that their vitality was not appreciably affected by their passage through the. stomach. Having found that the comma bacillus was so destroyed, Koch thought that if the organism could be intro- duced directly into the large intestine, so as to escape the action of the gastric juice, it might be able to retain its vitality and multiply; the results obtained, however, were in all cases negative, even when purgatives were previously given to induce an altered condition of the intestine. The only cases in which partially successful results were obtained were those of a series of rabbits into which pure cultivations of the bacillus were injected directly into the circulation, and of a number of mice, in which the cultivations were injected into the abdominal cavity ; the rabbits recovered in one or two days, after suffering from marked symptoms of intoxi- cation ; but the mice died at the end of the first or second day, bacilli in these experiments being found in the blood. Koch argued from these observations (1) that the gastric juice of the healthy stomach killed the micro-organism ; - (2) that even when it found its way into the intestine, it was passed along it so rapidly that it could not take any effect on the healthy or even slightly irritated mucous membrane, or that it was actually very rapidly destroyed in the intestine itself. In guinea pigs, in which he attempted to produce cholera, he found that there was great acidity of the gastric juice, and that the peristaltic movements of the intestine were very strong and rapid. It will have been concluded from what has been stated that both naturally and in artificial cultivations there is some poison developed during the growth of the bacillus, which, when introduced in considerable quantities, produces 164 BACTERIA. a condition of intoxication, which would account for the death of those animals in which partially successful results were first obtained. Further, it is evidently quite possible that this intoxication may account for the very rapid deaths which occur during certain epidemics, and also for the pre- liminary diarrhceas by which the intéstine, in some cases at any rate, is prepared for the reception and multiplication of the cholera organism itself. It must, indeed, be looked upon as one of the common causes of this diarrhoea, If these facts be borne in mind it is possible, even without analyzing the further experiments on animals, to understand the immunity against cholera experienced by Bochfontaine and Klein, and the susceptibility of the doctor who was attending the cholera course, whilst he was suffering from indigestion and diarrhoea. It may also be understood how infection occurs by milk and through water after what has been described, and in accordance with these facts is the experience of all those who have had to deal with cholera epidemics. Utilizing the experience gained by Koch in his experiments, Nicati and Rietsch performed a series of operations, by means of which they claimed they were able in a certain proportion of cases to produce typical cholera symptoms. They introduced pure cultures of the comma bacillus into the upper part of the intestinal canal, having previously tied the bile duct ; but as Koch pointed out later they were successful in producing true cholera, probably, only in those cases in which the intestine was somewhat injured during the manipulation to which it was necessarily subjected, and its peristaltic action interfered with, and it is very naturally objected that death might be due not to the action of the cholera bacillus at all but to this rough manipulation. The observers themselves believed that it was the presence or absence of bile in the small intestine which determined the success or failure of their experiments. In order to avoid the operation of opening into the duodenum, Koch thought that the two great factors in bringing about the destruction of the bacilli might be thrown out of court, first by neutralizing the acid reaction of the stomach by means of 5 c.c. of a 5 per cent. solution of carbonate of soda ; and, secondly, by interfering with the peristaltic action of the bowel. He found in test experi- ments that the intestinal contents remained distinctly alka- line for six hours after the introduction of such a solution. But he also found that the organism still passed through the stomach alive, and that it failed to produce any changes in the small intestine, the food and the bacilli passing from the . CHOLERA. 165 stomach to the cecum in a few minutes; the peristaltic action of the intestine causing such rapid passage of the bacillus that it was completely powerless to produce any characteristic symptoms of cholera, a fact the importance of which was accentuated when it was found that the only guinea pig in which any choleraic symptoms were observed, and in which there was any increase in the number of bacilli in the small intestine, was probably suffering from an attack of peritonitis, due to the animal having aborted just previous to the experiment—a condition in which, as has long been recognized, there is invariably interference with the peristaltic action of the intestine. It was possible, then, that this second factor might be neutralized by the introduction into the peritoneal cavity of some reagent, which would by its action cause partial or complete paralysis of the small intestine. For this purpose he first used opium, but he afterwards found that alcohol was equally efficacious. After administering the soda solution, and injecting into the stomach of the guinea pig 10 c.c. of broth, to which one or several drops of a pure cultivation of bacilli had been added, he injected into the abdominal cavity tincture of opium, in the proportion of I c.c. to every 200 grammes of the animal’s weight ; the results he obtained were indeed startling. Of course objections were raised to the method, and it was argued that some of the animals died from an overdose of opium, some from blood poisoning, and so on. Subsequent observers found, indeed, that the dose of opium was somewhat too large, as some animals experimented on never awoke from the opium sleep. Macleod, of Shanghai, found that he obtained the best results by giving ‘‘ repeated doses of 1 c.c. or less of the tincture till the animal was stupefied sufficiently to lie on the side or back for ten minutes when placed in that position.” ‘‘ Several times,” he says, ‘‘the full dose recommended by Koch had to be given, but usually a smaller one sufficed.” . Control-animals treated, according t- Macleod’s method, and in all respects in the same way, with the exception that they received sterilized broth instead of cholera material, always recovered. The contents of the bowel or dejecta from cholera patients passed into the stomachs of guinea pigs so prepared, pro- duced cholera symptoms and death. Giving the results of of his experiments, Macleod states that from one of the animals that died after a dose of cholera material, the small gut contents were collected into a sterilized vessel, and injected (by means of a fine indiarubber flexible catheter) in doses of 2 c.c. into the stomach of two other animals. 166 BACTERIA, These two animals died, and the contents of their small intestine were used in the same way, and so on through ten generations. Of twenty-one animals thus treated, two recovered, nineteen died. The doses varied from 0.5 to 2.5 C.C. It is sometimes argued that in consequence of the difference of thé symptoms in animals (especially guinea pigs), and in man, undet these conditions of infection by the stomach, the disease cannot be the same, and it is pointed out that vomiting and profuse diarrhoea are entirely absent in cholera, experimentally produced. Against this objection may be put the fact that there is usually in the guinea pig a large accumulation of fluid transudation in the small intestine and even in the stomach; whilst in the very acute forms of cholera the pathological appearances presented in the intestines are almost identical with those found in man. In exceedingly acute cases the peritoneum presents a dark colour and a peculiar glistening mucous appearance, which is very characteristic, and seems to be associated with the tarry condition of the blood. The mucous surface of the intestine is usually congested, and in the intestinal canal is a considerable quantity of serous fluid, in which are floating the small white rice-like bodies which are merely portions of desquamated epithelium and mucus. There is usually, at this stage, marked injection of the vessels of the solitary glands, and at” the periphery of the Peyers patches, and the longer the patient remains alive the more accentuated become these appearances. It is only in the later stages of the disease, or during the period of reaction, that the swelling of the follicles is very marked, and that more or less extensive ulceration of the mucous membrane may be observed. The dejecta, of course, are similar in character to the contents of the bowel, are watery owing to the great amount of serous effusion, and contain the rice-like bodies in which are found the comma bacilli. Bacilli are also found lying free in the watery fluid. The dejecta have little or no odour, and when they are allowed to stand they separate into two layers—an upper slightly grumous layer, and a lower grey deposit. ‘‘ Neutral, or slightly alkaline, they con- tain a very small proportion of organic or inorganic salts—one to two per cent.—which consists of chloride of calcium, carbonate of ammonia, potash, salts, and a small quantity of urea. They contain little or no albumen, and during the first day or two no bile pigments.” If microscopic sections of the lower part of the small intestine be made, and stained according to Léffler's method,' the bacilli may be seen with the aid of a high « First stain in a solution of Leonhardi’s Dresden methyl violet ink, and then in an alkaline solution of fuchsin, which is made up as follows : 100 c.c. aniline water. 1 c.c. of a solution of one per cent. of caustic soda. 2 grammes of solid fuchsin. The whole is well shaken, after which the slo ag may be left in it for twenty-four hours ; the sections are then washed in distilled water acidu- lated with a drop of acetic acid, dehydrated in absolute alcohol, cleared up in cedar oil, and mounted in Xylol balsam. CHOLERA. 164 fiagnifying power (x 800) beneath the loosened epithelium of the villi, in the follicles, and in the surrounding connec- tive tissue. They are especially numerous near the ileo- cecal valve, and are distributed, not in masses, but usually in ones, twos, and threes, scattered thickly through these tissues.* On post-mortem examination of the guinea pigs, in which cholera had been experimentally produced, ‘‘the blood was fluid, thicker and darker than natural, the tissues of the thoracic and abdominal walls were re- markably dry, the small intestine was throughout distended, congested, and paralyzed-looking, and occupied a much larger proportion of the abdominal cavity than usual. The czecum was distended with fluid or semi-fluid con- tents. If the animal died early the fluid was not quite clear in the small gut, there being present traces of food; still the watery character was very manifest, and mucous flakes were abundant. If the animal died on the second or third day no food remains were to be seen, and the fluid in the small gut was the counterpart of the typical cholera stool of man. In either case the comma bacilli were demonstrated microscopically, and by cultivation as in man.” While the organisms in the broth injected could be frequently counted in a microscope field, in a drop of the small bowel contents from an animal having received such broth, the bacilli might be so numerous that counting them, without dilution of the fluid examined, was an impossibility. On floating the bowel in water, the stripping of the epithelium could be well demonstrated ‘‘immediately after death,” a fact which appears to dispose of Macnamara’s assertion that this separation of the membrane is always a post-mortem appearance. In animals which died about the eighth day the appearances were very similar to those met with in cases of Asiatic cholera, when the patients have succumbed during the stage of reaction, and no bacilli can be found in the intestinal canal or in the surrounding tissues. Inthe earlier stages the bacilli can be demonstrated in sections lying under the partially-detached epithelium, or in the lumina of Lieber. kuhn’s follicles. In fluid cultivations, as has already been mentioned, the comma bacilli, or little groups of them, assume various forms, all of which, however, may be looked upon as made up of more or less perfect spirals. At the point of division, just before division take places, there is a small clear space which, by some authorities, has been looked upon as aspore. In some of Macleod's preparations this appearance was so marked that it was difficult to convince oneself that it was not due to actual spore formation, but as drying for twenty-four hours completely destroyed the activity of the bacillus in which these clear spaces were most marked, and as they could not * Itisnotmy intention to give the complete pathological anatomy of cholera, except so far as it is associated with the presence of the cholera bacilli. 168 BACTERIA. be stained by any of the ordinary spore-staining methods he was convinced that he was not dealing with true endospores. On three occasions Hueppe-was able to demonstrate in moist chamber cholera cultivations on agar-agar small brilliant spore-like bodies which he called “ Arthrospores.” These were placed sometimes at the extremities, sometimes in the middle of the comma bacillus. They appear to develop where the conditions of nutriment, temperature, or moisture, are somewhat unfavourable to the development of or to the very existence of the bacillus. This is proved by the fact that the protoplasm in the bacilli, in which these arthrospores are present, is stained very imperfectly with methyl blue; the clear bodies themselves taking on a brownish- red tinge. Hueppe maintains that he has observed these bodies germinate out into comma bacilli, and he holds that they are much more resistant to various germicidal reagents and unfavourable conditions than the vegetative form of bacillus. Several observers have tried to repeat these ex- periments, but, as any apparent spores that have been formed invariably failed to resist the action of drying, such “‘ Arthrospore ” containing bacilli, must, for the present, be ei ‘upon as involution forms, similar to those described below. When these are formed the ordinary dimensions and forms of the comma bacillus as previously given are sometimes departed from, and in old agar- agar cultivations, and in old gelatine cultivations in which the surface has become somewhat dried, spirals are frequently found of which the con- stituent bacilli are considerably thicker than ordinary comma bacilli, the large curved forms remaining attached by delicate terminal filaments. In most cultivations there are slight modifications in both form and size of the cholera bacillus, which appear to be associated with the difficulty or facility with which they obtain food, water, and oxygen, and also with the temperature at which they are grown, For example, if ten per cent. of alcohol be mixed with the gelatine, or if the gelatine medium contains but little beef extract or peptone, and if the temperature at which the media are kept be very high or very low, say 45° C. on the one hand or 20° C. at the opposite extreme, a large proportion of spiral fila- ments is formed; whilst if the medium be well adapted to the nutri- tion of the organism, and the cultivation be kept at a temperature of 36° C., short comma bacilli are almost exclusively developed. It is a curious fact, however, that, once developed, these various forms may persist cr pass on their characters unchanged for one or two generations, even though they are now inoculated into ordinary fresh peptonized gelatine medium, and Cornil and Babes say that if there are inoculated simul- taneously, on peptonized gelatine, a fresh culture of filaments, another of ordinary comma bacilli, and a third with the short and only slightly curved CHOLERA: 169 bacteria which are formed under various conditions, the same typical naked- eye appearances of the cholera bacillus are always obtained in each case, but on microscopical examination it is found that the first still develops in the form of spiral filaments,:‘the second as comma bacilli, and the third as short bacteria, and that these characteristics are carried on to the fourth generation. . With all these modifications in form they still produce typical cholera symptoms when injected into the alimentary canal of animals. In addition to these changes there frequently appear at one extremity of the bacillus small cystic dilatations which are due, according to Virchow, to a kind of cedematous degeneration. As these dilatations make their appearance in certain bacilli, others of them shrivel up, and tadpole and spindle-shaped organisms are formed which may break down into small granules and frag- ments. In the later stages of degeneration these various involution forms are completely sterile. In the earlier stages, so long as they take on the aniline colouring matter pretty freely, successful inoculations may usually be made, but if the unfavourable conditions are continued in the new cultivations, the organisms soon lose both their power of taking up the staining reagents and of reproduction. : It has been stated that the comma bacillus is entirely an zrobic organism, and there can be little doubt that its vege: tative activity is much more marked when the organism is grown in contact with the oxygen of the air. It is certainly more resistant to the action of germicidal agents, and exhibits movements in a much more marked degree. It had been observed, however, that comma bacilli do not cease to multi- ply even when their supply of free oxygen is entirely cut off, but under these conditions, as Wood has pointed out, the bacteria are much more sensitive to external influences, and are very readily destroyed by acids and other germicidal agents. He has found, however, that where free oxygen is cut off, as Pasteur had observed in the case of other ferments, the bacilli produce a relatively much larger quantity of the specific toxine, or poison, than when oxygen is present, and by a number of ingenious experiments he showed that when they were grown on albuminous substances with com- plete exclusion of oxygen, and of substances from which oxygen could be easily derived, the cholera poison was pro- duced more energetically and more rapidly than under the ordinary conditions of zrobic cultivation ; probably because, under these conditions, much larger quantities of albumen must be split up to meet the energy requirements of the organism. Wood and Hueppe from this argue that the bacillus gives rise to such important changes in the intestine because there 176 BACTERIA. we have quantities of albuminoid material which can bé rapidly broken up by the action of the bacilli even in the complete, or almost complete, absence of oxygen, which is supposed to rule in the intestinal canal, as a result of which we have the formation of large quantities of toxine and rapid in- toxication. But as they are voided in the dejecta after growth under these conditions, the cholera organisms are more easily destroyed than at any other time or under any other condi- tions. It is very significant that in cholera, yellow fever, and typhoid fever, three diseases in which the manifestations are chiefly in the intestinal canal, and in which the evacuations apparently contain the living poison, dzvect infection from the stools appears to be the exception rather than the rule, and Wood explains this as due, at any rate in part, to the state in which the * specific organisms associated with these diseases are present in the stools, and are dependent upon the conditions present in the intestinal canal. Should they find their way during this stage into the stomach of the living person they, being more susceptible, would be much more liable to be destroyed by the acid gastric juice, or to undergo attenuation ; but if they are allowed to live outside the body, even for a short time, they become more resistant in character, they multiply more readily, they are less par- ticular about the nature of their food, and consequently they are much more dangerous. CHAPTER IX. CHOLERA (continued). Pettenkofer’s researches—Saprophytic and Parasitic Stages of Cholera Bacillus—Temperature Conditions—Relation to Epidemics—Moisture —Ground Water—Flushing—Cholera in Shanghai Endemic but Intermittent—Cholera Endemic at the Mouth of the Ganges— Vitality of Cholera in Old Cultures—Relation of this to Quiescent Periods during Parts of the Year— Gastro-intestinal Irritations prepare for Cholera—Chinese Vegetables—Cholera Poison Formed in the Intestine absorbed into Body—Inoculation against Cholera—Gamaleia’s Experi- ments—Germicides useful in attacking the Cholera Bacilli—Water Supply, Pilgrimages, Feasts predisposing to Cholera-—Quarantine except in Harbours Useless—Time and Place Dispositions. PETTENKOFER’S indefatigable researches on the relation of ground water and the drying zone to cholera epidemics have thrown much light on many obscure points, and have opened up the way for further work. He holds that the increase of cholera is due entirely to the increase of the “ drying zone” near the surface of the soil—z.e., the lowering of the level of the “ ground water,” and that the rise and fall in the level of this ground water is the principal factor in the production of conditions necessary for the outbreak of epidemics of various kinds. On these grounds he has itaken up a very strong position against the spread of cholera directly from patient to patient. There can be little doubt that many of Petten- kofer’s observations are entirely in accordance with this view, but equally can there be little doubt that all his interpreta-. tions of the facts he has collected are not entirely accurate, although, as Hueppe points out; Wood’s observations offer solutions of questions that have hitherto been unanswerable. The instances are almost innumerable in which there has been a most remarkable immunity against the passage of cholera from individual to individual ; where the dead bodies have been buried immediately the disease has afterwards occurred, not in those who have had to carry out the actual burying of 172 BACTERIA. the dead cholera patient, but in those to whom has fallen the duty, a day or two later, of washing the soiled linen used by the patient during life. In the first case the attendant is exposed to the action of the bacillus as it comes from the patient, but at a time, it is argued, during which the organisms are more readily destroyed, either in the dejecta, or by drying ; but the attendant who has to wash the soiled linen may be attacked by the bacillus after it has had ‘time and opportunity to develop on the damp sheets, in the presence of air, and when it has acquired a greater power of resistance, and has become much more dangerous. It has now, in fact, lost its anzerobic habit, and has become adapted to its new surroundings, with the result that itis much more resistant and is better qualified to live outside the body and to resist the action of ordinary germicidal reagents, or of the acid gastric juice, should it find its way.into the stomach of a fresh host. In similar fashion may be explained the fact that when the drying zone becomes limited, the cholera bacillus appears to die out more readily. It appears that the micro-organism passing directly from the feces into very damp soil contain- ing insufficient oxygen to satisfy its saprophytic requirements is, on accountof its feeble resisting powers, “suffocated” at once, or within a very short period. Where, however, the depth of the drying zone increases, there is more air (oxygen) in the soil, the organisms are more able to multiply in its presence, and, taking the field against other putrefactive organisms, gradually become more and more hardy, acquire the zerobic and sapro- phytic habit, and thus become more dangerous to the inhabi- tants of the locality in which all this occurs. It must be re- membered, however, that the term “drying zone” is entirely a relative one, and that it may still contain, as it usually does, sufficient moisture for the wants of the cholera organism. That the organism takes some little time to pass from the zrobic to an anzrobic condition is evident from Koch’s early experi- ments, in which he made plate cultivations of cholera bacillus, and then, before the gelatine was perfectly set, covered about one-third of the surface with exceedingly thin glass—cover glass thickness—or split mica. He then found that colonies grew as usual, and became visible to the unassisted eye in the uncovered portion of the gelatine and for a very short distance under the covering plate (2 mm.). He observed, however, that where the air (oxygen) was cut off, the colonies did’ not CHOLERA, 173 increase in size sufficiently to allow of their being distin- guished without the aid of a lens; the power of growth to the size of a colony visible to the naked eye being attributed by Koch to the presence of the small quantity of available oxygen that remained in the nutrient gelatine—growth ceasing as soon as this oxygen is exhausted,—the colony remains of small size. A second experiment was made by inoculating~ nutrient jelly contained in a small glass with comma bacilli ; this “was placed under the receiver of an air pump, another glass pre- pared in a similar way being placed outside the air pump as a control experiment. It then appeared that those bacilli under the air pump did not grow, while those outside the ; receiver did well. But if those which had been under the receiver were afterwards exposed to the action of the air, they then began to grow. They therefore had not been destroyed ; they only wanted the necessary oxygen to be able to continue their growth. A similar result is obtained when cultivations are placed in an atmosphere of carbonic acid ; whilst those cultivations placed for control purposes outside the carbonic atmosphere grow in the usual manner, those subjected to the action of a stream of carbonic acid remain inactive and undergo no development. Here also, however, they do not die; for after they have been exposed to the carbonic acid for a con- siderable time, they again begin to grow as soon as they are taken out of it.” In place of carbonic acid, hydrogen may be used, and the result is much the same. It cannot be assumed from these experiments that the organisms require to obtain free oxygen from the air in order that they. may continue their growth, for, as has been pointed out by Hueppe and Wood, if they are grown on a suitable medium, such as unchanged normal albumen, they are able not only to exist, but to produce unusually large quantities of their specific poison, as they are able by the dissociation of such a medium to obtain all that they require for their growth and development. All that can be argued from Koch’s experiments is that the bacillus has a saprophytic stage (although this was apparently not at first recognized by Koch), during which it grows and multiplies, but only under ordinary con- ditions where free oxygen can be obtained from the atmosphere. During its parasitic existence it becomes so far modified, that, still growing with great vigour, it produces its toxine more easily or in greater quantities in the absence of oxygen, other conditions being favourable. During this stage, however, it is much more readily affected injuriously by acids and other germicidal reagents, and is therefore much more easily destroyed than when it has had time to acquire its full saprophytic faculties, 174 BACTERIA. Mention has been made of the conditions as regards temperature that are necessary for the growth of the cholera bacillus when other conditions are slightly adverse. As the result of an extensive series of experiments, first carried on by Koch, and afterwards repeated by other observers, it has been found that the comma bacillus flourishes most luxuriantly, and is most productive, at a temperature ranging between 30° and 4o° C., although it can grow at a much lower tem- perature, and even at several degrees above the higher point mentioned. At 20° C. it flourishes luxuriantly upon a peptonized gelatine medium. As early as 1884 Koch described the growth of the cholera bacillus at a temperature of 17° C., but as might be expected the growth is not nearly so prolific, and it is certainly considerably less rapid than at a slightly higher temperature. A temperature below 17° C. seems to be inimical to the growth of the bacillus, as at 16° C. develop- ment may be said to have almost ceased. It is, however, remarkable that an intense degree of cold does not deprive the organism of its power of growing, for, if after being ex- posed to great cold for some time, it is again placed under favourable conditions, it appears to regain its powers of rapid multiplication. Koch, to test this, submitted a culti- vation of the bacillus for one hour to a temperature of 10° C. (10° C. below freezing point), with the result that the nutrient medium was completely frozen. This was now thawed, and a cultivation made at a higher temperature and under favourable conditions; the bacillus began to grow again almost immediately, and appeared, indeed, to have lost none of its vitality. These experiments accord well with, and verify the observations that have been made on, the appearance and spread of cholera epidemics in the hot and cold seasons. Most of the cholera epidemics seem to have attained their maximum virulence during or at the end of the hot season of the year. In those regions that are periodically visited it usually breaks out in the autumn, when the external temperature is most favourable to the growth of the bacillus as a saprophyte, and when in consequence the micro-organism is enabled to live for a longer period outside the body, to give rise to numerous progeny and thus to multiply the possible sources of infection. On the other hand epidemics are of frequent occurrence, even in the CHOLERA, 175 coldest seasons of the year ; nay, in some cases the appear- ance of the cold damp season is the signal for the outbreak of a cholera epidemic, because not until the return of the cold weather, after the hot dry summerand autumn months, is there that dampness of soil and atmosphere which 1s essential for the existence of the organism, in addition to which the soil remains at a comparatively high temperature for some time after that of the atmosphere has fallen, Again, the external cold, though it may paralyse the organism for a time, does not destroy its vitality, but, keeping the organism in a passive condition, actually enables it to retain this vitality for a considerable period, so that, when it finds its way into dwellings, in which the atmosphere, owing to bad ventilation, is not only raised to a high temperature, but also contains a considerable quantity of moisture and organic matter, it is at once introduced to conditions that are essentially favourable to its saprophytic existence; it again begins to thrive luxuriantly, and becomes a possible source. of infection. The fact must not be ignored, also, that seasonal temperature plays not only a most important part in the determination of the amount of moisture in the air and in the soil, but also in the production of currents of air by which the organisms may be carried from point to point (though these currents, when the air is dry, or in hot, clear weather, play but a small part in the dissemination of the disease), and by its effects on certain kinds of vegetation, which indirectly have bee proved to play an important part in the distribution of the cholera organism. On the effect of heat on the amount of moisture in the atmosphere, and consequently on the depth of the ground water, it is scarcely necessary to speak at any great length, but there can be little doubt, from Pettenkofer's observations, that such factors do play a most important part in condition- ing the growth and multiplication of the cholera organism. The rapid removal of stagnant and upper ground water (if complete) is inimical to the saprophytic growth of the cholera organism, as even organic matter in a state of dust is absolutely useless as a nutrient material for the cholera bacillus. Where, on the other hand, there 1s a considerable quantity of moisture in the atmosphere, even though the temperature be high, stagnant water is often found. Koch says: ‘* On the surface or in the ground, in marshes, in docks, which have no outlet,in places where the ground is formed like a trough, in sluggish rivers and the like . . . there a constant nutrient solution can be formed and 176 BACTERIA. may accumulate in the neighbourhood of animal and vegetable decaying matters most readily and give the micro-organisms opportunities for growth ;” and he further says, ‘* that whenever the water has a swilt current, or is in a constant state of change, both on the surface and in the ground, these con- ditions occur less easily, or sometimes not at all, for the continuous current prevents a localized concentration of nourishment in the fluid sufficient for the pathogenic. bacteria. The connection between the sinking of the ground water, and the increase of many infective diseases, we might explain thus : that with the sinking of the ground water, the current which exists in it becomes very much lessened. Besides, the mass of water lying superfici- ally at disposal will be considerably diminished, and therefore such a concentration as I have described as necessary for the growth of the bacteria will be produced much sooner.” These conditions are necessarily closely associated with temperature, with the nature of the sub-soil, and the pre- vailing winds and air currents, and the consequent affection of the quantity of moisture in the atmosphere, and the pro- duction of rain must also be taken into account, as even the transmission of the cholera organism is affected or interfered with by great dryness of the atmosphere. Where there is great dryness of the atmosphere the small quantities of cholera dejecta, which are likely to be left unnoticed, and therefore left disinfected by the attendants of the patient are so rapidly dried that they are speedily rendered inert, and cannot convey the infection further ; whilst the materials on which, in ordinary circumstances, they would thrive, are so dried on the surface that although the organisms. may find their way on to them in a living condition, there is not sufficient moisture left to allow of the development of the probably somewhat weakened organism. As Fliigge points out, such conditions “ only occur where there is a very great deficiency in the saturation of the air with moisture—for example, in a desert . . . It is conceivable that in.a desert climate such as is present in Mooltan and Lahore during the greater part of the year, and where every- thing dries up, as it were, under one’s eyes, the conditions favourable for the spread of the cholera may only be present at most during the some- what moister or so-called ‘rainy’ season (July to October).” Extreme wet may, however, exert an influence in interfer- ing with the saprophytic growth of the organism, especially if the rains be heavy and continuous. The conditions men- tioned by Koch, as inimical to the collection of organic material on which the organism may grow, are brought into play, infective dejecta are removed from the surface soil, from the surface drains and from the sewers, rapid and con- CHOLERA. 177 tinuous currents are created, organic matter of all kinds is washed away, air is driven out from the subsoil owing to the rising of the ground water, and all the conditions become unfavourable for the growth of the organism. The fact that vegetation has been mentioned, as associated with climate in the production of cholera, requires some explanation, which will be given later. Dr. Macleod, writing on the subject of climate itself, says : ‘* Cholera makes its appearance in Shanghai every summer with start- ling regularity, Before the end of July it is hardly met with, by the end of August it is well marked, in September it is in full swing, not quite so virulent in October, and in the beginning of November an occasional case may be heard of, after which time it disappears entirely till the following late summer. For twenty years this has gone on with unfailing regularity under the observation of medical men now resident, and for how long previously no one can estimate.”’ He then goes on to describe the weather in June as damp and hot, in September as hot, damp, and muggy, in October as cool and wet during the first part, and as frosty towards the end, “at Christmas there is usually ice, there may be snow.’ He then says, ‘* so far as temperature of the air is concerned, we enjoy tropical heat for nearly a couple of months before the disease breaks out ; it is most virulent in the hot, damp September, and does not disappear until after the hoar-frosty mornings are experienced ” (the end of October). Dealing with the same subject Koch has stated that in no part of the world, of which the climate, the geology, and the epidemiology are known, does cholera occur all the year round except in the province of Bengal, and he says : ‘* All authors are agreed, that the delta of the Ganges is the true home of cholera, and I have come to the conclusion that this is the case, and that there are no other places of origin of cholera in India. For the only district in India, where cholera prevails continually year after year in a uniform manner, is the delta of the Ganges. In all other places it shows marked variations, or it may even disappear altogether for a shorter or a longer time. In certain places, for example in Bombay, it never entirely disappears, but it is highly probable, that on account of the unusually active trade with the rest of India it is constantly being imported there afresh.” He then describes this tract as a perfectly flat country only slightly raised above the sea level, which, during rainy seasons, is almost entirely under water in the lower part of the delta, where the ‘Ganges and the Brahmapootra break up into a network of water courses, in which the sea water, mixing itself with the river water, flows hither and thither with the tide, and at flood time places large tracts of the Sunderbunds under water. A luxuriant vegetation and an abundant animal life have developed in this uninhabited region, which is inaccessible to man not only on account of the floods and the numerous tigers, but is avoided principally on account of the pernicious fever which attacks everybody who remains there even for quite a short time. One can easily imagine how dense the vegetable and animal matter 13 178 BACTERIA. . is which is given up to decomposition in the marshy districts of the Sunderbunds, and that here an opportunity is afforded for the development of micro-organisms, such as exists in scarcely any other place on the globe. Peculiarly favourable for this are the regions between the inhabited and the uninhabited parts of the delta, where the excrements of an unusally thickly populated country are washed away by the current, and, flowing here and there, are mixed with the brackish water of the Sunderbunds, already teeming with decaying matter. Under these peculiar conditions quite a distinct fauna and flora of micro-organisms must be developed there, to. which in all probability the cholera bacillus belongs. For every- thing points to the cholera having its origin in this district. All the greater epidemics have begun with an increase of cholera in the southern portion of Bengal. Jessore, from which the first intimation of the epidemic of 1817 came, lies on the borders of the Sunderbunds ; and Calcutta, which is now the fixed home of the cholera, is connected with the neighbouring Sunderbunds by a marshy and sparsely inhabited tract of land. Now the comma bacillus finds in this district, contiguous to its presumptive home, the most favourable conditions imaginable to implant itself and to spread from one individual to another.” In the large towns in this cholera region there appears to have been no diminution in the mortality from the disease during the British occupation ; and until a new water supply was obtained and it was no longer necessary for the natives and others to take their water from tanks, from the Ganges, or from the Hoogley, even a better sanitary and drainage system appeared to have little effect. Once this supply was obtained, the mortality fell to about one third, and even a considerable portion of that third is to be accounted for by the fact that it was, and is, very difficult to impress on the natives the necessity of avoiding the old contaminated sources of water supply. Although Koch brought out these remarkable statistics he is still, as he says, “ not a supporter of the exclusive drinking-water theory,” and he considers “ that the ways in which cholera can spread itself are extremely varied, and that almost every place has its own peculiarities which have to be thoroughly investigated, and the regula- tions which are to serve for the prevention of infection in the place in question must be drawn up accordingly.” It is a remarkable fact that Koch never speaks or appears to think of the possibility of the existence of the cholera bacillus as a saprophyte. If it enters food or water it is the result of an accident and the organism can remain there, capable of development only for a short time and under exceptionally favourable conditions. Thus he contends that: cholera is endemic in the Ganges only because there is a complete CHOLERA. 179 chain of cases from one year’s end to the other by which the infection is handed on. Hueppe, on the other hand, main- tains, as above stated, that the bacillus can have a distinct saprophytic existence, and. points to the fact that in places analogous to the delta of the Ganges it has a marked tendency to become endemic. As an example of this we may summarize the results of Macleod’s observations on cholera in Shanghai. He says that “the remarkable regularity of the time of outbreak, period of duration, and time of cessation of the disease, so far as I am aware, has no parallel on record.” The country round Shanghai is strikingly like that of the deltas of the Ganges and the Nile ; “in each there is the alluvial deposit, rich in organic matter, and the high ground-water level, yet in the Ganges delta the disease is prevalent all the year round ; at the mouth of the Yang-T'sze it occurs with the regularity of a crop at the same season yearly ; in the delta of the Nile it occurs only occasionally but at the same season as at the mouth of the Yang-Tsze. The two latter regions have at least weekly communication with India. Some causes or combination of causes are prevalent all the year round at the mouth of the Ganges; at one season every year, viz, late summer and autumn at the mouth of the Yang-Tsze ; and at the same season, but not every year at the mouth of the Nile.” There must then in these three regions be perfectly distinct but local conditions which must determine the difference in the behaviour of cholera in these various regions. The general characters of the soil, the ground water, and position are much the same, but there are well-marked differ- ences as regards temperature, population and methods of cultivation. In the delta of the Ganges cholera is absolutely endemic, it is never absent ; the poison, whatever may be its nature, is therefore always present. ‘“ From the position and climate, the temperature of the soil varies little throughout the year, and is never so low that some vegetable growth is not active. At the mouth of the Yang-Tsze (where cholera is prevalent only during certain parts of the year), the poison is endemic or is introduced shortly before the time that cholera breaks out each year, in which case it is curious that it should always be at the same time, there being no means of communication with a cholera infected country opened up specially at that time, communication wlth India being weekly. Here the climate admits of a greater range in the soil temperature than at the mouth of the Ganges, there being great extremes of both heat and cold. The soil heats more slowly than the air, hence perhaps it is the determining cause for the spread of the disease in the late summer and autumn, as already described, and of its absence in winter and spring.” It is evident from this statement 180 BACTERIA. that cholera does not show itself during the summer when the air tempera- ture is at its maximum, but only when sufficient time has elapsed to allow of the rising of the ground temperature or that of the earth, and as a matter of fact the disease occurs in Europe during the autumn months often when the general or atmospheric temperature has actually begun to fall, but when the earth temperature has attained its most equable maximum, when of course we have the optimum temperature conditions for the growth of the cholera organism in the soil. ‘At the mouth of the Nile the con- ditions are much the same as near Shanghai, except that the extremes of heat and cold are greater at the latter. Both have at least weekly communi- cation with India, both communicate through the tropics; whilst Shanghai is at a distance of at least three weeks in time, Egypt is but two. The regularity of the yearly outbreak at Shanghai points to an endemic pvison, the irregularity at the mouth of the Nile to the occasional introduction of the poison. Late summer is also the period for the Egyptian epidemics of any extent.” ; Bearing on this, we have the fact that a cholera epidemic may remain dormant for months or a whole winter ; especially as the organisms retain their capacity of reproduction if they .are kept moist and are supplied with oxygen. According to Nicati and Reitsch, cholera bacilli were found alive eighty-one days after they had been placed in the harbour water of Marseilles. Koch found cholera bacilli capable of reproduction after they had grown one hundred and forty-four days on agar, but in one hundred and seventy-five days these cultivations were found to be dead. Macleod found that pure culti- vations renewed but once a month retained their virulence for a year. Taking all these facts into consideration, it is easy to understand how in Shanghai the organisms may remain dormant for a certain period, and then under favourable conditions begin to grow again with increased activity. As Koch puts it—‘‘ One can easily imagine that in superficial layers of earth, in marshes, and so forth, the cholera bacilli may find conditions in which they can exist preserved from death for five months or even longer, just as well or even better than on our moist agar jelly.” Here then are three places which, although they haye certain features in common, are characterized by certain differences, all of which appear to depend on climatic conditions. In the one case the cholera is prevalent during the whole year ; it is in fact endemic and continuous. In the second case cholera appears with the utmost regularity at certain seasons of the year, and then as regularly disappears. In this case the disease appears CHOLERA. 181 to be endemic, but on account of some local circumstances connected with temperature and vegetation it is developed only in crops--at harvest time, as it were. In the third case, in the Nile delta, the disease is extremely intermittent in its outbreak, and occurs only in those years in which the condi- tions as regards temperature, moisture, &c., are specially favourable for its development. In connection with local peculiarities as favouring the growth of the bacillus and the spread of the cholera, Macleod makes several very interesting observations, some of which bear out in a most remarkable manner Koch’s statement as to the general laws that govern the spread of the disease, and also as to the minor local factors that determine individual outbreaks. - The population in Shanghai he divides into three classes : resident foreigners, amongst whom deaths from cholera average less than 2 per 3000 per annum ; amongst the seafaring foreign population the average is very much higher, as of the sailors who come to port there die from cholera from 15 to 30 per 1000 yearly ; whilst among the Chinese, even in the settlement, there are rarely fewer than from 200 to 300 deaths in a single season. What part then do local peculiarities and customs play amongst these three sets of people? It is a fact, generally recognized, that any disturbance of the digestive function is the principal predisposing cause of the disease in a cholera outbreak—a fact that is explained by the absence of the ordinary amount of acid from the gastric juice in . these cases ; such gastric derangement is specially met with after a bout of drinking. Macleod says, “ Among the sailors a night ashore is the usual precursor of the disease, and commonly that indicates a large consumption of liquor.” This, how- ever, does not account for all the cases that occur, and he says, “Occasionally men who have not been ashore are attacked. A sailor belonging to an American ship was attacked two or three days after arrival ; he had not been ashore, the ship had touched at no port for weeks before, and the water supplied on board had been in use during the voyage. The captain reported that he had specially en- couraged the men to eat fresh vegetables largely, and that he had seen the man referred to sitting on deck munching lettuce the day before he became ill (three of this ship’s crew died of cholera). These vegetables had been supplied by Chinese bumboats.” 182 BACTERIA. The custom amongst the Chinese “‘is to carefully collect human excreta and add these to water ; this mixture is then used for watering vegetables, &c., so that the leaves come in for a fair share. All European and Chinese night soil is collected daily and carried away to be used for agricultural pur- poses. No night soil finds its way into drains, and there are no water closets,” and all refuse not used directly for watering vegetables is carried into the country and used for agricultural purposes, so that if the poison be in the excreta, in China at least, its deposition in the ground is secured, and it has certainly been discovered empirically by European residents that vegetables cannot be taken with impunity. ‘‘ Chinese vegetables are regarded with suspicion during the cholera season, more especially such as are uncooked.” When the relation of the comma bacillus to the dejecta from cholera patients is borne in: mind, there can be little wonder that the consumption of, Chinese lettuces should be followed by an attack of cholera. In connection with the conveyance of cholera by means of drinking water, it may be objected that the Chinese do not drink water as a rule, but indulge in weak tea ; but here again it is stated that water for household use is stored in an earthenware or wooden vessel placed in or near the kitchen, and into this vessel: others are dipped from time to time. The water is obtained from creeks or wells. Vegetables may be seen hanging over these vessels or lying on tables, so that though the vegetables are cooked, tables, dishes, cloths, and all that come in con- tact with uncooked vegetables furnish abundant opportunity for contamination of food after it has been cooked, where such filthy habits prevail as amongst the Chinese. They do not use milk as do foreigners, but they supply it, and from what has been said of vegetables and Chinese habits, unless the supply is known to be well cared for, it cannot be regarded as beyond suspicion of forming a vehicle for the distribution of cholera, typhoid, and other poisons. It is evident, then, that although no one set of factors alone can be looked upon as determining or explaining all outbreaks of cholera, the bacillus, so far as our present knowledge goes, must be looked upon as the essential factor in the causation of the disease ; this bacillus, like every other organism that we know throughout the whole animal and plant kingdoms, being, to a great extent, dependent on its environments for its very existence. In considering this question let it be understood most distinctly that though the bacillus is proved to be the cause of the disease, it is not necessary to assume, as some people seem to suppose, that the careful, and in CHOLERA. 183 themselves complete, observations of the great epidemio- logists of this and other countries are valueless ; and as in time a more perfect knowledge of the conditions favourable or unfavourable to the growth of the organism is obtained, it may be expécted that all observations made: with care by those whose only desire is to get at the truth, will fit in and take their place in a large and comprehensive scheme through which there will ultimately be a possibility not only of understanding the disease more thoroughly, but of re- meving or combating its causes. It has already been pointed out how Pettenkofer’s obser- vations may be made to agree with Koch’s ; it has been seen that the apparently contradictory statements on the subject of the spread of cholera along the lines of pilgrimage and trade have been more or less satisfactorily explained and reconciled on the assumption that the bacillus is the cause of the disease. Then, too, the well-known facts of the association between cholera and gastric and intestinal disturbances of various kinds ; between it and consumption of various articles of food and of water from sources which are probably contaminated, or in which the presence of the cholera organism has been de- monstrated, and the facts connected with climatic conditions, ground water and subsoil drainage, temperature and special local conditions, may all be satisfactorily explained on Koch’s comma bacillus theory. These explanations, taken with the fact that the comma bacillus is invariably found during cer- tain stages of the disease, that it has never yet been found in any but typical cases of cholera, that with it the disease has been produced in animals, experimentally, and one might also say in man, though accidentally (at the cholera course in the Hygienic Institute in Berlin), make it impossible for us to shut our eyes to the fact that in the cholera bacillus we have the only suggested causal agent that will allow of a satisfactory explanation of the mass of observations made up to the present. It had already been demonstrated that the bacillus was found only in the intestinal canal, when it naturally suggested itself to Koch that the symptoms of cholera were to be explained by the theory that in this disease there was absorption from the intestine of some soluble poison produced by the bacillus 2 sztw ; that there was, in fact, a local formation of the poison, but a general absorption into the system (the “ Intoxication” theory). This 184 BACTERIA. theory was at once accepted as being from every point of view more far-reaching and more satisfactory than the older one which explained all the symptoms by referring them to loss of water through the excessive intestinal discharges ; a symptom or effect was, in fact, looked upon as the cause of other symptoms. The earlier observations on ptomaines and sepsines, and Pasteur’s and Hansen’s later observations on ferments, naturally led to a search being made with the object of finding out any specific products of the cholera organism. Koch himself records the fact that he succeeded in preparing cultures of the comma bacillus which were so intensely poisonous, that when injected into animals, either subcutaneously or into a peritoneal cavity, there were set up in a few minutes all the symptoms which occurred in animals suffering from cholera a day or two after infection— “paralytic weakness of the hinder extremities, coldness of the head and legs, and prolonged respiration, a condition which usually leads after some hours to death.” Buchner, who demonstrated, as he believed, the formation of butyric acid during the growth of pure cultivations, was unable to corroborate by any experiments of his own this view of Koch’s, but Pouchet and Villiers were both able to extract from the dejecta or from the organs of cholera patients cer- tain products which they deemed to be characteristic. The former, with the aid of chloroform, extracted from cholera dejecta an extremely toxic oily liquid which, as it becomes oxydized in the presence of air and light, takes on, first a rose, and then a brown colour. It readily combines with hydrochloric acid to form a chloride, but again breaks down, im vacuo, or when the temperature is raised, or on the addition of an alkali. It gives the reactions characteristic of the alkaloids, and gives the blue re- duction coloration with ferro-cyanide of iron and perchloride of iron. Villiers succeeded in separating from the organs of a single cholera patient a couple of centigrammes of a peculiar alkaloid. On treating this with hydrochloric acid, there separated out a number of acicular crystals: these crystals have since been described by various observers, and it is stated that in this form the basic substance, whatever it may be, exerts comparatively little poisonous action, but that as soon as it is again set free from the acid combination, by the addition of an alkali, such as soda or potash, it exerts not only an extremely caustic local action, but also when injected into a guinea pig produces muscular tremblings and very great irregularity of the heart’s action, the animal-dying at the end of about four days. It is im- possible, however, to be quite certain that these reactions were obtained with Villiers’ pure acicular crystals, ang not with one or other of the sepsines or toxines, as there is some little doubt as to the exact nature of the poison obtained in several of the series of experiments, Brieger, going beyond CHOLERA. 185 these researches, was able to isolate, especially from old cultures, substances analogous to, or identical with, cadavarine, putrescine, and choline. Carrying his experiments further, and cultivating the comma bacillus in media con- taining creatine, he produced a toxine or specific poisonous product which produced, when injected into the animals, dyspnoea, muscular tremors, cramp, and death. To this he gave the somewhat formidable name of Methylguanidin. Carrying his observation still further, he succeeded in separating from a precipitate obtained by the addition of a mercurial salt, two other toxines, both of which appeared to be more or less characteristic of the cholera growth. It will be noted that in all the experiments made, in which the toxines were obtained even from pure cultures of Koch's comma bacillus, the time required for their production and the quantities separated are out of all proportion to what occurs in actual cases of cholera, where, from the rapidity of the course of the disease and the severity of the symptoms, a large quantity of the poison must be developed in a very short time. Most of the artificial experiments, however, have been made on different media (usually bouillon contain- ing peptone) and under very different conditions from those which obtain in an animal. In a series of investigations carried out by Wood in Hueppe’s laboratory, an attempt was made to grow cultures under precisely those conditions that are met with in the human intestine, with the result that a very rapid toxine production, and an additional proof of the bacillary origin of the cholera virus were obtained. He took for his nutrient medium normal albumen, and thus obtained the earlier products such as the albumoses that occur in the breaking down of albuminoids. The presence of these would of course account for the greater toxicity of his cultures under such conditions. One of the earliest observations made by epidemiologists was that one attack of cholera protects for a certain time and to a certain degree against a second, and from this it was now argued that it should be possible to obtain a system of pre- ventive inoculation ; and Gamaleia, basing his work on what was already known of preventive inoculation in other diseases, commenced a series of experiments by means of which he hoped to construct such a method of inoculation against cholera. In this he was ultimately successful. He found that if, from a culture of the vzbrz0 Metschnikovz, an organism almost identical with the comma bacillus, both morphologically and physiologically, in beef broth, he took 186 BACTERIA. the pellicle which formed on the surface at the end of five days, sterilized it in an autoclave at 120°C., and then injected from two to six c.c. of the fluid expressed from the sterilized mass into the muscles of a guinea pig, the animal was rapidly and completely protected against the action of Koch’s comma bacillus, introduced in the ordinary fashion. By further experiments he found that by simply keeping for several days the sterilized mass in which the dead bacilli still remained a much larger quantity of the poison could be obtained— sufficient, in fact, to kill the animal when given in the same dose as before, but still protecting the animal when exhibited in smaller doses. (Here the organisms could no longer be producing the poison, but it appears as though there was stored up in their body a considerable quantity which could only diffuse out into the fluid after a certain lapse of time.) To Gamaleia also we. owe the knowledge that this organism may become very much modified in various ways, and some of the experiments he carried out provide us with an explanation of some.of the most important facts connected with the increase and decrease in virulence of type, in cases that occur during the “ rise and fall” of an epidemic. He was able to increase the virulence of the special comma bacillus in a most remarkable manner. After obtaining a growth of the organism in broth he introduced a smadl quantity of the culture into the lung of a white rat ; this was followed by an-acute form of croupus pneumonia accompanied by marked pleurisy, the animal rapidly succumbing. With the fluid that ac- cumulated in the chest a second animal was inoculated in a similar fashion, with the result that this animal died more rapidly than the first. This inoculation was continued through « whole series of animals, until finally the rats succumbed very rapidly indeed, and an organism was found not in the intestine, but in very large numbers in the blood." In addition to drying, acid, and the other destructive reagents already mentioned, it. has been found that a very considerable number of chemical reagents arrest or prevent the growth of the cholera bacillus. Koch’s list contains alcohol, 10 per cent., sulphate of iron, 2 per cent. (this latter acts first as an acid, and secondly as a precipitant of the * That this may have a bearing on Wood's observations as to the viru- lence of the organism in the zrobic and anzerobic conditions is most evident, and is well worth further investigation, CHOLERA. 187 albuminoid materials on which the organism lives), alum, i per cent., camphor, .33 per cent., carbolic acid, .25 per cent., essence of peppermint, +5 per mille, sulphate of copper, 4 per mille, quinine, .2 per mille, corrosive sublimate, 1 per cent. “ : Babes has found that the organisms will not develop in a gelatine nutrient medium which contains .5 per mille of corrosive sublimate, .1 per mille of carbolic acid, .2 per mille of sulphate of copper, .8 per mille of salycilic acid, .1 per mille of thymol, 2 per mille of iodine, 2.2 per mille of bromine, 7 per cent. of alcohol, .8 per mille of sul phate of quinine, .5 per mille of acetic acid (the action of which, however, depends toa certain extent upon its interference with the degree of alkalinity of the original gelatine, as the bacillus cannot grow in a medium in which there is more than a merely appreciable trace of acidity). He also tried a very interesting experiment _in which he inverted a gelatine plate on which was placed a cholera culture over a watch glass or shallow vessel containing a drop of essential oil of mustard; no development of the organism took place, and if the plate was left for twenty-four hours the organisms.were all killed, as there was no subsequent development ; so that oil of mustard is an excellent disinfectant. Other active volatile principles act in very much the same manner. The experiments with gelatine cultures are of special value because they allow demonstration of the effect that disinfec- tants have on the bacillus as it grows on specially favour- able nutrient media. Reference has already been made to the fact that putrefactive organisms and their products exert a prejudicial effect on the cholera organism, but it should be remembered that when the conditions are favourable there is usually such rapid proliferation of the cholera organism for the first day or two, that the others are left behind in the race for existence. It is, however, a case of the hare and the tortoise, for after a time the cholera organisms die out, and never again obtain a permanent footing unless a fresh supply of nutrient material becomes accessible. It is im- portant to remember this in connection with the spread of cholera by water supply, as it explains the immunity enjoyed, after a time, even in the most cholera-stricken regions. In India, in the regions in which the cholera is endemic, the wells, as a rule, are merely surface tanks into which sewage and surface water may be drained, and which are frequently on the same level as, and connected with, the cesspools, so that even the water supply contains a considerable quantity of organic matter in which organisms of all kinds can flourish most luxuriantly; whilst these same wells, being merely -dug-out pits beneath the slightly raised houses, are open for the reception of sewage and excreta 188 BACTERIA. of all kinds, especially in times of illness, when neither patients nor nurses have strength or time to see that these are properly removed. ‘This source of danger is so evident, and is so in accordance with what one would expect, that efforts have been made to remove, as far as possible, all organic material which might serve as nutrient material for infective organisms from the soil and ground water, and also to remove as rapidly and as completely as possible not only dejecta, but also the water employed for cleansing linen, clothing, and utensils “ without allowing them to come in contact with the surface of the soil, with wells,” with vegetables, and the like. Most ‘hygienists are agreed that it is necessary not only to have a pure water supply—z.e.,a supply free from all pos- sible contamination, derived from wells so deep, or from reservoirs so far from human habitations, that there is no possibility of contamination by sewage, dejecta, or surface drainage, and so carefully conveyed by conduits and pipes that no such contamination can take place during distribu- tion—but that a water supply should be so ample that dirtiness is heavily discounted. With all the improvements that have been made in the drainage system and water supply of Lower Bengal, cholera has only diminished about 60 per cent., so that there still remain certain factors that favour the spread of cholera, and every now and again such a spread or outbreak may take place with extreme rapidity, and may involve a very wide area. Cleanliness, however, both general and personal, may be said to be the most important factor in the prophylaxis of cholera. Fligge, for example, states most emphatically and explicitly that “the average cleanliness of the population has the greatest influence in this respect. The more cleanly the method of handling the sick and the infected clothes, the more care- fully contamination of the soil, of the water, and of various other objects with the dejecta is avoided, the fewer will be the sources of infection. The more carefully the hands are cleansed and the articles of food prepared, the more will the paths of spread from existing sources of infection be diminished. It is evident that in this respect marked dif- ferences must exist between more and less civilized countries ; between new and well-built, and old and cramped cities ; between poor and wealthy neighbourhoods; between the CHOLERA. 18g portion of a city inhabited by the poor and that in which the better classes dwell.’’ The habits of people, then, es- pecially as regards their food, play a most important part in the propagation or restriction of cholera. -The organism is almost invariably introduced by the mouth, so that in addition to passing into the alimentary canal by con- taminated water directly, it may also be introduced by utensils, or food washed or sprinkled with such water, and Koch gives an excellent example of this when he describes the market women of Marseilles as being in the habit of sprinkling the vegetables exposed for sale with water from. the street gutter, into which he proved that comma bacilli were constantly making their way, so that any one partaking of these vegetables uncooked, just as in the case of the American sailor at Shanghai, was taking in an unknown quantity, but a very appreciable and deadly dose, of the cholera organism and its poisonous products. Not only do these uncooked vegetables offer a nidus and an excellent sub- stratum for the growth of cholera organisms, but they often produce that condition of slight indigestion which along with an overloaded stomach. is one of the most favourable for the development of the comma bacillus in the human alimentary canal. For the same reason feasts, fasts, Saturday night carousals and Sunday dyspepsias, pilgrim festivals, arrivals in port, and similar events are all predisposing causes of cholera, as in all cases there is a disturbance of the digestive function and a lowering of the system, due either to excess in drinking or in eating, or to gastric disturbance and lowered vitality resulting from abstinence from the use of food for too long a period. It is found, too, that wherever people assemble in Jarge numbers in excess of the ordinary popula- tion, the strain on the sanitary arrangements is always excessive, and further, is necessarily accompanied by care- lessness in the selection and preparation of food. Cholera depends for its existence, outside those places in which it is endemic, on these fairs and pilgrimages, and only by controlling them and by attending most thoroughly to the sanitary conditions at the points where people are massed together can there be any hope of preventing the outbreak and spread of this insidious and deadly disease. It is not necessary here to do more than mention the pre- 190 BACTERIA, disposing effect of autumnal gastric disturbances and slight diarrhoea, which have, in many epidemics, been the in- variable precursors of the true Asiatic or Indian cholera. It is known that during an epidemic one attack of cholera, especially a severe one, exerts a great protective influence on those who survive, and as these consist almost ex- clusively of the people who under ordinary circumstances are again most exposed to infection, there may be natural breaks in the line of extension of an advancing epidemic ; breaks which have been used, by some authorities, as evi- dence of the sporadic outbreak of the disease. So much has the study of Koch’s comma bacillus tinged and affected all our ways of looking at cholera epidemics that we now consider the conditions under which the bacillus can multiply and be carried from point to point, and the conditions that favour its development and mul- tiplication, instead of dealing with cholera itself as the entity with which we have to contend. The epidemiologist has now assumed the ré/e of biologist in the widest sense of the term. The bacteriological hygienist agrees with the “localist” that it is necessary to get rid of all conditions in which the cholera poison can be propagated and dis- tributed, and that to this end the ground: water should be kept as free as possible from sewage, that all areas should be properly drained, and that everything should be done to render the “drying zone” as little congenial to the bacillus as possible ; but he goes further, and insists that the poison should not be allowed to be introduced into localities in which it does not already exist, for he believes that however favourable the conditions may be, there will be no outbreak of cholera until the bacillus is introduced and gains a foothold. He therefore insists on careful in- spection of all ships coming from India or from other cholera- stricken regions, though he is firmly convinced that it is only in seaport towns that inspection or quarantine can be of any value ; that if cholera makes its way beyond the seaport no quarantine or sanitary cordon can stop its spread ; that once beyond port the most careful isolation of all patients and disinfection of every article of clothing, feeding utensils, &c., should be rigidly carried out and insisted upon; that the dejecta should be mixed with large quantities of carbolic acid, concentrated hydrochloric acid, or strong cor- CHOLERA. 191 rosive sublimate solution; that the comma bacilli in rooms should be thoroughly dried and aired by throwing open the windows for several days, and that instructions should be issued as to the necessity for thoroughly personal, culinary, and household cleanliness, as to the avoidance of all water except that known to be pure, as to careful boiling and cooking of drinking water and food, as to the necessity of paying attention to the slightest gastric derange- ment, and as to the avoidance of all excesses in both eating and drinking. He pays attention then, first, to all relating to the power of the individual to resist any attack of the organisms. All place dispositions favouring the production. of conditions of gastro-intestinal disturbance are to be care- fully neutralized, and in the same way all time dispositions, such as autumn diarrhoea, determined by the unripe or overripe fruits so abundant in the latter half of the year are to be met and counteracted; and second, to everything relating to the rendering of all the environments unfit for the development of the comma bacillus, so that the number of centres from which infection may spread may be kept down as much as possible. LITERATURE. Authors already referred to. Cornil and Babes. Bazses.—Virch. Arch., Bd. xcrx., p. 148, 1885. BocuFonTAINE.—Comptes rendus, t. xcix., p. 845, 1884; t.c., p. 1148, 1885. Borum.—Die kranke Darmschleimhaut in der Asiatischen Cholera mikroskopisch Untersucht. Berlin, 1838. BrieGer.— Untersuchungen iiber Ptomaine. Berlin, 1885, 1886. Brittan AnD Bupp.—London Med. Gazette. Sep., 1849. Bucuner.—Arch. f. Hygiene, p. 361, 1885. Bujywip.—Centralbl. f. Bakt., Bd. 1v., p. 494, 1888. CanTani.—Deutsch. Med. Woch. _ 1886. Cueyne, Watson.—Brzt. Med. Journ. April 25, May 2, g, 16, and 23, 1885. CunNINGHAM.—Scientific Memoirs Med. Off. Indian Army, Pt. 11., 1886, “on Cholera,” Calcutta, 1885. 192 BACTERIA. DavaineE.—Comptes rendus de la Soc. de ‘Biol., 2me Serie., t. 1, p. 129, 1859. Denexe.—Deutsch. Med. Woch., No. 3, 1885. EMMERICH.—Deutsch. Med. Woch., No. 50, 1884. ERMENGEM (VAN).—Recherches sur le Microbe du Choléra Asiatique. Paris, Bruxelles, 1885. FINKLER UND Prior.—Deutsch. Med. Woch., No. 36, 1884; Centralbl. f. Allg. Gesundheitspflege, Bd. 1, Heft 5, u 6. *FLUEGGE.—Deutsch. Med. Woch. Jan., 1885. GarrKy.—Arbeiten a. d. k. Gesundh, Bd. u., 1 2 Heft, p. 39, 1884. GaMALEIA.—Annales de |’ Institut Pasteur, t. 1, 1888, t. m1, 1889. Haiier.—Das Cholera Contagium. Bot. Unters., Aerzten u. Naturforschern. Mitgetheilt. Leipzig, 1867. Huerpe.—Berlin Klin. Woch., Nos. 46-47, 1889. Kuess.—Ueber Cholera Asiatica. Basel, 1885. KLEIN AND H. Gispes.—Rep. English Cholera Commission. 1885. 7 KLEIN.—The Bacteria in Asiatic Cholera. London and New York, 1889. Kios.—Path-anat. Studien u. d. Wesen des Cholera Pro- cesses. Leipzig, 1867. Kocu.—Deutsch. Med. Woch., No. 45, 1884; Berlin Klin. Woch., No. 374, 1885. Lewis.—Med. Times and Gazette. Sept. 20, 1884. Mac gop AND Miies.—Lab. Reports, R.C.P., Edin., vol.1., p. 161; Proc. Roy. Soc., vol. xv1., p. 18. MacnamarRA.—2&rit. Med. Journ., vol. 1., p. 502, 1884. MiLterR.—Deutsch Med. Woch., No. 9, 1885. NicaTi uND RietscH.—Comptes rendus, t. xcix., pp. 928- 929, 1884. Pacini.—Arch. de Med. Militaire de Bruxelles, 1855. PETTENKOFER.—Zum Gegenwartigen Stand der Cholera- frage. Munich, see Lancet, July 3 and 1oth, 1886. PreirFeR.—Deutsch. Med. Woch. 1885, 1886. PoucHEet.—Comptes rendus., t. xxvu., p. 555. April 23, 1849. Roy, Graig BROWN AND SHERRINGTON.—Proc. Roy. Soc. No. 247, p. 173, 1887. ScHOTTELIUS—Deutsch. Med. Woch., No. 14, 1888. CHOLERA. 193 Straus, Roux, Nocarp anp THUILLIER.—Comptes rendus de la Soc. de Biologie. Paris, t. 1v., 1883. SwaineE.— Lancet, pp. 368, 398, 1849. VILLIERS.—Comptes rendus, t. c., p. 91, 1885. WEISSERZUG, FRANK.—Zeitschr f. Hygiene. 1886. WoopHEAD AND Woop.—Edin. Med. Journ. April, 1890. * Full lists of references given. 14 CHAPTER X. TYPHOID FEVER. Typhoid Fever a Bacterial Disease — Recklinghausen’s Observations— Klein—Eberth—Klebs—Coats—The Bacillus—Method of Staining— Position in Tissues—Gaffky’s Observations—Pure Cultures—Excretory Products—Experiments on Animals—Mixed Infections--Action of Light and Heat on Typhoid Bacilli—Pseudo-typhoid Bacilli. From the curious nature of the symptoms of typhoid fever, and from the fact that after complete recovery from an attack there appears to be a certain immunity (for a certain period at any rate) against a second, although relapses are of comparatively frequent occurrence, it was early supposed to be the result of the presence of some specific micro-organ- ism within the body, probably in the deeper tissues of the wall of certain parts of the intestinal canal, and in those organs, such as the spleen and lymphatic glands, that are specially connected or associated with that canal. Although, as early as 1871, Recklinghausen described in abscesses that were formed during the course of an attack of typhoid fever, microbes which he considered to be specific, and although Klein in this country found several varieties of micro- organisms in typhoid lesions, the specific organism was first accurately described, and distinguished from others, by Eberth and Klebs abroad and by Coats in this country, all of whom give very exact descriptions of the typhoid bacillus, These bacilli are short, somewhat thick rods, about 2 to 3# in length and .3 to .5# in breadth ; they are usually distinctly rounded at the ends, where the protoplasm is always rather more deeply coloured by aniline dyes than the central portion, which was at one time supposed to be a spore, though more recently this lighter coloured portion has been looked upon as evidence of a process of degeneration. These bacilli are said to be stained with difficulty, but I have found that if the sections in which they are present are first TYPHOID FEVER. 195 allowed to remain for about ten minutes in a 1-5th per cent. solution of corrosive sublimate and then stained by Gram’s method, the bacilli are most deeply stained, although Fraenkel and others state that the colour is invariably discharged if Gram’s method be used. They may also be prepared by Kihne’s method of first allowing them to remain in a con- centrated watery solution of oxalic acid, washing them carefully and afterwards staining with methyl blue dissolved in a 1 per cent. solution of ammonium carbonate. Sections may also be stained for twenty-four hours in L6offler’s alkaline methylene blue, after which they are rinsed in water, which removes sufficient of the colour ; the water is driven out with aniline oil, the sections are allowed to dry on the slide and mounted in Xylol Balsam. The bacilli are found in the adenoid follicles, or lymphatic tissue of the intestine, in the mesenteric glands, in the spleen, and in the liver, and more rarely in the kidneys. They are usually collected in little clumps, and single bacilli are seldom if ever met with. These clumps, although readily enough recognized when seen, are as a rule so sparsely scattered through the tissues that it is often a difficult matter to find them, even in characteristic cases, and as Fliigge says, “ It is only after the examination of a large number of sections that one or several of these deposits can be found.” Gaffky, working in the Hygienic Institute in Berlin, was first able to make pure cultivations of this bacillus, and in 1884 he gave a very complete description of the bacilli that he was able to examine or to cultivate in twenty out of twenty-two cases of typhoid fever of which the examination was committed to his charge. The bacilli, when obtained pure, and cultivated in fluid, grew out into very long.threads, both threads and short bacilli apparently being motile, having a peculiar wavy motion ; quite recently this motion has been found to be due to the presence of groups of lateral flagella which, waving back- wards and forwards, impart to the organism its peculiar snake-like movement. The bacillus can grow perfectly well both in the presence of free oxygen and also when oxygen is cut off, but, as in the case of the cholera organism, it appears to-have somewhat different functions and different powers unde¢r the two sets of conditions ; outside’ the body in the presence of oxygen it. appears, to. develop. great “ resistant ”’ power and a saprophytic habit, whilst in the anzrobic con- 196 BACTERIA. dition, especially in the intestine, although its power of breaking up the albuminoid substances presented to it and of developing its specific toxines is greatly increased, its capability of resisting antiseptic substances is considerably diminished. In plate cultivations made from the organs of typhoid patients, Gaffky found that the bacilli developed in the deeper part of the gelatine as small white points, whilst Photo-micrograph of Typhoid bacilli in Lymph follicle of Intestine of a child in which the Typhoid lesions were very characteristic. x 500. on the surface they grow as moist-looking greyish colonies with irregular margins. Under a low magnification the small rounded points are seen to be slightly granular ; they have a sharply-defined margin and are of a dirty yellow colour ; the superficial growths, although spreading somewhat rapidly, are thin, sometimes almost transparent, and have a yellowish tinge when seen in the sunlight ; the margin is TYPHOID FEVER. 197 irregular and is marked by large and small indentations. There is no liquefaction of the gelatine around the growth. In a test tube cultivation the growth appears all along the line of the puncture and also on the surface. The surface culture has a peculiar mother-of-pearl look, it gradually spreads over the whole of the gelatine, forming a kind of bluish-grey film, whilst down each side of the needle track there is a delicate zone of the same bluish-grey colour, surrounded in turn by a peculiar opalescent milkiness. The most characteristic growth, however, occurs on sterilized potatoes. It is characteristic in that, even when there is a most luxuriant growth of the typhoid bacillus, it cannot be recognized by the naked eye, even at the end of three or four days, except by a peculiar moist appearance of the potato, which, taken along with the appearances in milk and on gelatine so far as is at present known, distinguishes the growth of this organism from all others. It will be remembered, however, that the potato is slightly acid, and it appears that this acidity is necessary for this typical growth, for on potatoes rendered slightly alkaline there appears a yellowish or dirty grey growth with sharply defined margins —a growth quite different from that above described. Chantemesse and Widal utilized the power of the micro- organism to grow in acid to help them to obtain pure cultivations. They prepared special tubes of gelatine by adding to each 10 cc. of the nutrient medium, 4 or 5 drops of 1 to 20 per cent. solution of carbolic or phenic acid ; they did this in order to prevent the development of those microbes that bring about liquefaction of gelatine. They were perfectly successful in their attempts, although, as we have seen elsewhere, it does not follow that their expla- nation was absolutely correct ; at any rate they were able to separate pure typhoid bacilli which had all the characteristic appearances both on the gelatine plates and under the microscope. The organism itself, unlike many other bacteria, seems to form an acid and not an alkaline excretory product. It grows in a variety of media, and as I have already said, it appears under certain conditions to become quite saprophytic in habit, this being evidenced by the ease with which it may be cultivated outside the body, and quite recently Wolffhigel found that it may develop readily in both milk and water to 198 BACTERIA, which it has gained access. This bacillus, then, has been found in the body ; it has also been found by Pfeiffer in the dejecta of typhoid patients, and now it has been found in the water supply, so that the chain of evidence is, so far, pretty conclusive. It is a somewhat important fact that the typhoid bacilli may remain active for a considerable length of time in the stools, for the bacilli in feces kept in a sterilized tube for fifteen days remain alive at the end of that period, and vigorous cultivations could be made from such material. Experimenters were now. confronted with the difficulty that typhoid fever is seldom met with except in the human subject. Experiments were made on animals by injecting cultures of the typhoid bacillus into the aural vein of rabbits, with the result that about 5o per cent. of the animals died, and on post-mortem examination it was found that the spleen and those glands usually affected in typhoid were somewhat swollen. A. Fraenkel was able to kill monkeys with in- jections of the bacillus, and Chantemesse and Widal point out that they can produce a septicemia by injecting a con- siderable quantity of a culture of the bacillus into the peritoneal cavity of a mouse. They also found, repeating Fraenkel and Simmonds’ experiments of injecting cultiva- tions into the vein of the ear, that this was followed by diarrhoea and rapid emaciation at the end of several days, although the animals frequently recovered. When they were killed at the height of the disease, lesions corresponding to those met with in typhoid fever were found in the intes- tine, and bacilli were also found in the organs. The method that was used in the inoculation of cholera was afterwards resorted to, the contents of the stomach were rendered alkaline, the peristaltic movement of the intestine was paralyzed by means of full doses of opium, and the typhoid bacilli were injected into the alimentary canal ; most of the animals died, and numerous bacilli were found in the intestines and even in the glands, but none could be demonstrated in the blood. It was observed, however, that it was not neces- sary to have an active bacillus present in order to cause very serious symptoms, and even death, with typhoid cultivations ; and it soon came to be recognized that these symptoms were due to regular poisoning or intoxication by toxines and tox- albumens, both of which were described by Brieger as being present in typhoid discharges and in pure cultures of the TYPHOID FEVER. 199 typhoid bacillus. It was in typhoid fever, in fact, that this investigator carried on some of his earliest experiments on the poisonous metabolic products of pathogenic organisms, growing in albuminoid substances. As regards the relation of | this special bacillus to the disease, it stands on exactly the same footing as that between the cholera organism and cholera, and it follows that most of the points that have been accentuated when we were considering cholera may also be accentuated in this instance. Klein pointed out that in typhoid lesions, especially in the intestinal canal, several organisms were usually associated, and other observers have agreed that in typhoid there is, very frequently, what is known as a “mixed infection _—z\e., in addition to typhoid bacillus other organisms appear to be present and to play an important part. Streptococci, and septic organisms, are frequently found in the tissue of the spleen, liver, and wall of the intes- tine. It is supposed that some of these organisms play their part in preparing the intestine for the reception of the typhoid bacillus, and it is maintained that a condition of irritation and a removal of the epithelium, brought about by the action of other micro-organisms on the wall of the intestine, may be necessary to prepare the way for the entrance of the typhoid bacillus. The intestine is, in fact, pre- pared just as a field is prepared by the farmer by ploughing and manuring for the reception of the seed that he intends to sow. The fact that the bacilli can grow on potatoes without becoming evident to the naked eye indicates the possibility of a similar growth occurring on other articles of diet, which, taken into an alimentary canal that has been previously prepared by gastro-intestinal disturbances—diarrhcea and similar conditions—may set up the disease. It may be appropriate here to consider the action of light on typhoid bacilli, as although the first observations on the germicidal action of light were made on other organisms, in this country by Downes and Blunt, and these experiments were continued by Tyndall and a number of other workers both in this country and abroad, the more recent experiments on the action of light on bacteria have been carried out on typhoid bacilli. That bacteria are influenced by the action of light either to their advantage or their harm is very evident. In the one case it will be found that certain of the colour-producing organisms cannot exert this function 200 BACTERIA. unless they are very well supplied with both air and light, whilst on the other hand such organisms as usually grow in the body appear to become markedly weaker as regards their power of growing and of giving rise to their special dele- terious products if they are freely exposed to the light. Recently Dr. Janowski has made a number of experiments by exposing growths of the typhoid bacillus to the action of light, and has found that it exerts a distinctly depressing action on the typhoid organism, an action entirely inde- pendent of any oxidation of the food material that might occur under the action of the chemical rays, these chemical tays acting directly upon the protoplasm and rendering it incapable not only of further development but of continuing alive. In order to prove his thesis he took a gelatine tube in which typhoid bacilli had been sown and exposed it to the action of the light on a cold winter day; a similar tube inoculated with the same bacillus was wrapped up in a layer of black paper and then in one of white paper, this also was exposed in the same position. The light in this case delayed the development and the multiplication of the organism in a somewhat marked manner, as in the two pro- tected from the light, growth took place in three days, whilst in that exposed to the light, it did not commence for five days. Of course the growth here referred to is measured by the size of the colony that can be seen with the naked eye and although both were probably growing during the whole time, the rate of multiplication in the one was very consider- ably greater than in the other. In order that there might be no doubt as to the identity of the organism and the quantity sown in each tube, a U-shaped tube (a double Pasteur tube) was taken and the inoculation was made; the fluid was thoroughly mixed by passing from one limb of the tube to the other, then one limb was protected as above and the other was exposed to the light ; similar results were obtained, the bacilli in the limb that was exposed to the light being considerably delayed in their development. He found that direct sunlight acting on fluid cultures of the typhoid bacillus kills the organisms in the short space of from four to seven ‘hours, but diffused light requires a considerably longer period to entirely arrest the development and multiplication of the organisms. Instead of analysing the rays of light by means of a prism, as Englemann and others had done, qeaowikl ' TYPHOID FEVER. 201 made use of solutions of alum, bichromate of potash, Bismarck brown, fuchsine, methyl blue, gentian violet, &c. He found that the yellow and brown solutions filtered out the chemical rays and prevented the action of light upon the organisms almost as efficaciously as the black and white papers, but found that the other fluids—fuchsine, methyl blue, gentian violet, &c.—had little more effect in preventing the injurious action of light on the bacilli than distilled water or alum solution. He therefore comes to the con- clusion that the hurtful action of both diffused light and direct sunlight is due in very great measure to the chemical rays of the solar ‘spectrum—the rays at the other end of the spectrum exerting comparatively little influence on the organism. This entirely accords with Englemann’s observa- tions ; he found that certain chromogenic bacteria when examined in a drop of water and illuminated by the rays from a micro-spectral objective, invariably made their way to that part of the spectrum furthest away from the violet end, thus indicating that they were attempting to evade the chemical rays which appeared to be hurtful tothem. This question of the action of light, especially on pathogenic bacteria, is one of very great importance, and Duclaux’s dictum that fresh air and sunlight are two of the most powerful agents that we have with which to combat the onslaught of the bacteria of disease cannot be too strongly insisted upon. Bacteria, especially those of disease, seek out the dark places for their habitation, and as the exclusion of light to a very great extent necessitates the exclusion of fresh air, they find in these holes and corners places of rest whence they may go out to do all the harm of which they are capable. Janowski also made an elaborate series of experiments on the effect of high temperature on the typhoid bacillus. He found that a temperature of 55° C. continued for ten minutes was quite sufficient to render sterile cultivations of this bacillus, but if this same temperature were continued for only five minutes he could not rely upon obtaining complete destruction of the organism. He also came to the conclusion that an extreme degree of cold, especially when continued for some time or where frequently repeated, had a moderately injurious effect upon the vitality of the typhoid bacillus ; a temperature of 14° C. being sufficient to kill the bacilli when 202 BACTERIA. growing in a fluid medium. When they were allowed to dry, however, this did not appear to hold good to nearly the same degree. These experiments are interesting in their bearing on the outbreaks of typhoid fever at certain parts of the year, especially in countries where the cold appears to be exceed- ingly intense, and where one would naturally expect the development of the bacilli to be interfered with, but where as a matter of fact such is found not to be the case. Quite recently Cassedebat, examining the drinking water supplied to Marseilles, which is a very hotbed of typhoid fever, was not able to find the characteristic bacillus in any one of 250 cultivations made of seventy specimens of water, but curiously enough he found three other bacilli which in many respects resembled the true typhoid bacillus most remark- ably, although they differed in certain essential character- ‘istics. He points out that they all grow in the phenic acid gelatine, and he further states that several other organisms offer quite as great resistance to this acid as the typhoid bacillus itself. They all present clear spaces or deeply stained masses of protoplasm which may easily be mistaken for spores, but these, like those in the true typhoid bacillus in which as we have seen similar bodies occur, are all killed at a tempera- ture of a little over 45°C. The pseudo-bacilli are very im- ‘perfectly stained by Gram’s method. They exhibit a lateral and oscillatory motion as well as a forward motion. The plate cultivations are so much alike, that unless all four can be examined simultaneously, it is a very difficult matter to distinguish one from the other ; their growths on potatoes, in broth and in milk resemble one another in a most re- markable manner, except that they develop with different degrees of rapidity, and vary somewhat as regards the alkali- nity and acidity of their products at the end of about thirty days, and also as to the degree and time of appearance of turbidity that is produced when these organisms grow in broth. In consequence of these slight differences the use of the various aniline staining reagents, added to such culture media as broth or milk in which the colours undergo changes under the different reactions, has been resorted to and described by Cassedebat, who was able by their use to distinguish one organism from the other. The ordinary cultivation methods are quite sufficient to distinguish these TYPHOID FEVER. 203 four forms as a group from most others, for which the typhoid bacillus itself has at different times been mistaken, whilst in addition to the differences above mentioned none of the pseudo forms are quite so toxic to white mice as the Eberth-Gaffky bacillus, and one of them is quite innocuous. Although Cassedebat was not able to find the true form in water taken from a supply that was open to contamina- tion, he found that this was not because the bacilli could not live in water, as in distilled water to which a cultivation was purposely added he could easily distinguish its presence at the end of forty-four days, and when added along with half a-dozen other forms he still found it living and active at the end of seventeen days. As the result of his observations he comes to the conclusion that the true typhoid bacillus does not occur in water so frequently as is sometimes represented, and that one or other of the pseudo-typhoid bacilli has in certain cases been mistaken for it. LITERATURE. Authors already referred to. Brieger, Fligge, Fraenkel. Atmguist.—Typhoid feberns bakterie. Stockholm, 1882. Baxses.—Journ. de l’Anatomie, p. 39, Jan., 1884. BEUMER UND PerpeR.—Zeitschr f. Hygiene, p. 489, 1886. Birco-HirscHFELD.—Zeitschrift f. Epidemiologie, Bd. 1, 1874. Hanes GND Exrucu.—Berlin Klin. Woch., No. 44, p. 661, 1882. CassEDEBAT.—Annal. de l'Institut Pasteur. Oct. 25, 1890. CHANTEMESSE ET WipaL.—Arch. de Physiol, p. 217, 1887 ; Annal. de l'Institut Pasteur, t. 1, p. 54, 1888 Coats.— Brit. Med. Journ., vol. L, p. 421, 1882. Crooxe.— Brit. Med. Journ., vol. u., p. 15, 1882. Downes anp BLunt.—Proc. Roy. Soc., vol. xxvr., No. 184, Dec. 6, 1877. Downgs, BLUNT, AND TYNDALL.—Proc. Roy. Soc., vol. xXVIII., No. 191, Dec, 19, 1878. Exsertu.—Virch. Arch., Bd. -xxxt, p. 58, 1880; and Bd. LXXXIIL, p. 486, 1881 ; Volkmann, Klin. Vortrage, 1883. FRAENKEL UND Simmonps.—Centralbl. f. Klin. Med. p. 737, Oct. 31, 1885; Der aetiologische Bedeutung des Typhus Bacillus, 1886. 204 BACTERIA. GAFFKY.—Mitth. a. d. Gesundheitsamt, Bd. 11, p. 372, 1884. JAmMIESON.—Vature, vol. xxtv., No. 620, Sept. 15, 1881. Janowski.—Centralbl. f. Bakt. u. Parasitenk., Bd. vut., Nos. 6-9, 1890. Kuess.—Arch. f. Exp: Path., Bd. xim., p. 381, 1880. K EIn.—Reports of the Med. Officer of the Local Gov. Board, 1874. Kitne.—Zeitschr. f. Hygiene, Bd. 1, p. 553, 1886. Lérrier.—Centralbl. f. Bakt. u. Parasitenk, Bd. v1, Nos, 8-9, and Bd. vi., No. 20, 1890. PFEIFFER.— Deutsch. Med. Woch., No. 29, 1885. RECKLINGHAUSEN.—Wurtzburger Zeit., June 10, 1871. SIROTININ.—Zeitschr f. Hygiene. 1886. SocoLorr.—Virch. Arch., Bd. txvi. CHAPTER XI. TUBERCULOSIS. Tuberculosis a widespread Disease—The Tubercle Bacillus—Koch—Baum- garten—Spores seen by Watson Cheyne—Relation of Organisms to Tissues—Bacillus in Tuberculosis of Animals—Tubercle Bacilli as Saprophytes—Bacilli cultivated outside the Body by Koch—Methods —Temperature Relations—Cultivation on Different Media—Channels of Infection—Ransome— Williams—Cornet—Conditions of Infection— Methods of Disinfection—Tuberculosis at Different Ages—Tubercle in Milk—Diagnosis of Tuberculosis in Cattleh—Tuberculous Meat— Koch's Method of Treatment—Nature of Virus and Mode of Action Is Immunity conferred?—Koch’s Method a New Departure—Sterile ‘Products cause Marasmus Maffucci—Indications for Treatment. TUBERCULOSIS, one of the most widespread and deadly diseases with which we have to deal, not in this country only, but in the whole of Northern Europe, has now certainly been proved to demonstration to be due to the presence of a specific micro-organism. Almost innumerable researches have been carried on with the object of finding out and piecing to- gether the various facts in the life-history of this organism, the products to which it gives rise, the conditions under which it can multiply in the human body and in the animal body, the nature of the very grave changes produced in the tissues, the mode of transmission directly and indirectly from one body to another, and, last, but not least, the possibility of combating the ravages made on the body by this organism, by interfering with its growth or retarding its development, either outside the body or after its introduction into the tissues. Tuberculosis and Phthisis or Consumption account for such an enormous percentage of deaths in our colder north- ern latitudes, that the subject has come to be one of intense interest, not only to physicians and surgeons, but to all well- educated people, and the subject of the treatment of tuber- culosis is—unfortunately perhaps for patients—taken up 206 BACTERIA, almost as exhaustively in the daily papers as it is in special treatises and in the medical journals. It has long been known that tuberculosis was an inoculable disease, but it was only quite recently (1883) that Koch and his pupils were able to demonstrate that a specific organism could be separated from tuberculous tissue and cultivated outside the body—the cultivated organism having all the characters of the organism found in the tissues—and that when introduced into certain animals, this organism was capable of producing tubercular disease, the organism in turn being again demonstrable in the new tubercular growth. It was for long found to be an exceedingly difficult matter to demonstrate any specific micro-organisms in tubercular tissues by means of aniline or other nuclear stains and Baumgarten’s method * was introduced after he had failed to attain his object by any of the ordinary methods. The difficulty he had, however, was that, although the tubercle bacilli un- doubtedly resisted the potash solution, other organisms were also more resistant than were the animal tissues, so that there was no great differen- tiation except in size and form between the tubercle bacillus and other bacteria. While Baumgarten was working out his method, Koch had completed a series of investigations, the outcome of which was that he proved that by tlie addition of a small quantity of an alkali to the aniline stain the dye was rendered capable of penetrating the resistant outer membrane of the tubercle bacillus. It was afterwards found that aniline, thymol,. turpentine, or carbolic acid, added to the stain, bring about the same results; these substances, acting, apparently, as mordants on the tissues. The next step in the process of demonstration of the tubercle bacillus was taken when it was found that this organism differed from others in the fact that it retained the staining reagent most tenaciously, even the strong mineral acids, which readily discharge the colouring-matter from nuclei and other bacteria having little effect, if acting for a short time only, in taking out the stain from tubercle bacilli; in sections stained in an aniline colour mixed with one of these substances and then treated with an acid, a most beautiful differential staining was obtained, the stained bacilli standing out most prominently frdm the unstained tissues. Tubercle bacilli when stained are seen as delicate rods or threads 1.5 to 3.5#in length and about .2# in thickness, though these dimensions are by no means constant even in the same preparation, and under different conditions the variations in size are sometimes very marked. As in the case of anthrax and cholera bacilli, the methods of staining and preparation exert a marked influence in determining the apparent size * The sputum, after being dried on a cover glass and then heated to coagulate the albumen, was simply soaked in a solution of caustic potash. Sections were treated in the same manner. TUBERCULOSIS. 207 of the organism. The length is sometimes given at 2.6 and the breadth as from .2 to .S. They are usually described as from a quarter to a half the diameter of a red blood corpuscle and longer than the bacilli of mouse septi- cemia, or 0.0015 to 0.0035 m.m.in length. These bacilli always appear to have the larger dimensions when treated by Baumgarten’s liquor potasse method. The bacilli are Photo-micrograph of Sputum, from a phthisical patient, containing large nucleated epithelial cells and characteristic tubercle bacilli. x 1000. usually slightly curved, or two are arranged end to end so as to contain an angle. At first they were described as not containing spores, but it was demonstrated by Watson Cheyne that from two to six spores may frequently be seen in these rods; the spores occurring as small ovoid or rounded clear spaces placed at intervals in the stained thread. In some cases they are so prominent that they appear to 208 BACTERIA. project beyond the straight outline of the bacillus ; sometimes this is so much the case and the spores are packed so closely together, that when examined under a sufficiently high power the spore-bearing thread has been described as a chain of cocci; by some it is maintained that this appearance is present only if the specimen is imperfectly stained, too much heated, or too long treated with a strong acid ; whilst, on the other hand, certain observers assert that the tubercle bacilli also occur in the form of regular chains of cocci. The bacillus is non-motile. Fliigge holds, however, that it is ‘always possible, in carefully prepared specimens and with the aid of good lenses, to convince one’s self that the pecs chain of cocci does not exist, but that the delicate contour of the bacillus can be for the most part traced through its whole length, and that it is only within this contour that the alternation of stained and unstained zones gives the deceptive appearance of stained cocci separated by narrow intermittent spaces.” The association of this organism with tubercular disease is undoubted ; it is found in the lungs and sputum in various forms of consumption, it is found also in tubercular ulcers of the intestine, around the vessels in tubercular inflamma- tion of the membranes of the brain, a condition which occurs frequently in children, in tubercle of the liver and of all other organs of the body, and in tubercular eruptions of the skin such as lupus. In all these cases the bacilli are found most abundantly at those points where the disease appears to be spreading into the surrounding tissues, and especially where there is the formation of large multi- nucleated epithelioid cells. If these bacilli are present in considerable numbers in such an area, there will usually be found in the immediate neighbourhood a small portion of tissue that has undergone marked degenerative changes, the cell protoplasm is somewhat hyaline or glassy looking, and takes on any staining reagent, except perhaps picric acid, very badly; the nucleus is also considerably altered, especially in that it no longer stains, or is stained very imperfectly, with carmine or the aniline dyes. At such a stage there can frequently be demonstrated, in these cells, imperfectly stained tubercle bacilli, though in some cases the bacilli stand out sharply and very brilliantly from the glassy or homogeneous looking cell. After a time the cells lose their outlines; they become more and more TUBERCULOSIS. 209 indistinct, until eventually nothing but the ghost of a cell is left. This occurs where we have what is known as the cheesy or caseous degeneration, in which condition nothing but broken-down nuclei and very rarely a tubercle bacillus can be distinguished. It would at first sight appear as though the tubercle bacilli had disappeared, root and branch, from this caseous centre, but if a small portion of the cheesy material be inoculated into an animal susceptible to tubercle, flourishing groups.of tubercle nodules all con- taining tubercle bacilli are produced, first near the seat of inoculation, and then at points situated at some distance from the point of primary infection. The results of such experi- ments naturally led the opponents of the bacillary theory of tubercle to assume that the poison of tubercular virus was not associated with the bacillus, which they contended was merely of accidental or sequential and not of causal occurrence, as it could not be found in material which undoubtedly produced the disease ; but after the demon- stration of spores in the bacilli, and bearing in mind what was known of spore formation in other organisms, most observers were very naturally led to the conclusion that, although the bacilli are not to be demonstrated in these infective caseous masses, spores in enormous numbers are probably present, and that from these, bacilli are developed as soon as the sur- rounding conditions of nutrition, moisture, and heat are again sufficiently favourable. The fact that it is so difficult to stain spores made it no easy task to demonstrate the accuracy of this theory, but it may now be held, as the out- come of inoculation and other experiments, that there can be no reasonable doubt on the subject. It is a curious fact that whilst in the human subject tubercle bacilli appear in many cases to be actually contained within the giant or other large cells, in some herbivora, and in fowls, the bacilli sometimes occur single or in small masses, apparently outside the cells. There are several explanations given for this, but the most rational appears to be that where the bacilli are few in number, and where they are being rapidly destroyed by the tissue cells, by far the larger proportion are taken up by, and are seen in, these cells; this being specially observable in cases of chronic tuberculosis, whilst in those cases where the tubercle bacilli are relatively numerous, as in cattle, and even more markedly in the fowl and in the horse, the 15 210 BACTERIA. destruction of the tissue cells takes place so rapidly, in con- sequence of the invasion of each cell by a large number of bacilli, that nucleus and protoplasm break down com- pletely and the little group of bacilli is set free, or at any rate is not embedded in a mass of protoplasm, but is merely mixed up with the granular débrzs of the cell, from which, Micro-photograph of Tubercle bacilli, found in the scraping from the lung a cow suffering from Perlsucht. x x000. or along with which, it may be carried to other parts of the body. , Having found the tubercle bacillus almost invariably accom- panying tuberculous disease, Koch, to complete his proof, wished to separate the organism in the form of what is called a pure cultivation, in order that he might study its life-history, and that he might determine whether the organism when introduced alone into the animal body, TUBERCULOSIS. 2it cculd give rise to tuberculosis. With all the ordinary nutrient media then at his command he entirely failed to obtain any growth of the organism outside the animals in which it led its parasitic life. It depended so much upon these conditions of parasitic life that when removed from them it was no longer able to grow and multiply : it might still remain alive; in fact, Cornil was able to demonstrate that at the ordinary temperature of the room the tubercle bacillus, kept in ordinary Seine water, continued to exist, but not to multiply, for seventy days. At length, however, Koch overcame the difficulties with which he had to contend, in a most ingenious manner, and he succeeded in growing as a saprophyte what had hitherto been demonstrated only as a parasitic organism. He argued that as the tubercular process developed but slowly, he would have to obtain a medium which would remain unaltered for a considerable length of time when placed in a temperature at which the organism could grow and multiply. He was satisfied too, from his early experiments, that only special substances would serve as nutrient material for this fastidious organism, and he ultimately found that solidified blood serum was by far the best medium on which to cultivate it, as it alone of the many substances which he had then tried supplied all the requirements of the organism. This blood serum contains all the elements necessary for the nourishment of the organism ; it remains solid at the normal temperature of the body, at which temperature it may be kept for a long enough time to allow of the development of the slowly growing bacillus, whilst a small amount of water might be left in the test tube along with the serum without dis- solving it, thus serving to supply the moisture requisite for the perfect growth of the bacillus. In consequence of - the slowness of the growth above referred to, it is an ex- ceedingly difficult matter to obtain pure cultivations of the tubercle bacillus should it once become mixed with putre- factive bacteria, and it was for long deemed almost an impossibility to separate it from these other forms: this difficulty has now, however, been overcome. Most of Koch’s earlier pure cultivations were obtained by taking as seed material, tubercular lymphatic glands from freshly-killed guinea pigs, which had been inoculated, some three or four weeks before, with tuberculous material. 212 BACTERIA. As this method may be looked upon as almost classical it will be well to give the description in Koch’s own words. ‘ A number of knives, scissors and forceps are heated in a flame sufficiently to free them from any adherent bacteria. They are then laid ready in such a manner that no further con- tamination of the instruments can take place. Meanwhile the animal immediately after being killed is fastened to a dissecting board. In order to avoid the flying off of particles of dirt, hairs, &c., when the skin is incised, the fur of the animal is freely moistened with a 1 to 1000th solution of corrosive sublimate. With a pair of scissors and forceps, both still hot, the skin is now divided and turned back on each side sufficiently to free the lymphatic glands of the axilla and groin ; but the glands, if they are to be used to start cultivations, must not be touched with the instruments em- ployed for cutting through the skin. With another pair of scissors, also heated, a piece I to 2 c.c. cube is cut out of the side wall of the thorax, and the surface of the lung laid bare. A number of tubercular nodules are thus rendered accessible, and a few are removed as quickly as possible with fresh instruments, which must, however, be cooled for this purpose. In order to set free the bacilli contained in the nodules, the latter are cut in pieces or crushed with the scissors, or, better still, between two scalpels that have just been heated and allowed to cool. The substance thus subdivided and crushed is removed by means of a platinum wire fused into a glass rod (which, immediately before use, has been heated and allowed to cool), introduced into the test tube, spread out on the surface and well rubbed about. During this operation the test tube must be held obliquely or almost horizontally between the thumb and forefinger, and the cotton wool plug held meantime between the other fingers of the same hand in such a way that no contamination of it by other objects can take place. The transference of the substance into the solidified serum, which may for brevity be designated inoculation, must take place as quickly as possible in order that no germs of extraneous organisms from the air may alight on the inoculation material or enter the test tube. It is desirable also to conduct the experiment in a room where no dust is flying about, and in the same way all unnecessary movements by which dust from the clothing, &c., is mingled with the air are to be avoided, as experience has shown that it is to particles of dust that the germs of micro-organisms suspended in the air adhere. “ In spite of all these precautions we cannot be perfectly sure of preventing the entrance of a few solitary germs, and it is necessary in each case to inoculate several (five to ten) test tubes, so that if we fail to obtain a pure cultivation in one or two tubes, we shall yet have others that are free from impurity. af **The process is the same as that above described for obtaining seed from a pulmonary tubercle, when lymphatic glands, tubercles from the spleen, &c., are to be used to start a culture. The process must always be carried out with heated instruments, which must be changed every time a fresh stratum is laid bare. All preparatory incisions which do not come in contact with the inoculation substance itself are to be made with hot instruments, but the inoculation material is to be cut out with a cool pair of scissors and forceps. It is necessary to change the instruments constantly in order that impurities adhering to them after the division of the skin and superficial layers, may not be carried into the cultures. ‘ ** When the organs of a recently killed or dead animal could be obtained, TUBERCULOSIS. 213 and the inoculation with substances containing tubercle bacilli was done in the way just given, I invarjably succeeded in obtaining pure cultures. The result was uncertain, on the contrary, when material from human corpses or from cattle with ser/sucht was used, as it was always impure on the surface, and, moreover, was not always quite fresh when it reached me. In these cases I first rinsed the surface of the object repeatedly with a solution of corrosive sublimate (1 to 1000) and then cut away the upper parts in layers with red-hot instruments, which were changed repeatedly ; finally, I took the material for inoculation from a depth which justified me in concluding that it would be free from the bacteria which had entered the tissue after the death of the animal. In this way I generally succeeded in obtaining pure cultures even from this kind of material, particularly from small superficial pulmonary cavities, the outer wall of which, after treatment with solution of corrosive sublimate, was removed with hot instruments. “After the inoculation of the solidified serum with material containing bacilli has been accomplished, the vessels are placed in the incubator and kept constantly at a temperature of about 37°C. Every incubator is not suitable for the culture of tubercle bacilli. Growth takes place but very slowly, and the vessels must therefore remain in the incubator for weeks. So that if the incubator is so constructed as to favour rapid evaporation of liquids from the culture vessels, the serum gets dry before visible colonies of tubercle bacilli have developed. For example, an apparatus cannot be used in which the heat is unequally distributed, so that the vapour constantly present condenses in the cooler parts, ¢.g., on the glass cover, and has to be continually replaced by moisture given off from the culture glasses. D’Ar- sonval’s thermostat is very convenient ; the warmth is equally distributed in it, and the blood serum remains almost unchanged. ‘For the first few days no alteration is to be observed in the cultures in the incubator. If, however, there is a change, and drops or spots of white or other colour form on the surface of the serum, increase more or less rapidly in size, render the fluid at the bottom of the glass turbid or cause the serum to liquefy, it is a sign that the culture is not pure, and that foreign bacteria have entered and choked the growth of the tubercle bacilli. If these drops or spots are examined they are found to consist of bacilli or micrococci which, by Ehrlich’s method of staining, assume a different colour from the tubercle bacilli, and are distinct from them also in size and shape. In the tubes free from these impurities, the first signs of the growing colonies of tubercle bacilli are not visible to the naked eye for ten to fifteen days. They then appear as whitish points and small spots lying on the surface of the serum; they have no lustre, and consequently stand out clearly from their moist surroundings. They are best compared to tiny dry scales adhering loosely to the surface of the serum. The number of the scales and the extent of surface covered by them vary with the richness of the infecting material in bacilli, and with the extent of the surface over which it was rubbed or spread out. “The individual scales attain only a limited size, so that if few are present they remain distinct ; but when numerous and closely packed, they coalesce finally and form a very thin, greyish-white, lustreless covering on the serum. After a fragment of the tubercular lung of a guinea-pig has been rubbed on serum, small whitish colonies of tubercle bacilli appear close to the greyish- red bit of lung, and also in its neighbourhood wherever it has been pushed over or pressed on to the surface of the serum by means of the platinum 214 BACTERIA. wire used to distribute the bacilli as widely as possible. The colonies in some cases are relatively few in number, because only a few bacilli were present in the pulmonary tubercles, as the examination of sections shows. In other cases the little colonies are much more numerous; in many, as especially after inoculation with the contents of cavities very rich in bacilli, they soon coalesce and form a coherent membranous mass.’ From the cultivations so made other tubes were inoculated by lifting off the small scales with a needle bent at right angles, breaking the scale up somewhat so as to cover the point of the needle with the bacilli, and then drawing it along the surface of the solidified blood serum. At the end of ten or eleven days there became visible to the naked eye, and very much earlier if examined with a lens, a regular thin superficial layer spreading along each side of the track of the needle. This organism does not bring about the slightest liquefaction of the blood serum ; the scales are somewhat dry ; they are exceedingly thin, and spread only over the surface, no growth making its appearance in the deeper layers of the nutrient medium. Tubercle bacilli cultivated in this manner through seventy generations still continue to act on animals in the same way as the original or first culture. Similarly the bacillus grows only on the surface of a fluid, forming avery delicate, thin film, the organism being essentially zrobic in its habit. The film growing on solidified serum is loosely attached to the surface, and any fluid intro- duced, however gently, floats off a considerable portion in larger or smaller flakes, which do not break up, but gradually sink tothe bottom. Koch observed, from the behaviour of the films when growing on the surface of fluids, or broken down and introduced into fluid media, that the tubercle bacilli were non-motile. He described the appearances of these colonies under the microscope, and came to the conclusion that the growth was perfectly characteristic, as compared with any other organisms known up to that time. They have the appearance of lines or short threads thrown into curves like worms, or snakes, or flourishes of a pen, thinner or thicker according to the age of the colony, each thread being com- posed of a large number of individual bacilli, arranged with their axes in the long axis of the thread, running parallel to one another, but with a small space between each, these being apparently occupied by some zooglcea medium. This arrangement can be best brought out by pressing down a TUBERCULOSIS. 215 cover glass on to the surface of a colony as it grows on the serum, and then removing it without sliding it in any way and staining by any of the usually recognized methods. As regards the conditions under which this organism exists, we have already seen that a certain amount of moisture is absolutely necessary for its growth, and Paw- lowsky was able to cultivate it, even on potato, when he took the precaution to keep a considerable quantity of moisture in contact with the growing organism, and watched the potato for a considerable length of time, the growth not being visible to the naked eye for three weeks or a month. Koch’s great difficulty in his earlier experiments was, as we have already seen, to obtain a substance which, in addition to containing all the other elements necessary for the nutrition of the bacillus, would remain sufficiently moist for the requirements of the bacillus when exposed to a pretty high temperature for a considerable length of time. As regards temperature, it was found that, although there was an actual cessation of growth and development below 28° or 29° C., on blood serum the organism might be exposed to very low temperatures for a considerable length of time without losing its power of again becoming active when returned to favourable environments. It grows best at about 37° C., but as soon as the temperature rises beyond 38° C. the development of the bacillus in the cultivation tubes begins to diminish in activity, and at 40° C. (Koch originally gave this limit as 42° C.) it ceases entirely to grow and multiply. It is a remarkable fact that the temperature at which the bacillus develops best is exactly that of the human body (37.8° C.). In the cow and the horse, where the other con- ditions must also be favourable, the temperature is about 38.3° C., in the calf a little over 39° C., whilst in the hen we have the maximum temperature of 40° C. Klein, in a series of experiments reported in 1886, finds that it is possible to inoculate successfully with tubercle taken from the human subject, also that it is possible to inoculate from a guinea-pig to a cow, but when inoculation is made from a cow to a fowl the experiment breaks down, and there is no tubercle produced, so that, not only can one modify the activity, and the power of growth of these bacilli outside the body by altering the temperature at which they grow, but it is also within the range of possibility 216 BACTERIA. to modify the bacilli by introducing them into different animals whose normal temperatures and other general metabolic conditions are different. This modification has more than nominal value, for it has been proved experi- mentally that, although the organisms in human and in bovine tuberculosis are morphologically identical, they are. not absolutely the same in all their vital and pathogenic characteristics. For instance, tubercle bacilli taken from a phthisical patient and introduced into the tissues of a cow will soon set up an acute general tuberculosis, whilst bacilli taken from a case of per/sucht, or ordinary bovine tuber- culosis, almost invariably give rise to the per/sucht form of tuberculous disease, and rarely, or never, to the acute generalized form. It would appear that in these cases the microbe becomes adapted to the special conditions present in each host, and consequently becomes less suited to the con- ditions in others. It might be objected that in the case of the fowl the bacilli are present in enormous numbers, and that, therefore, their action should be more virulent; but, in answer to this, it may be pointed out that increased activity of growth is not necessarily always associated with increased virulence. In illustration of this fact, it may be pointed out that, after many experiments, Nocard and ‘Roux were able to obtain most luxuriant cultivations of the tubercle bacillus on agar-agar or on blood serum, to which 6-8 per cent. of glycerine had been added. The organisms on these media grow so rapidly that they are quite visible at the end of four days, and at the end of twenty days, and not four weeks, as on ordinary blood serum, the growth seems to have reached its maximum, when it appears as a pale grey, thick, mamil- lated or reticulated, mass. In the same way luxuriant growths may be obtained in bouillon to which a similar proportion of glycerine has been added, small opaque flakes or flocculi first making their appearance at the surface, and then sinking to the bottom, where they remain. This growth in glycerine broth may take place at a comparatively low temperature, 18°-20° C., though it then goes on very slowly. [Earlier generations of such cultivations produce typical tubercle nodules that grow with extreme and charac- teristic rapidity when inoculated, but after several generations of such pure cultivations have been made in these glycerine media, the virulence may become distinctly diminished, TUBERCULOSIS, 217 although the growths are as luxuriant as, or are actually more luxuriant than ever. We have in fact a kind of reversion to the saprophytic condition of the culture, a condition accompanied by diminished parasitic virulence. It is possible, therefore, that the higher temperature that is met with in cattle along with other, conditions there present may have a distinct effect in diminishing the virulence of the organism, whilst at the same time it may play an im- portant réle in causing its parasitic and vegetative activity to be increased within the body of these animals, though this is not necessarily accompanied by increased vegetative activity outside the body. As Koch pointed out at the International Medical Congress, of 1890, the tubercle cultures from fowls were quite distinct and could not be passed on as such from animals to animals of different species or by growth at different temperatures, and he con- cludes that although nearly related to the ordinary tubercle bacillus they are specifically distinct. It should be noted, too, that tubercle bacilli grown on glycerine agar are, according to Nocard and Roux, somewhat shorter than the bacilli met with in tubercular sputum, that they also contain numerous ovoid spores, but that otherwise they are exactly like those described by other observers in various animals and on other media. As has been already mentioned, there was necessarily a doubt whether any disease could be tubercular if it was not possible by special methods to demonstrate in histological preparations the presence of the bacillus. This histological demonstration is, however, after all, a clumsy method, and in many cases it has been found possible to obtain demonstrations of the tuberculous nature of a disease by inoculation experiments when the organisms have been so few that they have escaped the notice of the most careful observers. It may therefore be confidently anticipated that more and more proof of the tuberculous nature of lupus, scrofula, cold abscesses, and bone disease will be gradually accumulated. As early as 1843 it had been demonstrated that tubercular material from dead subjects when inoculated into rabbits produced tuberculosis; in 1865 these experiments were repeated and extended by Villemin, and other observers have from time to time confirmed the results that were then obtained. Koch’s observations and experiments have now, 218 BACTERIA however, placed the matter on a much surer footing ; he has, as we have seen, succeeded in separating a specific bacillus from tuberculous material, with which he has been able to produce tuberculosis with the utmost certainty, so that, instead of dealing with comparatively large fragments of diseased tissue, he has worked with particles so small that they can only be distinguished with the aid of the best microscope and the use of special’ methods of preparation, This, of course, has cleared up many points which hitherto have been very obscure, and, most important of all, it has enabled us to determine the channels by which human beings may become affected with the disease, especially in connec- tion with the respiratory passages and by way of the alimentary canal, and through wounds or damaged tissues. It has been demonstrated that in the sputum of patients . suffering from phthisis, in those cases where the softened lung tissue is breaking down and is being expectorated, an enormous number of tubercle bacilli may often be met with, though they may be present in comparatively small num- bers ; so frequently is this the case, that the presence (in different numbers from day to day) of tubercle bacilli in the sputum, or their absence, is relied upon as a diagnostic feature by attention to which the physician is enabled not only to determine the rapidity with which breaking down is going on, but even to obtain very considerable assistance in arriving at a decision as to whether a disease is tubercular or not.* Until quite recently not the slightest attempt has been made either to alae the diffusion of such sputum, or to disinfect it in any way, and it as been said, with some considerable degree of truth, that a very large quantity of virulent tuberculous material has been allowed to be freely dis- seminated, with the result that it must have been the lot of many individuals to contract tuberculosis by means of the inhalation of particles of dried tuberculous sputum in which active tubercle bacilli or their spores were necessarily entangled. That the sputum contained the elements which * In making an examination of sputa for tubercle bacilli, the fact that the bacilli are most numerous in the small yellow caseous points should always be borne in mind. Such points should be carefully searched for, crushed between two cover glasses, dried, stained, and examined. Single bacilli may be found elsewhere, but masses can, as a rule, be found in these disintegrating points only. It is only where disintegration has commenced that any reliance can be placed on the numbers of tubercle bacilli as affording any assistance in forming a diagnosis and prognosis, TUBERCULOSIS, 219 were the exciting causal agents of the disease had been experimentally proved, even before the actual discovery of the bacillus was made, and dogs, which had been in the habit of taking up the sputum of tuberculous _persons, had been known to contract the disease, an observation that was fully corroborated by further experiment. Similarly it had been related how barn door fowls in a country district, which for a long time were perfectly healthy, were suddenly attacked by an outbreak of tuberculosis after a phthisical patient had come to liveat the farm. The expectorations of this patient were voraciously devoured by the fowls, with the result that tuberculosis of a most virulent nature broke out in 2 most extraordinary fashion amongst the brood. Feeding experiments were also made to corroborate this accidental experiment ; and more recently similar acci- dental experiments have been recorded both in France and in this country. In the case of certain micro-organisms the products of putrefaction exert such a deleterious influence on them that they are destroyed very readily and rapidly. Again, in the case of the cholera bacillus, desiccation at once proves fatal, not only to its growth, but also to its actual virulence and power of infection. In the case of the tubercle bacillus, however, observers, both in France and in Germany, very early pointed out that putrefaction and drying could exert but little influence on the number of the bacilli, whilst dry- ing alone interfered only slightly with their virulence, as it was quite easy to inoculate rabbits with sputum that had been dried at a temperature of 30° C. Later, Galtier found that maceration and putrefaction for a period of five days, and even intermittent freezing and melting, did not interfere with the transmission of the disease by means of the bacillus. Other observers have demonstrated that the bacillus remains virulent after it has been exposed,in sputum, for forty days, and even after 186 days if it is carefully protected from the action of the air. It is, of course, concluded from these experiments that, difficult as it is to cultivate the specific micro-organism of tuberculosis outside the body as a saprophyte, the parasitic form (or its spores) still retains its vitality and power of development for a considerable length of time—and under what would appear to be very unfavourable circumstances— even when removed from its host. The way was thus being thoroughly prepared for Dr. Georg Cornet’s researches on infection in hospitals and rooms where phthisical patients were treated. Dr. Ransome had, early in the con- troversy, demonstrated the presence of tubercle bacilli in the air respired by tuberculous patients; and Dr. Williams, in 1883, suspended glass plates smeared with glycerine for a 220 BACTERIA. period of five days in one of the ventilating shafts of the Brompton Hospital for Consumption. Washing off the glycerine “with distilled water, the fluid was mixed with a little mucilage and evaporated down to one-half, and then examined for the bacilli,” and in this he was able to demonstrate bacilli in fair numbers. In a thoroughly purified ward a number of non-phthisical patients were placed, with the result that no bacilli were found in the air carried off by the extraction shaft ; whilst in another ward, filled with consumptive patients, the washings from a plate exposed for fourteen days in the extraction shaft contained numerous bacilli. Similar experiments have been made by other observers, but it was left for Cornet to make a systematic examination of the dust in rooms were phthisical and non-phthisical patients were treated. By numerous careful inoculation experiments, he demonstrated the fact that the expectorations from phthisical patients are a source of a very real and appreci- able danger. The bacilli are not only exhaled, in small numbers no doubt, but he finds that they are also con- tained in very considerable numbers in the dried sputum obtained from handkerchiefs, bed linen used by phthisical patients, and in the sputum that has made its way on to the floor and walls through the dirty habits of many of the patients. His experiments extended over a very considerable period, and to the rooms of private patients in hospitals, in lunatic asylums, &c. ; he even found bacilli in the streets and open spaces in a certain proportion of cases where tuberculous patients were collected together. These results have the greater value from the fact that in no case did he consider his experiments complete unless the dust with which he was experimenting, when inoculated into animals, produced the disease. It follows from all this that infection, as the result of inhalation of the dried virus, is one of the most common forms with which we have to contend, this being especially the case in older people, amongst whom pulmonary tuberculosis is most commonly met with, but in whom, as in children, pulmonary catarrh seems invariably to precede the tubercular disease. In children, the catarrhal inflammation of the lungs that so frequently accompanies such conditions as measles, scarlatina, diphtheria, whooping- cough, and other similar conditions, may very frequently become tubercular in character. TUBERCULOSIS. 221 ‘There seems, in these cases, to be, first, a weakened con- dition and impaired power of resistance of the epithelial cells lining the small bronchi, the smaller air passages and the air vesicles making up the spongy tissue of the lung. The bacilli in the air and dust then finding their way to a surface already weakened and specially prepared as it were for their reception, recommence their parasitic life, multiply, and make their way further into the tissues, where they set up the changes associated with tubercular disease. It is evident from all this that much work still lies ready to our hand in connection with the spread of tubercle, and ‘that if we could only persuade people to look upon tubercle as an infectious disease similar in character to scarlet fever, though not so rapidly developed, much would have been done to prevent its spread, and a great advance in preventive medicine would have been made. Through the work of Koch, Cornet, and others, the Ger- mans have come to look upon perfect cleanliness in the treatment of phthisical patients as absolutely essential. Pocket handkerchiefs and bed linen used by phthisical patients are most carefully sterilized by means of bichloride of mercury, hot air, steam, or other germicidal agents ; patients are strongly enjoined not to expectorate except into receptacles specially made for the purpose, receptacles that can be carried about, can be most readily cleaned, and in which expectorations can be easily disinfected. Of course, the results of all this are not yet manifest, but it may be confidently anticipated that within a comparatively short time a considerable diminution in the number of phthisical patients in Germany will have to be recorded ; not to be compared, perhaps, with the diminution of cases of other diseases, but still a very appreciable one. As a single example we may take the Grand Duchy of Baden, where there was a diminution of deaths from tuberculosis from 3.08 per 1,000 inhabitants in 1882 to 2.80 per 1,000 in 1887, or no less than .28 per 1,000. Were this to be equalled in the British Isles, and the patients were not carried off by other diseases, the saving to our community would be nearly 10,000 lives per annum. We may here mention the various disinfectants used. By far the best is heat, especially moist heat. Sunlight, or even ordinary daylight, will, ac- cording to Koch, kill tubercle bacilli in from a few minutes to five or six days. Koch has also proved that a number of ethereal oils, some of the so-called aniline or tar dyes, mercury in the form of vapour and silver and gold com- 222 BACTERIA. = pounds, all exert aninhibitory effect on the growth of tubercle bacillus ; the compounds of cyanogen and gold, especially, even in a solution diluted to one part of cyanide of gold to two millions of thesolvent substance, checking the growth of tubercle bacilli. Carbolic acid also kills the bacilli if it is allowed to act in considerable strength and for a lengthened period ; but none of these can, for a moment, be compared as regards not only efficiency and cheapness, but for facility of application, with steam or boiling water. All disinfection should be just as completely carried out when a phthisical patient leaves a room or ward as if a case of scarlet fever had been treated there ; the walls, floor, and even the roof should be thoroughly washed and disin- fected by means of hot water or lime. During treatment in such apart- ments no patient should be allowed to expectorate on the floor; and glass or porcelain spitoons, which can easily and safely be boiled in water, should be placed for the reception of all sputa. During the time that corridors, steps, and rooms are being cleaned and disinfected, they should be kept quite moist, in order that as little dust as possible may arise from the clean- ing operations. No room that has been occupied by a phthisical patient should be used until it has been thoroughly disinfected ; the bedding and curtains should be well boiled, the blankets steamed, the mattresses dis- infected, all the furniture washed with soap and water, the carpets and upholstering thoroughly beaten in the open air, the dust of which should be caught in straw (such straw and the paper taken from under the carpets should always be burned), the floor thoroughly washed with soap and hot water, and the wall paper rubbed down with freshly baked bread. In San Remo all these methods of procedure have been brought under the notice of the hotel-keepers, and they are advised to carry them out in connection with the whole of their rooms at the end of each season, not only the sleeping rooms, but also the public rooms. The alimentary canal is probably the next most important channel of infection. Evidence of infection by this channel was first obtained by the feeding experiments already referred to, but further evidence of it has been noted in the ulcera- tion of the intestine that is so frequently met with in phthisical patients, and which appears to be due to the action of bacilli contained in the sputum passing down the gullet through the stomach in cases where the gastric juice is not very active, and so to the intestine in which ulceration occurs in the course of the tubercular process as the result of the pathogenic activity of the bacilli. In children this mode of infection is comparatively common, and tubercular ulceration of the intestine, or tubercle of the glands connected with the intestinal tract, is of frequent occurrence. In 127 cases of tuberculosis in children that I examined this tubercular ulceration was found in 43; whilst in 100 cases, or nearly 79 per cent. of the whole, the glands were in some stage or other of tubercular degenera- tion. It would thus appear that tuberculosis connected with the intestine is of frequent occurrence in children, and we should therefore argue that TUBERCULOSIS. 223 the infection, in these cases at any rate, frequently takes place by the ali- mentary canal. The age at which these tubercular glands were found is very significant ; during the first year of life there were 4 cases ; from 1 to 2% years, 33; from 3 to 54 years, 29; from 6 to 74 years, 12; from 8 to 10 years, 133; and from 11 to 15 years 9 cases. In 14 cases these glands only were affected. Bolitz (Inaugural Dissertation, Kiel) gives similar figures, but on a more extensive scale. Out of 2,576 children whose bodies were submitted to a post mortem ex- amination in Kiel during the years 1873-1889, there were 424 cases of tuberculosis, or 16.4 per cent. of the whole mortality. The following shows the percentages of the whole of the deaths from tuberculosis at each of the diferent ages :— Still-born children . 0.0 per cent. { Up to2-3 years old 33. per cent. 6 Upto4weeksold 00 ,, » 3-4 5» 5 29. ” ” 5-10 ” 0.9 ” » 45 ” 31.8 ” » 3-5 months old 8.6 ,, »» 5-10 5, 55 3443 » G12 ,, » 18.3 ” »» 10-15 5, 29 30.1 ” » I-2years ,, 268 ,, Here, again, as where the lung is attacked, we must look upon the bacillus as the exciting cause, but tissue weakness as the predisposing cause. These conditions may be summed up as follows :— _(a@) The presence of the bacillus tuberculosis in such a position and for such a length of time that it obtains a coign of vantage, so to speak, from which to attack the tissues of the body. (6) Some weak point in the epithelial surface “made by disease, or due to irritation or bad food,” by which the organ- isms may attack the deeper tissues in sufficient numbers to ensure their being able to hold their own in the struggle for supremacy that ensues. (c) The comparatively low vitality of these deeper tissues brought about by imperfect nutrition or irritation ; the cells of which they are composed being no longer able to deal successfully with any large number of bacilli that can under ordinary circumstances find their way thus far. As regards the possibility of the bacillus tuberculosis being present in the intestinal canal, it should be remembered that the class of patients amongst whom abdominal tuberculosis is most rife, consists of infants, which during the first year of their life, and sometimes for a longer period, are suckled at the breast ; after this, however, the diet is extremely mixed, and as a rule it is extremely unsuitable ; but it is in by far the 224 BACTERIA. larger proportion of cases, even amongst the poorer classes, partially, at any rate, composed of cows’ milk. During this first year of their life, children with tuberculosis of the mesenteric glands, or of those glands connected with the intestine, form a very small proportion of the cases of infantile tuberculosis. Whilst the child issuckled by its mother there is little tubercle, but after this first year there is a very rapid rise in the mortality from tubercle. It isa somewhat singular fact that although tuberculosis is frequently met with in young married women, tubercular disease of the breast is extremely rare, so rare, indeed, that one observer, Dr. Huber- maas, who took great interest in this subject, was able to collect the records of only some eight cases. In cattle, on the other hand, where the mammary gland carries on its functions when the animals are placed under conditions which are far from healthy, or at any rate far from normal, this tubercular disease of the milk gland is not by any means of infrequent occurrence. Some of the earliest experiments from which actual proof of the infective nature of tuberculous material was obtained were those made by Gerlach and Chauveau, who used the milk of tuberculous cows to feed young animals ; though tubercu- losis was not produced in every case, the former was successful in a sufficient number to justify his conclusion that there was some specific virus in the milk of tuberculous cows which could, when ingested, produce tuberculosis of the alimentary tract, or of the glands associated with it. Numerous experi- ments on young pigs, some of them accidental, others de- signed, and others on calves and hens, have been recorded, in which tuberculosis has evidently followed their being fed with tuberculous milk. At the International Medical Congress held in Copenhagen in 1884, Professor Bang, of the Royal Veterinary School in that city, gave the results of a care- ful examination of twenty-seven cases of tubercular disease of the udder in cattle, in the milk of which he was able to demonstrate the presence of tubercle bacilli, both directly under the microscope and in the sediment obtained by means of the use of acentrifugal separator. With this milk, or with the separated sediment, he was able to produce tuberculosis both by inoculation and: by ingestion. Another observer, Nocard, was able to demonstrate the presence of the specific bacillus in milk in eleven cases, and Professor M’Fadyean and TUBERCULOSIS. 225 I also found tubercle bacilli in the milk from six cows out of six hundred examined. So certainly are the bacilli found in cases of tubercular disease of the udder that certain authori- ties maintain that it is possible to differentiate between the simple and the tubercular inflammation of the udder in the cow merely by means of a microscopic examination of the milk. (See Appendix). This question of tubercle in milk has now assumed such importance that much attention has been given to it abroad, and in Denmark a most complete system of inspection has been instituted in connection with one of the largest milk supply associations in the world ; and private enterprise, guided by Prof. Panum and Mr. Busck of Copenhagen, has indicated a way in which the State might tread with very great advantage. In connection with this association, six special veterinary surgeons, in addition to local practitioners, are constantly employed in keeping watch over the cattle that supply the milk to it. One of these veterinary surgeons alone, specially retained, examines eight hundred cattle fortnightly, and makes most careful notes (notes that I had an opportunity of examining) of the con- dition of every animal. Bang, to whose energy and observations the opening up of the tuberculosis question is very greatly due, contends that a diagnosis of tubercular disease of the udder can generally be made without difficulty during life and in the very early stages of the disease. With this conclusion it is somewhat difficult to agree, but much more may be done in the way of careful and systematic examination than is done at present, and in Denmark they have certainly systematized the examination of cattle to a far greater extent than we have succeeded in doing. As it may be of interest to mention the points on which the veterinary surgeon should depend in making his diagnosis, they may, so far as I was able to follow the routine of a number of inspectors, all of whom however differed slightly in their methods, be briefly summed up in the following :— (a) First of all the sub-maxillary glands are examined ; these are easily felt, and any change is readily made out. (4) The glands at the root of the neck and those in front of the haunch bones are always carefully examined. The glands in the flank should be equal in size, about the size of the middle finger, and not hard. Mere enlargement, even when considerable, is, however, not looked upon as of great importance if it is perfectly equal on the two sides. (c) The animal is made to cough by means of pressure on the trachea, and the lungs are carefully examined during and after the coughing. The condition of the skin over the flanks is carefully observed ; it should, in a healthy animal, be “ loose,” like that of a dog, soft and pliable ; any adhesion, hardness, or harshness, should be carefully noted. (d) The udder is carefully examined for inequality of size and for any induration. It is a somewhat curious fact that tuberculous disease usually affects the hind quarters of the udder, which become hard and knotty, but not painful ; whilst in acute inflammation of the udder, the anterior quarters are quite as much affected as the posterior ; the pain is usually very acute, and the process is accompanied by much more marked febrile symptoms. (e) Then the glands above the bar high up between the quarters, are I 226 BACTERIA. most carefully examined. In cases of tubercular disease of the udder these glands are invariably affected, are unequal in size, and the larger one, corresponding to the affected quarter, is usually considerably indurated. ) Careful auscultation is carried out at least once a month. The fore- foot of the side that is being examined being always well advanced. The normal expiration sound lasts half as long as the normal inspiration, and if this rhythm is deviated from in any way, a further and thorough examina- tion of the lungs should always be made. ) The examination is continued still further if the slightest suspicion of tubercular disease is aroused by the above investigation, and an examination per rectum is made, with the object of determining whether there is any tubercle of the peritoneum or not. As the onset of the disease in the udder is so rapid, and as, as yet, it is held by most observers that the bacilli may make their appearance in the milk, even where the udder is not directly affected, it follows that if there is the slightest suspicion of the existence of tubercular disease in a cow, the milk from that animal should not be put into the milk supply, and as a matter of fact, on the Danish farms above re- ferréd to, it is not sent to town, but it is either thrown out, or after being most thoroughly disinfected by prolonged boiling, is given to the pigs. (4) The farmer keeps a record of the quantity of milk given by each cow, and a note of what is done with it; and any milk that is put out of the supply by the veterinary surgeon, or by the farmer himself, on account of suspected disease, is paid for by the company, or the difference between the full value and the value as pig food. other inflammatory condition of the udder is carefully noted, and even then the milk is withdrawn from the regular supply. (¢) A small quantity of milk is always drawn off by the veterinary sur- geon, who carefully notes its colour. If it is too thin and watery looking he immediately condemns it; whilst if it loses the peculiar blue tinge that freshly-drawn milk from a healthy cow almost invariably has, and takes on even a slight yellow tinge instead, the milk from the infected quarter is not used for any purposes, although the milk from the other quarters may be used, after being thoroughly boiled, for the feeding of pigs. The authorities of the association insist rigidly on the fortnightly in- spection, because it has been observed that very great swelling may appear as a sign of udder tuberculosis in from ten to fourteen days, as in this posi- tion the onset of the tuberculous disease is usually much more rapid than in the lungs, in which the process in a very large majority of cases appears to be far more chronic in character. In all cases, the condition of the glands must be systematically and carefully observed. In the light of recent events, too, it is to be hoped that Koch’s tuberculin may be utilized in the diagnosis of tuberculosis in cattle, and that satis- factory results may thus be obtained. In consequence of the rapid onset of the disease diag- nosis without regular inspection is almost impossible, and it is probable that the swelling of the udder is only one of the later manifestations of the disease, the glands above the udder apparently being a far more reliable index. Professor M’Fadyean and I were so much struck TUBERCULOSIS. 227 with this difficulty of diagnosis, that in a paper read at a meeting of the Pathological Section of the British Medical Association, held in Dublin, August, 1887, we stated that it appeared to us “that where, as is very frequently the case with cows kept in towns, a complete history of the diseased condition of the'udder is not obtainable, a differential diag- nosis of mammitis (inflammation of the milk glands) is by no means easy, except by microscopic demonstration of the Tutercle bacilli arranged around a closed up milk-duct in a case of Tuberculous disease of the udder of a cow. x 1000. bacilli in the milk ; which may also fail if a most careful search is not made” ; and Principal Walley, dealing with the same subject, says, “that he could not undertake to diagnose, with accuracy, tubercular mammitis in every case, nor even in a majority of cases”; and he states that in specimens of the udders of tuberculous cows he has ex- amined, after death, he has found good examples of tubercu- losis in the mucous membrane of the milk sinuses without 228 BACTERIA. the occurrence of any induration of the milk gland, “indeed,” he adds, “I may say that no veterinary surgeon could, during life, have diagnosed the existence of tubercular mastitis (tubercular inflammation .of the udder) without the aid of a microscope.” That tubercle bacilli make their way into the milk, when there is tubercular ulceration in the milk- ducts, can be readily understood, and has frequently been shown ; but it has also been demonstrated by Professor M’Fadyean that in cases of tuberculosis, bacilli may be found lying free in what are otherwise apparently healthy milk ducts. Fresh evidence is being accumulated every day, but these facts alone, when considered along with the occurrence of bacilli in milk, with the feeding experi- ments already recorded by so many observers, and taken in connection with the great prevalence of tubercle‘in certain classes of animals, afford strong presumptive evidence that milk is a source of tubercular infection, especially in children, and in those in whom, on account of imperfect nutrition and impaired digestion, the walls of the alimentary canal are less able to resist the invasion of the organism known as Koch's Bacillus Tuberculosis. The danger of contracting tuberculosis from taking meat from cattle affected with the disease is perhaps not so great as that associated with the drinking of tuberculous milk ; it is nevertheless one with which the sanitary authorities will have to deal, and one to guard against which it is necessary to take very considerable precautions. There can be little doubt that in those cases where the disease is localized to any one of the viscera at the time of the death of the animal, there is little danger to be anticipated from eating the well-cooked flesh from other parts of that animal; and if we could be absolutely certain that the localization was complete, all would be well. Even in cases where the flesh is taken from animals in advanced stages of tuberculous disease there would be a certain proportion of cases in which no evil results would follow, and one observer who made sixty-two experiments with such flesh boiled for ten or fifteen minutes, found that only 35.5 per cent. of the inoculated animals became tuberculous. Even in cases of generalized tuberculosis Nocard failed in thirty-nine out of forty cases to transmit the disease by means of raw muscle juice injected into the peritoneal cavity of guinea pigs, but TUBERCULOSIS. 229 he succeeded in the fortieth. Other observers, however, have been more successful, and in two rabbits I was able to produce tuberculosis by injection into the peritoneal cavity of the raw juice expressed from the intercostal muscles of a tuberculous cow after all tuberculous pleura had been carefully “stripped”; whilst the juice taken from the muscle of the thigh injected into two other rabbits was perfectly innocuous. The danger of infection by the consumption of meat from tuberculous cows may have been much exaggerated, but that there is a very appreciable danger must most certainly not be lost sight of by our Medical Officers of Health and the Veterinary Inspectors of the Board of Agriculture. Koch, as we have seen, had discovered a specific or- ganism which he had been able to cultivate outside the body ; he had inoculated and produced tubercle ; and he had found certain germicidal agents which were capable of destroying the organism outside the body. He, and the many interested workers who had investigated the subject, had found that it was more difficult to inoculate certain animals successfully than others. It had been observed even that certain individuals of the same species were much more refractory to the action of the virus than others, and it very naturally suggested itself to those workers, that there were two conditions which would have to be determined before any systematic and organized attack could be made on the tubercle bacillus within the body. The tubercle bacillus itself might be developed under such conditions that its virulence might be more or less modified. For example, Koch found that all his cultivations made on blood serum retained their power of growing in animal tissues in a most extraordinary degree, and for long this substance was the only nutrient medium used for the cultivation of the tubercle bacillus. It was found, indeed, that the other combinations used: for the cultivation of other organisms up to that time were valueless as nutrient substrata for the tubercle bacillus. Nocard and Roux, however, found, as we have seen, that on the addition of a certain percentage—6-8 per cent.—of glycerine to peptonized broth, solidified by the addition of agar, they could obtain a nutrient medium on which the tubercle bacillus would grow most luxuriantly. After it had passed through several generations of cultivations on this 230 BACTERIA. medium, the virulence of the organism, or its power of growing in tissues, was distinctly diminished—z.e., the organism was in a condition to be much more readily de- stroyed by the cells of the animal body, which appeared to keep the upper hand throughout the struggle, very few of the cells being destroyed, as is the case when virulent tubercle is used, where although a number of the tubercle bacilli are un- doubtedly destroyed, the degeneration of the proliferated cells is so extensive that caseous nodules are formed, and the typical appearance of caseating tubercle are presented in the inocu- lated area. The second factor, though equally important, and though recognized indirectly by all physicians, could not put itself so fully in evidence as the first, and it was only after Metschnikoff had made his beautiful observations on the daph- nia and on the separating tail of the tadpole and the phagocyte action of certain cells in these processes, that any direct evi- dence of the bearing of the activity of the cells of the tissues of the body on the destruction of micro-organisms within the body could be definitely adduced. Then, indeed, began the narration of the history of the battle between the cells and the bacilli. If the bacilli were weak, or were present in small numbers only, the cells were invariably the victors ; if the cells were degenerated, or were badly nourished, or if their vitality was low, the bacilli proved themselves the more powerful. How these results were brought about soon became the subject of most violent controversy, and in the controversy thus raised explanations of facts that had hither- to puzzled scientific workers were projected and forced home. Let us here, however, examine only those facts that appear to have a special bearing on the subject of tuberculosis. A perfectly healthy individual, placed under favourable con- ditions as regards food, fresh air, and exercise, is never attacked successfully by tubercle bacilli, the active, vigorous tissue cells being perfectly competent to destroy any bacilli that may make their way into the lungs, the pharynx, or the intestine ; whilst even in cases of direct inoculation into a wound, if the wound heals rapidly, no tubercular process may result, the tubercle bacilli, as we have said, being destroyed by the cells. It may be, indeed, that certain secretions of the cells—z.e., those substances that bear the same relation to the connective tissue cells that an enzyme bears to the yeast-cells—also exert a deleterious action on the TUBERCULOSIS. 231 bacilli in the healthy individual, and in a minor degree even in individuals with weaker tissues ; the activity of the bacilli is so interfered with or modified that they can be readily attacked and devoured by the tissue cells, which, as has long been known, have a most remarkable power of taking up into their substance many effete materials and particles of dead or inorganic matter. On the other hand, certain excretions, by accumulating in the blood and in the lymph spaces, may impair the activity of these tissue cells and so render them less able—(1) to secrete their protective material, and (2) to wage war directly against the bacilli; whilst, in turn, the bacilli on their side, as we have already seen, secrete a material that has a most injurious action on the tissue cells, causing them to swell up and eventually to become hyaline. . This material is only a poison when in large or comparatively large quantities; in smaller doses it acts in the first instance as an irritant or stimulant, stimula- ting the protoplasm to exert all its powers against the advancing bacteria, powers that are so strongly exerted (un- less the conditions of nutrition and excretion are specially favourable) that they are rapidly exhausted. It has been observed that in those cases where phthisis was curable the cure has been effected only by careful nutrition of the tissues, and that as soon as they have been brought up to a certain standard of health the disease has been checked, in many cases permanently ; on the other hand it has been found that when the tissues have again fallen below far there has been a fresh outbreak of the disease. These facts are in themselves sufficiently interesting and suggestive, but as we shall see they have a further important bearing on the question of the curability of the tubercular phthisis. It has been observed that a process of localization occurs even when large caseous patches have been formed, and it has been found that around these patches, just as around an abscess, there is always erected a kind of barrier, made up of vigorous connective tissue cells, small, round and larger epithelioid cells; the blood vessels in this cellular zone being comparatively numerous and of considerable size. We have, in fact, in this arrangement of the blood vessels and cells, a making of roads (the blood vessels) for the bringing up and massing of forces (the active cells) around the enemies’ camp (the tubercular or caseous mass with the con- 232 BACTERIA. ‘tained bacilli or spores), and by a process of close siege pre- venting the organisms from making their way outwards, and confining them entirely to their own territory, so that, when they have utilized what food material there is in the degenerated cells, they are no longer able to exist as vege- tative bacteria, and only the spores remain—which may, however, remain latent for a very long period awaiting a favourable opportunity for another attack on weakened tissues. These spores or hibernating germs are confined within the same area, and the aébrzs with its contained spores is gradually encroached upon by the surrounding tissues until, eventually, if the mass is not large it may be entirely absorbed, though, owing to the amount of fibrous tissue that is formed by the attacking cells after their activity is somewhat diminished, this process of absorption sometimes goes on very slowly. It was an easy enough matter, when the history of the development of the tubercle bacillus was known, to kill the organism outside the body, and Koch found that a very large number of germicidal substances were capable of interfering with its growth ; but unfortunately most of these germicides also exerted an injurious effect on the tissues, so that what was gained in one direction was lost in another. In most other diseases in which preventive or curative inoculation by means of vaccines—less virulent cultures of the microbes—to accustom the animal to the action of the poison and so enable it to resist the more virulent, has been attempted, the aim has been to render the bacilli less, and the cells more active, and to develop in these cells a special activity. By accident, as he tells us, Koch found that by injecting tubercle virus into the subcutaneous tissues of guinea- pigs which had been previously inoculated with tuberculosis, the tissue in which the tubercle bacilli were acting was actually destroyed and an eschar or slough was formed at the point of inoculation; whilst although the bacilli were not directly destroyed, they remained embedded in a mass of food material that was gradually but surely used up to supply the needs of these bacilli, and eventually they had to go into winter quarters, But the products of the tubercle bacilli appear to act in some way on the tubercles already formed; they do something more than bring about complete degenera- TUBERCULOSIS. 233 tion of the weakened cells. In the immediate neighbour- hood of the young tuberculous tissue, z\¢., in those cells that have been slightly affected by the products of the tubercle bacillus it sets up a further reaction ; it stimulates these cells, and at the same time causes a dilatation of the vessels and probably also an increase in their number (this latter may be only apparent); a larger amount of food material is brought up for the nutrition of these cells, excreted matter is more readily carried away both by vessels and lymphatics, and in consequence of this, cells that have been too far stimulated by the tubercle poison to recover, die off, whilst those that are still capable of living, even under the excessive stimulation, proliferate and help to form the barrier between the dead mass and.the surrounding normal tissues. In this way it would appear that in certain cases of tuberculous disease, Koch has produced an imitation of the natural process of cure. By a process of combined reasoning and experimentation, and basing his method of procedure on the one that had been already adopted in con- nection with the preparation of vaccines for other diseases, Koch succeeded in obtaining a substance with which, in a certain degree, at any rate, he was able to combat the advance or to modify the tuberculous disease in animals. As this advance in the treatment of tuberculosis marks a most important point in the history of the disease, and in order that there may be no misconception as to Koch’s exact position, it may be well to give in his own words the descrip- tion of the discovery, composition, and probable mode of action of the remedy. He says, in describing the observations, by the considera- tion of which he was led totake the lines of experimentation that ultimately led him to success: “If a healthy guinea-pig be inoculated with a pure cultivation of tubercle bacilli, the inoculation wound generally becomes glued over or sealed, and appears to heal up during the next few days. It is only in the course of from ten to fourteen days that a hard nodule "is formed, which soon opens, forming an ulcerating spot which persists until the death of the animal ; if an animal that is already tuberculous be inoculated the course of events is very different. The most suitable animals for this experi- ment are those which have already been successfully inocu- lated four or six weeks previously. In the case of an animal so 234 BACTERIA. treated the small secondary inoculation wound becomes sealed at first, but no nodule is formed ; a peculiar change takes place at the point of (primary) inoculation. As early as the first or second day the point becomes hard and dark- coloured—a condition that is not confined to the point of inoculation—and spreads around for about 0.5 to 1 centi- metre. During the next few days it becomes more and more clear that the epidermis thus changed is necrotic, and finally it is thrown off, and a flat ulcerated surface remains, which generally heals quickly and completely, without infec- tion being carried to the neighbouring lymphatic glands. Thus the inoculated tubercle bacilli act quite differently on the skin of a healthy guinea-pig, and on that of a tuberculous one. But this remarkable action does not belong exclusively to living tubercle bacilli, it also be- longs in the same degree to dead ones, whether they be killed by low temperature of long duration, which I at first tried, or by boiling heat, or by the action of certain chemicals. “This peculiar fact having been ascertained, I followed it up in all directions, and then further found that pure culti- vations of tubercle bacilli thus killed, after they are ground down and suspended in water, may be injected under the skin of healthy guinea-pigs in large quantities without producing anything but local suppuration. Tuberculous guinea-pigs, on the other hand, are killed by an injection of very small quantities of suspended cultures within a time varying from six to forty-eight hours, according to the dose ; a dose which is just insufficient to kill the animal being sufficient to produce a widespread necrosis of the skin in the region of the point of (primary) inoculation. If the fluid - with its suspended matter be still further diluted, so that it is scarcely turbid to the eye, the animals remain alive ; and if the injections be continued at intervals of one or two days a noticeable improvement in their condition soon sets in ; the ulcer at the point of inoculation becomes smaller, and finally cicatrization takes place. This is never the case when such treatment is not resorted to. The swollen lym- phatic glands become smaller, the condition as- regards nutrition improves, and the progress of the disease is arrested, if it is not already so far advanced that the animal dies of debility. TUBERCULOSIS. 235 “These facts formed the basis of a therapeutic method against tuberculosis. But an obstacle to the practical em- ployment of fluids containing in suspension the dead tubercle bacilli was found in the fact that the tubercle bacilli are not (readily) absorbed; they disappear, and remain for a long time unchanged zz situ, producing larger or smaller suppurating centres. It was clear, there- fore, that in this method the curative effect on the tuber- culous process was obtained by a soluble substance, diffused so to speak, into the fluids that surround the tubercle bacilli, and transferred without delay to the circulating fluids of the body ; whereas that which has the pus-forming quality seems to remain bound up in the tubercle bacilli, or at any rate to be only very slowly dissolved or washed out. Thus the only important thing that remained to be done was to carry out the process that takes place within the body— outside of it also—and if possible to extract and isolate the curative substance from the tubercle bacilli. This problem required long and continued experimentation, but at last I succeeded, by the help of a 40 to 50 per cent. solution of glycerine, in extracting the active principle from the tubercle bacilli. My further experiments on animals, and finally on human beings, were made with liquid thus obtained ; and in this way, also, the liquid which I supplied to phy- sicians and surgeons in order that they might repeat the experiments, was obtained. Zhe remedy with which the new therapeutic treatment of tuberculosts zs carrted out, 1s, there- sates a glycerine extract of pure cultivations of tubercle acills. “Tn addition to the active principle there pass from the tubercle bacilli into the simple extract all other substances soluble in so per cent. glycerine, and therefore it is found to contain a certain quantity of mineral salts, pigment, and other unknown substances—extractives, &c. Some of these substances can be readily separated, as the active principle is insoluble in absolute alcohol, by which it can be preci- pitated, not pure certainly, but in combination with such other extractive matters as are also insoluble in alcohol. The colouring matter, too, can be separated out so that it is possible to obtain from the extract a colourless dry substance, which contains the active principle in a much more concen- trated form than does the original glycerine solution. 236 BACTERIA. “This purification of the glycerine extract has, however, no advantages as regards practical application, as the substances removed have no action on the human organism, so that the purifying process would only involve un- necessary expense. The constitution of the active principle can, as yet, be only a matter of conjecture. “Tt appears to me, indeed, to be a derivative of albuminous bodies, and to be closely related to them, but it does not belong to the group of so-called toxalbumens, as is proved by the fact that it can withstand high temperature, and in the dialyzer it passes quickly and easily through the mem- brane. The quantity of active principle present in the extract is in all probability very small. I estimate it at a ‘fraction of 1 per cent. Thus, if my assumption be correct, we have to deal with a substance, the action of which on the tuberculous organism, far surpasses that of the strongest drugs known. “ Various hypotheses may of course be formed as to the specific mode of action of the remedy on tuberculous tissue. Without in any way affirming that mine is the best possible explanation, I imagine the process to be as follows: The tubercle bacilliin their growth produce in the living tissues— just as in the artificial cultivations—certain substances which exert various but always deleterious influences on the living elements surrounding them, the cells. Amongst these substances is one which, in a certain concentration, destroys living protoplasm, and causes it to undergo a transformation into the condition called by Weigert, ‘coagulation-necrosis.’ The tissue having thus become necrotic, the conditions are so unfavourable to the nutrition of the bacillus that it is unable to undergo further development, and finally, in some cases, it dies off. In this way I explain the remarkable phenomenon, that in organs freshly attacked by tuberculous disease—for instance, in a guinea-pig’s spleen or liver filled with grey nodules—numerous bacilli are found, whilst bacilli are rare or entirely absent when the enormously enlarged spleen is made up of whitish substance in a condition of coagulation-necrosis, such as is often met with in guinea- pigs that die of tuberculosis. A solitary bacillus, however, cannot produce necrosis at any great distance from itself, for, as soon as the necrosis has covered a certain area, the growth of the bacillus—and, in consequence, the production of the necrosis-producing substance—diminishes, and thus a sort TUBERCULOSIS. 237 of mutual compensation is set up, and to this is due the fact that the growth of isolated bacilli is so remarkably restricted, as for example, in the case of lupus, in scrofulous glands, &c. In such cases the necrosis extends only over a part of the cell, which then, in its further growth, assumes the peculiar form of a giant cell. I thus follow in this statement of my views the explanation of the growth of giant cells first given by Weigert. Now if the necrosis-producing substance were artificially added to that contained in the tissue surrounding the bacillus, then the necrosis would extend further, and thus the conditions of nutrition of the bacillus would become much more unfavourable than is usually the case. Then, not only would the more completely necrosed tissues disintegrate, slough, and—where this is possible—take with them the enclosed bacilli, carrying them outside the body, but the growth of the bacilli would also be interfered with to such an extent that they would die off much sooner than they do under ordinary conditions. It is in calling forth such changes that, to my mind, the action of the remedy seeems to consist. It contains a certain amount of the necrosis-producing substance, of which a correspondingly large dose has a deleterious influence—even in healthy persons—on certain elements of the tissues, probably on the white blood corpuscles or cells closely related to them, thus giving rise to the fever and the whole peculiar complex of symptoms that supervenes. In tuberculous persons a much smaller quantity suffices to cause, at certain spots—z.e., wherever tubercle bacilli vegetate, and have already impreg- nated their surroundings with the necrosis-producing sub- stance—a more or less extended necrosis of cells with the production of the accompanying conditions that affect the entire organism. In this way it is possible to explain—at least for the present—in a provisional manner, the specific influence which the remedy, in certain well-recognized doses, exercises on tuberculous tissue, as well as the possibility of increasing the doses in so remarkable a fashion, and, finally, to explain the curative effect which the remedy undoubtedly exerts where the conditions are at all favourable for its exhibition.” The substance to which the name of Tuberculin has been given has been analyzed, and is found to be ‘‘a syrupy, slightly foaming liquid (sp. gr. 1015?) of brown sherry colour, its aqueous solutions 238 BACTERIA. showing a greenish florescence. In odour it resembles elder yeast or leaven, combined with a sweet aromatic admixture, such as honey. If slowly heated, the smell of yeast gives way to an agreeable odour resembling fruits ; on further heating, the smell- becomes like that of fresh bread crust,” but without the acid fruit odour. If the heating of the material is continued, ‘‘ the smell assumes the empyreumatic character exhibited by burning albuminous matter and carbonizing horny substances. Only an extremely small quantity of ash (under 1 per cent.) was obtained. The liquid shows a neutral reaction.” It was found to contain a small quantity of mucine, indicated by a turbidity on the addition of dilute acetic acid, which is increased on the addition of potassium ferrocyanide, indicating the presence of albumen. Peptones are present in considerable quantities. There is a slight reducing action obtained when the fluid is heated with Fehling’s solution ; there was no reaction with acid bichromate of potassium, so that acids and ptomaines are absent ; but it was assumed, as would now appear incorrectly, that toxalbumens, or albumoses, globulins, or enzymes must be the substances to which the material injected by Koch owes its special properties, Any description of the lymph other than that given by Koch himself must be the result merely of guess-work, but even guesses will sometimes afford indications as to the lines on which researches are at present being carried out, not only in connection with tuberculosis, but with several other most deadly and wide-spread diseases. The peculiarity of this method of treatment is, that it is not known to pro- tect against an attack of tuberculosis, though Koch states that he hopes his guinea-pigs will be protected from future attacks ; it is used solely as a therapeutic agent to check or stop the disease after it has once.obtained a foothold in the body. Itis thus in principle more like Pasteur’s inoculation against hydrophobia, for the inoculation is made after the patient has been inoculated with the disease virus; and it is also similar in certain points, though the principle is different, to the protective inoculation obtained by Hankin, who, by means of albumoses obtained from anthrax cultivations, has been able to produce immunity against anthrax, though not to cure, after that disease was once induced ; and that Dr. Cart- wright Wood and I carried on with the pyocyanin products in rabbits, to tide them over an attack of anthrax the bacilli of which were introduced into the subcutaneous tissue shortly before, or immediately after, the injection of the pyocyanin. We, however, considered that the pyocyanin had not acted directly on the anthrax bacillus, but that it acted by stimu- lating the cells ; whilst Koch’s “lymph” acts (1) by destroy- ing the tuberculous tissue, and rendering it unfit for the | | TUBERCULOSIS, 239 nutrition of the bacillus, and (2) by setting up a localized reaction in the neighbourhood of the bacilli, by means of which the cells are so stimulated that they are able, as we have already seen, to prevent the extension of the bacilli into the surrounding parts. Koch's fluid may not accomplish all that is expected from it ; it may, in fact, be found that Koch has not completed his ex- periments, but he has made a wonderful advance in our know- ledge of the conditions necessary for the combating of micro- organisms ; and has extended the observation of the earlier workers at the globulines and albumoses who really opened up the way for the advance of the numerous workers who have re- cently come into the field. It would, however, take us beyond the scope of the present work to say more about this marvellous discovery of Koch's, Let it always be remembered that tubercle destroys the tissues in which it grows, and that the treatment by Koch’s method completes the process of destruction, if this is not already accomplished; so that, under the very best conditions, phthisis can only be stopped, and, although ‘a comparative cure may be obtained, highly differentiated tissues once destroyed can never be restored or replaced. It will thus be seen that although Koch has selected a special irritant material as that which it was found necessary to separate in order to obtain the results that he wished, and has obtained the sequence of specific (?) stimulation of the cells, he has departed from the usually accepted methods, in that he acts directly on the tissue cells, and leaves the bacilli to die of starvation. It is not now a case of the cells destroying or modifying the activity of the bacilli; it is simply, in the. first instance, a cutting off, or rather a rapid exhaustion, of the substances required for the nutrition of the bacilli; the bacilli remain, and although they may eventually undergo retrogressive changes, it appears probable that their spores remain for some time, at any rate, ready to break out should favourable conditions again pre- sent themselves. Apart from this local action, however, the fact cannot be ignored that where the tissues are not already too far weakened, or the cells so imperfectly nourished that they cannot react to stimulus, the liquid injected by Koch may exert a general and specific action on the tissue cells through which they are acclimatized, as it were, to resist the poison ; so that it is quite possible that a partial immunity 240 BACTERIA. as regards the tubercle bacillus may be acquired. It is probable that the tuberculin acts as a stimulant on all cells, and that to the increased metabolic changes that are set up is due the general reaction that is described as following on the exhibition of this material, That this is not entirely a fanciful explanation may be argued from an example. It is a fact, well known to those who have charge of tuberculous patients suffering from diseased glands—scrofulous glands, as they are called—that so long as these glands remain uninjured and are subjected to no stimulation, and so long as the nutrition of the patients keeps fairly good, they remain as a rule comparatively free from pulmonary phthisis and other forms of tuberculosis ; whilst other members of the same family, existing under the same conditions, both as regards hygiene and nutrition, become affected with the commoner and more fatal forms of the disease. A further illustration may be taken from the difference that exists between children of the lower classes and those met with in other grades of society—children in a sick children’s hospital frequently having every organ crammed with, or almost replaced by, tubercular nodules and cavities, so that it seems marvellous that the unfortunate children could have remained alive at all ; whilst children better nourished, but not previously affected by any form of tubercle, appear to succumb very easily, when comparatively localized tubercle has been developed. In these cases, however, it is usually found that some vital organ such as the brain is affected. It would appear that, in these cases, the tissues may de- velop greater resistance in consequence of the circulation in the fluids of the body of the soluble products; but that when this acquired resistance is once lost, or is over- come, the stimulation exhausts the cells, and they now more easily fall a prey to the active tubercle bacilli. It must, however, in this connection, be pointed out that the products of the tubercle bacillus, or sterilized cultures of the organism, when introduced subcutaneously into healthy fowls or guinea-pigs, produce a condition which is spoken of as “marasmus ;” and as early as 1879, Maffucci, as the result of a series of experiments, concluded that these sterilized cul- tures when left in the body, exerted such a marked influence on the tissues, that they induced emaciation, atrophy of the liver TUBERCULOSIS. 241 cells, and of the cells of the different parts of the spleen, and that they also set up certain changes in the circulation, the result of which was seen in marked congestion of the lungs, kidney, spleen, &c. These experiments were no doubt sug- gested by the similar changes that are met with in the human subject in the course of tubercular disease, even in those organs that are not directly affected by the tuberculous infiltrations. Recently he has confirmed and extended his former observations ; and it will be interesting to see whether the conditions met with in patients injected according to Koch’s method, are similar to those observed in animals experimented on by Maffucci’s method. , How far the object of Koch’s endeavours will be attained still remains to be seen, and his method has been, and will be, put to most severe tests for it involves the question of life or death to thousands, nay, tens and hundreds of thou- sands. ‘There appears little doubt that in lupus (a tuber- culous skin disease) the process has been checked, and, in some cases, at any rate, it has not again broken out for a considerable period after the treatment had been stopped. There also appears to be pretty reliable evidence in favour of his contention that there is an amelioration of the condition of the patient and an improvement in the disease in certain other forms of tuberculosis ; but the use of the remedy has not been sufficiently prolonged to allow of our arriving at any very definite conclusion, however favourable our opinions may be. Virchow, the greatest pathologist of the age, has found, in a number of cases that have come under his obser- vation—a comparative small number when the enormous number that have been injected is taken into consideration —that the characteristic degeneration of the tissues of the young tubercle is not always brought about, that the locali- zation of the disease is not by any means perfect, that there is a tendency of tubercle material that should be “thrown off” to continue the infection and even increase its rapidity of spreading, especially in the lungs, and that in some cases the bacilli, instead of being rendered inert, appear to take on greater activity, and to be carried in the various currents in the body, even to parts situated at some distance from the original tuberculous focus. We must bear in mind, however, that almost all the cases that up to the present have been brought to the post-mortem 17 242 ' BACTERIA. table have been cases in which the disease was far advanced, and in which Koch’s inoculative treatment has been sought as a last resource by physicians and patients alike. A most important point to be remembered in connection with tuberculosis is that under favourable conditions it is an extremely curable disease ; such conditions, however, must be very far-reaching and must include a sufficient and suitable supply of food to the patient, perfect oxidation of the tissues, and facilities for the excretion of effete products. The effects of climate, of exercise, of pure mountain air, free ventilation in houses and of generally favourable hygienic conditions, are all due to the promotion of the healthy condition of the tissues and of the increased vitality of the cells ; this vitality increasing their power of resistance and enabling them to cope successfully with the bacilli and their products with which they may be brought in contact. It should not be left out of count, however, that we may, by the use of suitable drugs, be able to exert an antidotal influence by means of which, acting directly on or through the cells, these cells may be put in a still better position for resisting the attacks of the tubercle bacilli and their products. This antidotal system of treatment, if it could be carried out by means of drugs, should prove far more efficacious than the use of any means of treatment that we have at present at our disposal, especially if it could be combined with some of these latter. This is not the place to discuss the medical aspects of the question ; but the above facts are mentioned in connection with the biological problems associated with the action and inter-action of the bacilli and of the cells of the body. LITERATURE. Banc.—Proc. Internat. Med. Congress, Copenhagen, vol. 1, Path. Sec., p. 1, 1884. BAuUMGARTEN.—Zeischrift f. Wissensch Mikroskopie, Bd. 1, 1884 ; Deutsch. Med. Woch., Bd. vill, p. 305, 1882. Bo.itz.—Journ. Com. Path. and Therapeutics, p. 370. Dec., 1890. Boiiincer.—Baier Aerztl Int. Blatt., 1883 ; Centralbl. f. d. Med. Wiss., Bd. xx1., p. 600, 1883. CHEYNE, WaTSON.—Practitroner, vol. XXX., p. 241, 1883. CouNHEIM.—Uebertragbarkeit der Tuberculose. Berlin, 1877. - TUBERCULOSIS, 243 Cornet.—Ztschr f. Hyg., Bd. v., p. 191, 1889; Ueber Tuberculose, Leipzig, 1890. FLtecr.—Ztschr f. Hyg., Bd. 11., 1882. GarFFxy.—-Mitth. a. d. k. Gesundheitsamt, Bd. 1, p. 126, 1884. GERLACH.—Comptes rendus. Feb. 10 and 24, 1868. HusperMAaS.—Beitrag z. Klin. Chir. Mitth. a. d. Chir. Klinik z. Tiibingen, Bd. 1, p. 45, 1886. Joune.—Die Geschicht. der Tuberculose. 1883. Kocu.—Berlin Klin. Woch., Bd. xv., p. 221, 1882; Mitth. a. d. k. Gesundheitsamt, Bd. 11, p. 1, 1884; Deutsch. Med. Woch., No. 10, 1883. M’Fapyean AND WoopHEap. Brit. Med. Assoc. Dublin, Aug., 1887. Marrucci.—Centralbl. f. Allg. Path. u Path. Anat., Bd. 1, No. 26, Dec. 15, 1890. MeErscunikorr.—Annales de l'Institut Pasteur, 1888-9. Nocarp.—Amer. Vet. Rev., N. Y., vol. 11, p. 258, 1884-5. Nocarp anp Rovux.—Annales de J'Institut Pasteur, t. 1, p. 19, 1887. Paw.Lowsky.—Ann. d. l'Institut Pasteur, t. 11, p. 303, 1888. Toussaint.—Comptes rendus, t. Xcll., pp. 281, 322, 350, 741, 1881. VILLEMIN.—Etude sur la Tuberculose. Paris, 1868. Watey.—Lain. Med. Journ., p. 1088, 1888. WEIGERT.—Deutschen. Med. Woch., No. 24, 1883 ; and No. 35, 1885. Witurams, C. T.—Lancet, vol. 1, p. 312, 1883 ; Pulmonary Consumption, 1887. WoopHEAD.—Tabes Mesenterica and Pulmonary Tubercu- losis. Reports from the Lab. R. C. P. Ed., vol. 1, p. 179, 1889. : Worzpure.—Mitth. a. d. k. Gesundheitsamt, Bd. u., p, 89, 1884. WESENER.—Futterungstuberculose. 1885. See also Comptes rendus et Mémoires de la Congress de la Tuberculose. Paris, 1888-1890. For the more recent controversy on Tuberculosis and Tuber- culin, see Lancet and British Med. Journal. The earlier literature is given very fully in CorNEL and BaBEs “ Les Bactéries.” CHAPTER XII. LEPROSY. Distribution of Leprosy—Similarity to Tuberculosis—Description of Dis- ease—Tubercular, Anzesthetic, Mixed—The Leprosy Bacillus—Method of Staining—Position—Leprosy Cells—Bacilli Resistant and Grow Slowly—Cultivation Experiments Mostly Negative—Theories of Cause of Leprosy. Wiruin the last few years leprosy has, metaphorically speaking, returned to life in this country. The occurrence of a case in one of our market attendants hascreated morecommotion, and has put in train a more complicated machinery, than phthisis, with its thousands and tens of thousands of victims was for long able to set going. Nevertheless, we can now afford to think of leprosy as almost a thing of the past, as far as our own country is concerned, although we still come across traces of its sojourn amongst us in our Libertons, or Leper towns, or Leptons, that indicate only too surely that this disease was looked upon with the greatest dread by our ancestors of the Middle Ages, who evidently took pains to keep in their own regions, and within their own asylums, the lepers of that period. With the exception of certain isolated areas along the shores of Spain, and in Portugal, in Norway, some parts of Sweden and Iceland, in Italy, Roumania, and Hungary, in the Balkan Peninsula, and in Greece where the disease is still endemic, leprosy is now rarely seen in Europe; but in certain parts of Asia and Africa it is still frequently met with. It is found doing its fell work in the Sandwich Islands, in Mexico, in Cuba, in some parts of Central America, in the north-east of South America, and in the Argentine Republic, in the north-east and north- west of Africa, in Guinea, and in Cape Colony, and in Mada- gascar, along the shores of the Black Sea, in Persia, Arabia, India, China, the Malay Archipelago, Japan, in Asia, and in New Zealand. LEPROSY. 245 From the whole nature of the symptoms, and from the course of the disease, it was long considered to be a disease somewhat similar to tuberculosis, and the discovery, by Armauer Hansen, in 1880, of ‘a specific leprosy bacillus, which was found to be present in enormous numbers in the lymph channels of the skin in cases of leprosy, paved the way, for the reception of Koch’s discovery of the tubercle bacillus. It was for some time supposed that the tubercular and anesthetic forms of leprosy were different in their origin, and that they might possibly be due to the action of different bacilli, although it was of course known that in some cases there was a kind of mixed leprosy. The fact that the two diseases were the same was, however, strongly accentuated when it became evident that the same kind of bacillus was found in all three forms of the disease—the tubercular, the anesthetic, and the mixed. Before a description of the morphological characters of the bacillus is given, the characters of the various forms of the disease may be briefly indicated, in order that the patho- logical changes produced by the bacillus may be more fully understood, and that the bacilli may be followed and localized in the tissues. In tubercular leprosy there usually appear small irregular spots, sometimes brown, sometimes some- what purple. These spots occur on the face or on the limbs ; they gradually project so as to form more or less marked tubercles, which vary very considerably in size. There is irregular thickening of the tissues under the skin of the face, which gives rise to a most curious facial expres- sion, the patient appearing melancholy or jubilant according to the folds affected ; the tissues about the various orifices of the face, mouth, eyes, nostrils become swollen, and are often studded with tubercles, the lower lip becomes heavy and hangs down in a curious fashion ; these alterations giving the face what is described as the “leonine” appearance. Sometimes sensibility is interfered with, or it may be alto- gether abolished. These tubercles are not confined to the tissues immediately under-the skin ; they may also be found in the submucous tissue of the mouth, larynx, and pharynx; and small nodules may even be met with—especially where the swelling is marked—around the eye in the soft tissue of the eyelids. These nodules frequently ulcerate, especially in 246 BACTERIA. the later stages of the disease. On the fingers the thickened patches and ulcerations may extend so far and so deep that part of the finger may be actually separated and thrown off, first, however, becoming dry and black or mummified. The patients may linger on in this condition for a considerable length of time ; or the tubercles may disappear, leaving dis- coloured patches, in which sensibility is entirely wanting, and- which, in consequence, very rapidly undergo ulceration, usually brought on by injury, the patient, unable to feel, allowing the parts to be injured without making any effort to save them. The second form—anzsthetic leprosy—commences in much thesame way, except that the discoloured patchesaresomewhat smaller, and may be red or brown ; in some cases the patches have a peculiar glistening or silvery-white appearance, The pigmented patches, which are often very widely distributed, have, especially where they attain any very great size, a dull, pallid centre, with a line of brown, or brown and red coloura- tion, surrounding it. Some of the smaller spots are anzs- thetic, whilst, in addition, there may be large areas in which. sensation is markedly diminished or altogether abolished; the hair falls out ; there is marked atrophy of the skin and the cellular tissue immediately beneath, this giving rise to a very wrinkled appearance of the face; and the patient, as in the other kind of leprosy, becomes dull and heavy-looking, as though he had lost all interest in external affairs. Here also injury to the extremities is of very frequent occur- rence, partly owing to the fact that the patient cannot feel, but partly also, because the nutrition of the skin is markedly interfered with. Other skin diseases, the formation of vesicles, and so forth, are often met with. Either form may gradually pass into the other, in which case we have what are known as mixed varieties of the disease. In order to demonstrate the presence of the bacillus in one of the nodules in a case of tubercular leprosy, it is only necessary to tie an india-rubber ring somewhat firmly around the base of one of them until it becomes pale from the blood supply being cut off, and then, with a needle-shaped lancet or the point of a sharp knife, to make a small puncture, from which a clear fluid exudes. In this may be found an enormous number of bacilli. These bacilli resemble the tubercle bacilli in a most remarkable manner, the only difference being that they are, if anything, slightly shorter. LEPROSY. 247 They may be stained by almost any of the methods that are used for staining the tubercle bacillus, although here an method that is used must be slightly modified, as the bacilli characteristic of leprosy are rather more easily decolorized by acids than are the tubercle bacilli, The Ziehl-Neelsen method, with a contrast stain of methyl blue, gives most admirable results, or Gram’s method may be used. The Bacilli from Juice of a Leprosy Nodule. x 500. bacilli so stained are seen to be from 4 to 6 in length and .3-in breadth ; they are more constant in size than are the tubercle bacilli, and, as a rule, are not marked by the curves that are almost characteristic of that organism ; their ends appear to be slightly pointed. It was at one time considered to be beyond controversy that these bacilli contained spores ; but more recent observations on these and other organisms have led observers to the conclusion that what were at one 248 BACTERIA. time considered to be spores are not spores at all, but are appearances due to the alterations that have taken place in the protoplasm during the processes of preparation and staining. As it is not yet certain that the bacilli can be cultivated, little evidence either for or against the spore theory can at present be adduced. These bacilli, to be seen in their characteristic form, should be examined as they lie in the thickened nodules of the skin or in the mucous membrane of the mouth and larynx, in the thickened nerves, in the lymphatic glands, in the spleen, or in the liver. Ifa thin section be made of a nodule of the skin, and the specimen be stained with fuchsin, and decolorized with a mineral acid, and a contrast stain of methylene blue be obtained (see Appendix), it will be found that, throughout the section, branching lines or small rounded points of bright red may be seen on a blue back- ground ; these bright red areas are nothing but masses of leprosy bacilli which fill the lymphatics of the skin, and, as one would expect, interfere very seriously with its nutrition. There seems to be a considerable difference of opinion as to whether these bacilli actually invade the cells or whether they lie free in the lymph channels ; there can be no doubt that a very large number of them ultimately are seen as free bacilli lying in these spaces, the difference of opinion being as to the nature of the so-called “lepra” cells. There are usually found in cases of leprosy a number of large proto- plasmic masses, which are said to be multi-nucleated, and are spoken of as giant cells, and in these are found numerous bacilli. In younger nodules there are smaller masses of protoplasm, in which nuclei and bacilli may be distinctly seen. At the meeting of the British Medical Association, held in Dublin in 1887, Dr. Unna, of Hamburg, placed before the members his views on many of these so-called lepra cells. He contended that the rounded outline was due in many cases tothe form of the lymphatics, and he showed in his specimens that the rounded section was equal in diameter to the longitudinal section of one of these lymphatics filled with bacilli ; that the longitudinal section proved that we have here to deal simply with a lymph channel blocked with leprosy bacilli, and therefore that the rounded masses are merely transverse sections of these stuffed lymph chan- nels. He also maintained that these so-called cells, with their contained bacilli, were merely masses of these organisms LEPROSY, 249 which were held together by gelatinous material ; and that even where there were apparently globular masses in the lymphatic channels, they were nothing but these zoogloea masses, whilst a great number of the organisms were lying entirely free, the nuclei of the so-called lepra cells again being the nuclei of the walls of the lymphatics or of the lymph cells that had become embedded in the gelatinous mass. Many competent observers, however, maintain that the lepra cells have a real existence, and there can be little doubt that bacilli may be found in large cells squeezed from a leprosy nodule. One of the most marked distinctions between tuber- culous and- leprous tissue is, that in tubercle the bacilli are comparatively few in number, especially when they are met with in the more chronic cases—the only cases that could possibly be mistaken for leprosy; whilst in leprosy the bacilli are almost invariably present in enor- mous numbers in the lymph channels of the tissues in which they are growing, on which they seem to exert com- paratively little destructive influence, as they remain for a considerable length of time in an almost quiescent condition, setting up little change, but undergoing no retrogressive changes themselves. Cornil and Babes record that in a small fragment of a leprous nodule that had been left in an envelope, forgotten for nearly ten years, they were still able to stain the bacilli very distinctly ; and sections that had been stained in picro carmine and kept mounted in glycerine for a number of years, it was found, might still’ be stained so as to bring the bacilli into prominence, Then, too, leprosy affects the skin and nerves specially, rarely the lungs and serous membranes. Tuberculosis, on the contrary, affects the latter very frequently, and very rarely the former. From the enormous number of bacilli that are found in the lymph spaces, it can be readily understood that when ulceration of a nodule takes place, these organisms are to be found in large numbers in the blood and serum that is discharged from the ulcerated surfaces. Although this is the case it is a somewhat peculiar fact that this organism is seldom found in the superficial layers of the epithelium covering the nodule, although in the ducts of sebaceous glands, and around the hairs of the skin, as Babes demonstrated, the bacilli may be present in considerable 250 BACTERIA, numbers ; they are seen in the internal sheath of the hair, from which they may, in some few cases, pass through the epithelium, and so to the surface; this is, however, a very rare condition. Asa rule, leprosy bacilli are not met with in the blood ; but in the febrile condition that occurs shortly before death, bacilli have been described as being found in the circulating blood taken at some distance from any nodule. As the leprosy nodules may be found in all parts of the body so also may the bacilli. In cases of anzsthetic leprosy the bacilli may usually be readily demonstrated lying in the dilated lymphatics of the thickened and nodulated nerves. Here, too, as in tubercle, the lymphatic glands are distinctly infiltrated. From its resemblance to tubercle, and from the. fact that its specific bacillus.is found so constantly associated with the disease, being most numerous in those positions in which the leprous processes are most advanced, but being present from the very commencement of the tubercle forma- tion, it is evident that the bacillus bears a constant—probably a causal—relation to the disease, and it was therefore supposed that leprosy might be carried from one individual to another through its agency ; that, in fact, the disease was a specific infective disease and was inoculable. Numerous experiments, made with the object of proving this thesis, have, however, failed. (Babes and Klarindero mention, in support of the contagious nature of the disease, the case of a child that developed leprosy on the lips and cheeks during the time that it was being suckled by a leprous mother, and Dr. Castor and many others insist on the communicability of the disease. A patient has recently been reported to be dying from leprosy, the result of inocu- lation in 1884. The patient was a condemned criminal in Honolulu, who elected to be inoculated with leprosy by Dr. Arning in place of being hanged). Even the inoculation of fragments of leprous tissue gave rise in all recorded experi- ments to no true leprosy, unless the patients were already the subjects of the disease. The cultivation of the bacillus has also proved to be a most difficult matter. Neisser observed a number of small pellicles that appeared to shoot out from small particles of tissue introduced into consolidated blood serum, kept at a temperature of between 37° C. and 38° C. Bordoni Uffreduzzi obtained growths from the marrow of a bone in which there were a number of free leprosy bacilli ; LEPROSY. 251 these appeared on serum (to which a quantity of glycerine had been added) that was maintained at a temperature of 37°C. These he described as delicate, thin, slightly yellow films with irregular borders ; on glycerine agar they are said to have developed as small grey rounded isolated points usually at the end of ten days or a fortnight ; secondary cultivations, however, made their appearance at the end of forty-eight hours, and after the first few cultivations the organism could be grown on serum or on ordinary gelatine and agar, but much more slowly than when glycerine had been added. From the general description, and the im- perfect staining obtained, some doubt must remain as to the true nature of these bacilli. Babes also was able to obtain cultivations on similar media, even from other organs; he described the growths as being very like those of diphtheria ; upon serum they appeared as pale yellow elevated plates, glistening and waxy-looking and surrounded by a transparent indented zone ; the cultures emitted a peculiar characteristic odour. They developed all along the track of the needle, especially in glycerine gelose ; the rods were elongated, but there appeared to be a number of involution forms, and many of the-bacilli were much more plump than usual, and, ‘bearing out Unna’s observations, they appeared to be sur- rounded by a clear capsule somewhat similar to that met with around Friedlander’s pneumonia organism. Babes was not successful in making secondary cultivations, nor was he able to produce the disease by inoculating with his cultiva- tions any of the animals that he had in his laboratory. Until these experiments are confirmed by other observers they can scarcely be accepted as conclusive, as it requires a much longer series of successful cultivations and more careful comparison of the organism as it appears in the tissues with that found in the cultures, than either Bor- doni Uffreduzzi or Babes have made, to set the matter at rest. Still they have indicated the lines on which future work may be done, and we may anticipate that before long Dr. Bevan Rake, Dr. Castor and others, may continue their experiments with the enormous amount of material which they have at their disposal and give to the world most im- portant results. None of the numerous non-bacillary theories as yet put forward to account for leprosy appear to be sufficiently well 252 BACTERIA. supported to be able to oust the bacillary theory from its present position. Leprosy is found in all climates ; it is not specially confined to the seashore, it occurs in regions where fish diet cannot be resorted to, and where other articles of diet such as pork and rice (to both of which have been ascribed a causal agency in the production of this disease) are not used at all. The only factor that is common to all forms of the disease, and that is met with in every case, is the leprosy bacillus, and in spite of the fact that we have not yet been able to trace the method of contagion or infection through the agency of this bacillus, we must, from what is known of the presence and action of bacilli in other diseases, assign to it the rd/e of leprosy producer, until much stronger evidence than we have yet obtained can Be adduced in favour of any other cause. LITERATURE. Authors already quoted. Cornil and Babes. Arninc.—Virch. Arch., Bd. XcviL, p. 170, 1884. Bases.—Arch. de Physiol. July, p. 42, 1883 ; Comptes rendus, t. XCVI, pp. 1246, 1323, 1883. Bases AND KLARINDERO.—Congrés Intern. de Dermatologie a Paris, 1889. Bevan Raxe.—Reports on the Trinidad Leper Asylum, 1888-90; Monats-Hefte f. Prakt. Dermatologie, Bd. vu., No. 12, 1889. Borpont UFrrepuzzi.—Zeitschr f. Hygiene, Bd. m1., p. 178, 1888. Fe.xin, R. W.—On the Geographical Distribution of some Tropical Diseases, p. 36. Edinburgh, 1889. Hansen.—Virch. Arch., Bd. Lxxix., p. 32, 1880. Hirscu.—Handbook of Geographical and Historical Path- ology, vol. 111., New Syd. Soc., 1885. Lrtoir.—Memoires de la Soc. de Biologie, p. ror, 1885. Nersser.—Virch. Arch., Bd. yxxxtv., p. 514, 1881. Unna.—Brit. Med. Assoc., Dublin, 1887; Deutsch Med. Woch., 1885-1886. Vircuow.—Berlin Klin. Woch., Bd. xu., No. 18, p. 123. CHAPTER XIII. ACTINOMYCOSIS. Nature of the Disease—Differences in Cattle, Man, Pig, and Horse— Methods of Preparation and Examination of the Fungus—Microscopic Appearances: of Fungus in Different Animals—Nature of Clubs— Cultivation Experiments—History of Actinomyces—The Actinomyces a Streptothrix. A DISEASE that was for long very imperfectly understood, and which has been described under very different names, is Actinomycosis, which, in 1876, was first recognized by Bollinger as being of parasitic origin. The disease mani- fests itself in cattle, frequently in the jaws, where it is known in various parts of the country as wen, osteo-sarcoma or bony sarcoma, malignant tumour of the palate, or in the tongue, where it may be recognized as “ wooden tongue ;” the pharynx or the loose tissues under the skin of the head and neck, or even the trunk, may also be affected. In the pig, abscesses with running sores are most frequently found in the milk gland and in the tissues around the pharynx ; whilst in horses it is usually found as “ scirri:ous cord,” a condition well known to veterinary surgeons, though as Professor M’Fadyean points out, it may also be met with in the tongue of the horse. This curious disease has also been known to attack the human subject, in whom the lesion resembles in its characters that met with in the pig, the parasite giving rise, by its presence, to abscesses in the lungs, to pus in the pleural cavity, and to similar conditions in other organs; the bones, especially those of the spinal column, are frequently attacked, and “cold abscesses” or chronic gatherings form in those cases where sinuses from which matter may escape are met with. It is interesting to note in this connection that even in the pig the lung is sometimes riddled with similar cold abscesses, and that there is frequently breaking down of areas of 254 BACTERIA. bone in the cervical and dorsal vertebra. Those who had examined the condition only in the human subject and in the pig describe the disease as consisting essentially of a sup- purative process, whilst those who have examined “ wooden tongue”’ in cattle, and “ scirrhous cord ” of the horse, maintain that the process, though sometimes leading to softening, is essentially of the granuloma or new young tissue formation type. These differences, however, appear to be due rather to the nature of the tissues in which the growths occur than to any difference in the character of the organism that produces them, for it is found that in many cases in which the bones are attacked in the cow the lesion takes on a suppura- tive character. It was, however, the non-suppurative form that was first described, and it will therefore be better to examine it in the first instance. In “‘ wooden tongue” of the ox, on superficial examination, firm hard points may be seen scattéred over the surface, varying in size from that of a millet seed to that of a split pea. On incising one of these, it is found to be firm and fibrous at the periphery and especi- ally in the small nodules ; the centre may be soft but not purulent, or, rarely, gritty and almost calcareous ; in the larger nodules little fibrous bands running through them form a kind of net-work, and in the centre of each mesh is a similar soft point. If this softened caseous point be removed with the point of a knife and examined under the low power of the microscope (x 50) it will usually be found to contain, embedded in a small mass of cells, a small core, which by transmitted light is yellow or brownish in colour, dashed with a tinge of green. This little mass is seen to be com- posed of a kind of star made up of a number of wedge- shaped rays, the apices of the wedges meeting in ‘the centre, the bases being rounded; looking down on the centre of this cord it appears as though there are rounded instead of wedge-shaped bodies (this is simply because we are looking down on the rounded ends of the wedges). If now one of the “ bony ” tumours, or one of the tumours in the fibrous covering of the bone, where the tumours are usually of larger size, be examined, it will be observed that the fibrous net-work is exceedingly well marked, especially as on section the softened caseous centre gives way very readily : in the softened material similar “ rayed ” or star-like organisms may be found. In the material that is ACTINOMYCOSIS. 255 discharged from the “cold abscesses” which are formed in the pig or in the human subject, similar small yellow points (which appear as green or greenish yellow grains, even when examined with the naked eye) may be found on examination under the microscope. The pus discharged from these abscesses has an exceedingly characteristic appear- ance; it is usually yellow or brownish yellow in colour, is extremely granular, of a peculiar slimy consistence, and contains the green points that may be said to be specially diagnostic of the actinomycotic condition. The small green points when taken from the cow are very frequently somewhat gritty, this being due apparently to the deposition of particles of lime in the core; but the particles that come from the pus in the human subject are usually soft and tallow-like, so that they can be readily flattened out between two cover glasses. The appearance of the actino- myces or “ray fungus” in cattle, as first described, was so exceedingly characteristic that it was thought it could not be mistaken for anything else, and the corresponding condition in the human subject was for some time over- looked, simply because the same typical appearances were not always developed ; and it was only after some time that, transition stages being found, first in cattle and then in the human subject, the real nature of this fungus was thoroughly understood. On examination of the fungus under a high magnifying power, when the sections have been properly stained, the organism is found to be like the capitulum of a daisy, the sterile flowers in the centre corresponding to the club- shaped rays, and if we conceive of two of these heads of flowers as placed base to base, or stalk to stalk, we may obtain an idea of the appearance of the ray fungus as a whole ; the organism in sections of course having the appearance of sections through the little ball formed by the two heads. The clubs, however, are not all simple, but in some cases branch, sometimes dichotomously, sometimes irregularly, compound clubs being thus formed. In a tumour examined by Professor M’Fadyean, the transi- tion stages between the forms sometimes found in the human subject, and those most frequently seen in cattle, are met with ; and as IJ have had the privilege of seeing Professor M’Fadyean’s specimens I shall follow pretty closely his descriptions, as 256 BACTERIA. I consider that the interpretation he gives of the appearances presented is perhaps the most satisfactory that has yet been published. The tumour from an ox was fleshy in consistence, had a faint pink colour, and was studded with minute softened pink points, and in each of these points was found an actinomyces colony, at the margin of which, however, only a few of the characteristic clubs could be found, the centre being finely granular. On making sections of these tumours, that had been hardened in alcohol, and staining by Gram’s method (see Appendix), and examining under a high magnify- ing power, Professor M’Fadyean observed that the colony consisted of three distinct elements, though, in many instances, the club-like bodies were absent. The first element is a coccus about .5 in diameter ; these cocci are usually arranged in chains consisting of ten or fifteen elements, a few of them are usually found in the centre of the mass, but immediately outside this they are exceedingly numerous, so numerous indeed that they appear to be more like masses than chains; as we approach the margin again the chains radiate outwards and are very distinctly seen. Where they are not very numerous, some larger cocci may be seen under- going regular vegetative division, so giving rise to the forma- tion of diplococci. The second element is a thread-like leptothrix or clado- thrix, a number of which, interlacing freely, form a kind of felted net-work, especially in the centre of the colony. As we pass outwards, however, they gradually assume a more regular radiate arrangement, and near the periphery “ they sometimes shoot out in a tendril-like manner beyond the coccus heaps already described.” These threads vary very considerably in length; sometimes they are divided into short bacilli or even into cocci ; in other cases long threads without any sign of division may be seen. The diameter of these threads is usually greater than that of the cocci. They are described “as some nearly straight, others gently curved, and occasionally they show short nodules almost like a spirillum.” Near the margin these threads sometimes become branched just as in the case of the club-like forms already mentioned. The club-like forms when met with here appear to bear a definite relation to the threads ; they are only found at the margin of the fungus mass ; they are arranged radiately with the ends of the clubs outermost ; ACTINOMYCOSIS. 257 they vary much in length, and are usually quite simple, “ but some carried lateral buds, and occasionally two appear to be carried by a common stalk.” These buds are best stained by the Ziehl-Neelsen method; some of them exhibit a very important relation to the leptothrix forms already described, the thread appearing to be continued into the centre of the club, the outer part of which is formed by a homo- geneous, somewhat faintly stained material. This axial thread may be divided into longer or shorter segments, corresponding apparently to the bacilli and cocci forms. They are usually most divided and are undergoing most marked changes in the larger clubs, whilst there are also small rounded bodies, which appear to be essentially of the nature of cocci, surrounded by the same material that forms the thickened club. In some of the smaller colonies of the actinomyces Professor M’Fadyean describes cocci only, which are usually arranged in short chains or in little groups. They are embedded in masses of leucocytes, some being actually within the cells, and appearing to be the points from which the larger colonies start, the cocci being carried by the leucocytes from point to point. Other colonies contain only cocci and thread forms, the threads in this case appearing to be developed from cocci, whilst those colonies in which clubs are present appear to be in a still more advanced stage of development. By some observers the club-like forms have been described as the spore-bearing parts of the organism, and are spoken of as Conidia or Basidia, but it appears from the above case, and from those that have been described in the human subject, that the thickened extremity is due merely to a kind of involution process, and occurs in the older thread- like organisms as the result of a growth and swelling of the outer sheath of the Cladothrix threads, this sheath corresponding, in fact, to the gelatinous material which is formed in zoogloca masses of other organisms, or to the capsule first described by Friedlander which is formed around the bacillus of pneumonia. When these clubs are once de- veloped the central part may be only partially active, whilst the periphery remains passive, but extremely resistant to the attacks of the surrounding cells, which attack the club- shaped masses very vigorously, the large thickened clubs being frequently found in various stages of degeneration 18 258 BACTERIA. within the large masses of protoplasm found in this region. In the human subject the felted network, the cocci and the bacilli are usually most numerous; they are, in fact, said to be typical of actinomyces in the human subject. Here again the involution or club-forms are frequently met with, especially in the pus that is discharged from the abscesses of the lungs or of bone. The slimy pus in these cases, however, appears to contain a considerable proportion of the “ mucine,” that in cattle goes to form the thickened sheaths above described. The process of evolution of the “Ray” fungus is much the same in the human subject as in the abnormal case already described as occurring in the cow. It is interesting to note that most of the experiments that have been made on the cultivation of this organ- ism have been attended with complete failure—a failure that in some measure, at any rate, appears to be due to to the fact that almost all experimenters have used for their inoculating material only those colonies in which the club- shaped organisms have become well developed. The first attempt that was at all successful was made by Bostrém, who, throwing aside the club-like processes, took for his inoculating material the central network, selecting as far as possible young growing colonies for his seed material. His method of procedure was as follows: With the utmost care he removed small colonies, which were at once in- troduced into sterilized gelatine, with which a “plate” cultivation was made. After a few days any points that were found to be pure, ze. around which other ovgan- isms were not growing, were removed from the gelatine with a sterilized platinum needle, and were crushed be- tween sterilized glass plates; with the platinum needle, stroke cultivations were made on ox blood serum, and agar-agar. A finely granular growth first made its appear- ance along the line of inoculation; this gradually became more marked, then small yellowish red nodules were seen, around which delicate branched processes spread out ; these yellowish masses soon began to run together, and at the end of seven or eight days were covered with a delicate fluffy white layer. This growth apparently went on best at the temperature of the body, and on microscopic examination ACTINOMYCOSIS. 259 of the structures, cocci, segmented threads, longer threads and clubs were all found ; threads and clubs alike in most cases being characterized by a branching similar to that met with in the fungus as it grows in the human body. Inocula- tion of this fungus into the peritoneal cavity of rabbits and other animals was usually attended with positive results. Later, by attending to the same details as regards the nature of the seed material, M. Wolff, J. Israel, and Babes have all succeeded in cultivating this organism on agar-agar, blood serum, and especially on the raw white of egg, according to Hueppe’s method (see Appendix), and with the cultivations so obtained actinomycosis has been produced experimentally in animals. ” From all these facts it will be gathered that actinomy- cosis is the result of the activity of a living organism introduced into, and existing as a parasite in, the animal tissues ; that the same organism may be found in animals and in the human subject, that the club-shaped organism is really an involution stage, and that the characteristic growth is the mycelial thread-like mass which appears to develop from the cocci. The fact that we are unable to cultivate from the clubs alone, affords ample evidence that, in place of being spore-bearing masses, they are merely encysted, or thick-walled, involution forms. , As to the mode of infection, it has been pointed out that the actinomyces has been found lodged in the crypts of the tonsils of the pig and of the human subject, and that the parasite evidently leads an epiphytic life on barley and other cereals. It may be introduced from without through wounds, as in cases of scirrhous cord in horses, or through inoculation, by means of accidental scratches of the skin, or of the mouth and pharynx, and it is recorded that a case of ptimary mediastinal actinomycosis in the human subject was in one case supposed to be traced to perforation of the back of the throat by a barley spikelet swallowed by the patient. In pigs the mammary affection is thought to be due to the entrance of the actinomyces into the teat ducts. In Denmark the farmers attach so little importance to infection from animal to animal, that cows in which these acti- nomycotic tumours are well developed are allowed to run with the rest of the herd, at any rate until suppuration 260 BACTERIA. commences; when this occurs, however, the animals are usually slaughtered. I may here summarize what is known of the history of a colony in the words of M’Fadyean, who has given the subject much careful study, and to whose authority in this matter I attach much weight. “yx. It has its starting point in one or more cocci trans- ported by the plasma currents, or by the agency of a carrier cell (leucocyte). “2, The cocci multiply by elongation and subsequent fission. When undisturbed by the surrounding leucocytes their growth and multiplication are after the manner of a streptococcus, but frequently they become irregularly grouped together (Staphylococcus heaps). “3. By elongation, some of the cocci give rise directly to short bacillary forms, and through these to long filaments. “4. The further extension of the colony is effected by the growth and multiplication of both threads and cocci. The former multiply by segmentation into bacillary elements, which may again elongate to leptothrix forms. “5. The leptothrix filaments may give rise by close segmentation to coccus forms. “6, The formation of clubs and similar forms is evidence of diminished vegetative power of the filaments (possibly also cocci), in connection with which they originate. “9, The growth of a colony may be arrested at any stage by the agency of the animal cells (leucocytes), or by failure in the supply of the necessary pabulum. Jn that event the majority of the threads tend to develop clubs at their outer ends (involution forms). The central cocci and the remainder of the filaments then disintegrate ; but the clubs which offer a greater (passive) resistance to the surrounding cells may persist for an indefinite period.” From what is here stated, and from what I have already said, it will be evident that we have here to deal not with an ordinary “mould” fungus, but rather with a form of streptothrix that undergoes dichotomous division at certain points. It must therefore be looked upon as possibly one of the Cladothrix alge, or more probably as one of the Schizomycetes in which the Cladothrix formation occurs, and which has'been described as closely allied to the strepto- thrix Forsteri. ACTINOMYCOSIS. 261 LITERATURE. The following works may be consulted : Acianp.—Brit. Med. Journ., vol. 1, p. 1159, 1886; Trans. ’ Path. Soc., vol. xxxvul., p. 546, 1886. BanGc.—Tidskrift for Veterinaer, 1883. Botiincer.—Centralbl. f. d. Med. Wiss., No. 27, p. 481, 1877. BostromM.—Verh. d. Congr. f. Inn. Med. Wiesbaden, p. 94, 1885. *CROOKSHANK.—Manuel of Bacteriology, London, 1890 ; Medico Chir. Trans., vol. Lxxur., p. 175, 1889. DELEpPINE.—Trans. Path. Soc. 1889. IsrAEL, J.—Virch. Arch., Bd. cxxiv., Heft. 1, p. 15, 1878 ; Bd. txxvitt., Heft. 3, p. 373, 1889; Bd. xcvi., Heft. 1, p. 175, 1884. P Joune.—Deutsch. Zeitschr. f. Thiermed. *M’FapyEan.—Journ. Com. Path. and Therap., vol. IL, p- 1, 1889. PERRONCITO.—Giornale di Anat. Fisiol. e. Pathol. 1875. Ponrick.—Die Aktinomykose des Menschen, Berlin, 1881 ; Breslauer Aerztl. Zeitschr., 1882. TrEvES.—Lancet, vol. 1., p. 107, 1884. Wotrr.—Virch. Arch., Bd. xcu., Heft. 2, p. 252, 1883. * Full references given. CHAPTER XIV. GLANDERS. Glanders—Farcy—Clinical Appearances of the Disease—Chauveau’s obser- vations on Glanders Poison—Loffler and Schiitz—Method of Demon- strating the Glanders Bacillus—The Bacillus—Methods of Cultivation —Glanders in Various Animals—Farcy in Man—Temperature relations of Bacillus—Desiccation—Germicides. ALTHOUGH one of the best known, and, from its anatomo- pathological point of view, best described of all diseases with which the veterinary surgeon has to deal, it was long before most observers in the veterinary schools could be brought to look upon glanders as a contagious infective disease. It is found especially in the horse and the ass, but as in the case of tetanus, it may also be encountered in other animals. When it occurs in or under the skin it is known as Farcy, and in this form is usually found in man. The primary disease is usually in the respiratory passages, the lungs and the skin, especially around the orifices of the nostrils and mouth ; the parts secondarily affected may be those in communication with the lymphatics from an in- fected area, or we may have distinct metastatic abscesses (or abscesses at some distance from the original focus of the disease), the material in such cases being carried apparently by the blood along the course of the blood vessels. In all cases the intensity of the disease appears to be in direct ratio to the number and rapidity of formation and softening of the small nodules, the process, as we shall find, being determined by the presence and activity of a specific bacillary organism that has made its way into the tissues. The small nodules, whether they occur in the skin or on the mucous membrane, say of the septum of the nose, appear as small grey, gelatinous-looking points about the size of a millet seed. In spite of this gelatinous appearance they are usually comparatively firm in consistence. Under GLANDERS. 263 the microscope each of these points is found to be made up of granulation tissue, consisting of small round cells very like leucocytes or lymph corpuscles, a few larger nucleated cells, with here and there fragments of disintegrating con- nective tissue. After a time, as the nodules become rather larger, the centre assumes a yellowish appearance, or small opaque points may be seen, and ultimately at these points we have the tissue breaking down into soft, pulpy, or caseous, purulent material. The small nodules met with in the early stages of the disease, are usually surrounded by a deeply- congested area, and if they occur in the nostril the mucous membrane covering them is greatly congested, and there is great discharge of water, or of very watery “ matter,” from the nostril. As the nodules become softened in the centre, ulceration of the tissues near the surface takes place, the softened centre escapes, and a “ punched-out” ulcer is left. These ulcers may gradually run into one another, and from the fact that the mucous membrane is congested and thick- ened, the loss of tissue seems to be very great—much greater than it really is. Along the lines of the lymphatics, and in the lymphatic glands communicating with the ulcerating surfaces, there is great inflammation and induration, what are known as “‘farcy-pipes ” being formed. Here just the same processes are carried on as when the disease occurs in the mucous membrane, in the lungs and in the glandsat its root, the lymphatics being of course similarly affected. It will thus be seen that glanders resembles, in a most remarkable man- ner, certain other infective diseases already described, such as leprosy, tubercle, actinomycosis, &c., and, as a matter of fact, numerous observations were early made with the view of proving, first, that this was really the case ; and, secondly, that the contagious element was a contagium vivum, and probably a form of micro-organism. Chauveau, as early as 1869, inferred from a series of interesting and most ingenious experiments that the poison was particulate in character, and was, very probably, present in the leucocytes. In December, 1882, Léffler and Schiitz made known the results of experiments by which they had been able to demon- strate the presence of a bacillus which appeared to have a distinct causal relation to the disease ; and about the same time Bouchard, Capitan, and Charrin also described an organism in glanders—an organism, however, the charac- 264 BACTERIA. ters of which were somewhat different from those ascribed by Léffler and Schiitz to their bacillus. As I have from time to time had opportunity of verifying a number of the points insisted upon by these latter, and as it is now generally accepted that the Léffler-Schiitz bacillus is the one really associated with the disease, this organism may be briefly described. The best method of demonstrating the bacillus is to take a small particle of the softened central part of a nodule and squeeze it out between two cover glasses ; all superfluous matter from the edge is carefully removed, and the covers are then treated in the ordinary way, after which they are stained in a mixture of concentrated alcoholic solution of methyl blue, one part to three parts of a 1 in 10,000 liquor potassz solution; the cover glass is rinsed for about a second in a one per cent. solution of acetic acid which has been tinged to the colour of Rhine wine by the addition of a watery solution of tropxolin. It is then quickly washed with distilled water, then with absolute alcohol, cleared up with cedar-oil and mounted in benzol- or xylol-balsam. Sections of a nodule that has been hardened in absolute alcohol should first be placed for a few minutes in - weak caustic potash solution, after which they may be transferred to the stain and treated as above. An even better method of staining is to use Ziehl Neelsen carbolic fuchsin or carbolic methylene blue, and then to decolorize the tissues with distilled water, or with a two per cent. solution of hydrochloric acid. Kiihne’s carbolic-methylene blue method may also be be used for staining sections of tissues. After preparation there may be seen minute rods from 2.5 to 5 in length, which are usually one-fifth to one- eighth of their own length broad. They are always more numerous where cell proliferation is going on most rapidly. From the fact that these organisms do not take on any stain at all readily, and also that they are very easily decolorized, it is often an exceedingly difficult matter to distinguish them from nuclei and nuclear detritus, both of which take on staining material in much the same manner as the bacilli, and it is only by the exercise of the greatest care and by the use of the very best optical appliances that these organisms can with certainty be distinguished in the tissues. It is, therefore, all the more necessary to obtain pure culti- vations of the glanders bacillus in order that its characters may be accurately described, and to determine whether it really plays an important etiological part in the production of the disease. The first successful attempt to cultivate this specific bacillus was made by Schiitz in 1882. Adopting the strictest GLANDERS. 265 precautions to prevent the entrance of extraneous organisms, he took small particles of the grey translucent material surrounding the caseous centres of some of the above described: nodules from the liver, lung, spleen, and lym- phatic glands of a glandered horse. These small particles carefully broken down, were inoculated on fluid and solid blood serum from the horse and from the sheep, and also in broths made from the flesh of the dog, horse, fowl, and ox, and even in various fruit and vegetable infu- sions. For two days no indication of the presence of any growth could be observed on any of these media, but on the third day the fruit infusion became slightly turbid, and on the gelatinized serum there appeared numerous small, clear, transparent, yellow, slightly elevated drops, like drops of a yellowish fluid that had been splashed on the surface of the serum. In eight to ten days these became slightly cloudy or milky.t On examining one of these small drops under the microscope it was found to consist entirely of masses of short, rod-like bacilli, similar to those already described as present in the glanders nodules, giving the same colour reactions, being perfectly distinct in this respect from the tubercle bacilli, which, as regards size and general appearance, they very closely resemble. Similar bacilli were found in the fluid media, and were usually in the form of pure cultures, that is, only this single kind of organism was present. In some cases there were impurities, but these were so evident to the naked eye that they could be detected at once, such impure cultivations being thrown aside. The bacillus is usually straight or slightly curved, is rounded at one end, and if any difference at all can be observed between it and the tubercle bacillus, it is slightly shorter and perhaps thicker than that organism, especially « In order to obtain growths on sterilized potatoes some of the nasal discharge from a glandered horse should be mixed with from 100 to 10,000 parts of boiled distilled water, and_a few drops of this mixture run over the surfaée of the potato. Three days after inoculation there appear spots of an amber-like growth; these points, as they increase in size, become redder and more opaque, the colour deepening until it becomes like ‘‘ copper oxide.” The manner of growth, according to Loffler, is quite characteristic ; there are only two which are at all like it, the bacillus of blue pus (the growth of which has a yellowish-brown tinge on potato but none of the amber transparency and a peculiar pearly iridescence), and the cholera bacillus. 266 BACTERIA. when it is grown ina fluid medium. It exhibits no movement. With pure cultivations, obtained in this way, and propagated for several generations outside the body, Loffler and Schiitz succeeded by careful inoculation in producing typical glanders in various animals, the artificially infected animals pre- senting on examination exactly the same appearances as the animals that had been naturally infected, and from the nodules artificially produced the typical glanders bacillus could again ‘be cultivated and passed on to other animals, where they, in turn, gave rise to the usual symptoms of the disease. In making these experiments they came across a most interesting point. Inan old horse which they inoculated, and that was apparently quite healthy at the time of the opera- tion, they observed that the disease remained perfectly localized, and that the ulcers very early evinced a marked tendency to heal. The animal, however, was killed, and on making a post-mortem examination it was found that it had already, in all probability, been affected with glanders,and that this had been running its course for some considerable length of time, as not only were there old scars on the septum nasi, but there were old caseous masses scattered through the lungs. We have here an exactly analogous condition to that recently observed by Koch in the case of guinea- pigs already afiected with tuberculosis, on which he made the observations that led him to the discovery of his protecting fluid ; this animal had been “ protected” against the general outbreak of glanders which usually results from an artificial inoculation by a previous chronic attack of the disease. In order to illustrate the method of procedure in such cases, we may give a short resumé of one of Loffler and Schiitz’ series of experiments. Two horses were inoculated—one twenty years old from the eighth cultivation, and the other two years old from the fifth serum cultivation that had originally been taken from a [guinea-pig which had in turn been inoculated from a fourth generation (also grown on serum), originally taken from another animal. Both these horses were inoculated on each side of the neck and on the breast ; and the younger animal was also inoculated in the posterior nares. This was purposely omitted in the older animal, in order to see whether the ulcers would occur in the nasal mucous membrane if it were left intact, as far as direct inoculation was concerned. At the end of a few days both horses had been attacked ; they ate badly ; diffuse boggy swellings made their appearance at the points of inoculation; they were stiff in the joints, the hair was ruffled, and on the eighth day farcy pipes corres- ponding in their distribution to the course of the lymphatic vessels and glands of the affected area could be distinctly felt under the skin. At this time GLANDERS, 267 the swellings at the points of inocitlation had ulcerated, and were dis- charging an opaque, greenish-yellow fluid. On the twelfth day an ulcer about the size of a shilling appeared on the skin of the forehead; its margins were thickened, and it was so deep that it extended down to the bone ; there was a discharge from both nostrils, at the margins of which it dried into thin yellow crusts or scales; then small ulcers with indurated and thickened margins appeared on the nasal mucous membrane in both animals ; in fact there were here-all the characteristic features usually associ- ated with glanders. These two animals died within twenty-four hours of each other, and typical glanders lesions were found in the tissues after death. Guinea-pigs inoculated with material from these cases exhibited similar characteristic symptoms and lesions ; they died in from fifteen to fifty days, and on post-mortem examination it was found that the characteristic macro- and microscopic appearances were present in all of the lesions. In the study of the specific infective diseases, it has been found that certain animals are especially susceptible to one specific virus, whilst others are but little affected, and that in the case of a second virus, things may be exactly reversed—the non-susceptible animal remaining entirely immune, or, in place of a constitutional disease being set up, only small local lesions making their appearance. It is therefore necessary for experimental purposes to determine in all cases what small and easily-kept animals are susceptible to a given disease, and what are unaffected or are only slightly affected by the virus of that disease. In the special case of glanders it is a matter of very great importance, from the diagnostic point of view, that the guinea-pig is very susceptible to the disease, the natural virus or pure cultivations of the glanders bacillus setting up a typical disease which eventually brings about the death of the animal. Similar inoculations into a rabbit produce merely a slight rise of temperature, some local irritation accompanied by swelling, and per- haps by slight ulceration, without any further constitutional or general symptoms. Having found that this is the case as regards these two ani- mals where the inoculations are made from animals that are undoubtedly glandered, it is evident that in a doubtful case of glanders, strong proof for or against the specific nature of the disease may be obtained by inoculating rabbits and guinea-pigs with material from the doubtful sources ; if the guinea-pig dies with characteristic symptoms, and the lesions remain local in the rabbit, there is strong presumptive evidence (it might almost be .said, definite evidence) that the suspected case is one of glanders In connection with this immunity (complete or partial) or susceptibility of different animals to this disease, it should be pointed out that the human subject may be inoculated either through wounds or scratches or through the application of 268 BACTERIA. the nasal discharge of a glandered animal to the mucous membrane of the nose or mouth. There are, undoubtedly, cases recorded of glanders occurring in the human subject, but these are not so numerous as they might be if it were possible to put all those cases described as acute or chronic bood poisoning under their proper heading. An old friend of mine, the late Dr. Howard Bendall, in a Thesis. presented for the degree of M.D. in the University of Edinburgh, 1882, described a case of acute farcy in man, and collected the records of 68 similar cases,a number that might now be very considerably added to. Of all the cases of acute farcy, 47 in number, only 6 were cured ; whilst of 21 more chronic cases no fewer than 15 recovered or were partially cured: the acute cases run a very rapid course, the duration of the disease, however, varying from 4 to 47 days, the average course of the disease being from 2 to 3 weeks. In chronic farcy the patients died in from 50 days to 14 months, whilst of those that recovered, the disease lasted as long as two and a half years. It has now been proved beyond doubt that this disease of farcy in man is due to the action of the same bacillus that is found in the glanders of the horse, this minute organism having been found both in the blood and in the contents of pustules taken from a man affected with farcy. Cattle are completely immune against glanders as regards spontaneous infection, and only localized ulceration, which rapidly heals, follows inocu- lation. The goat is somewhat susceptible to the disease, though it appears to occupy a position between cattle and the horse in this respect, Sheep are fairly susceptible, but the disease runs its course very slowly, and appears to resemble the chronic farcy in man. Lions, tigers, and cats may all become affected, the disease in such cases running a very rapid course. Dogs react to the poison very much as do rabbits. So markedly is this the case that it has been suggested that the glanders bacillus might be attenuated by passing it through the dog before it is inocu- lated into horses and asses. It should be pointed out, however, in connec- tion with all these experiments, that if a very concentrated virus, such as a pure cultivation of the bacillus, especially in considerable quantities, be inoculated even into a rabbit, a generalized disease may be set up, whilst a weaker virus, such as that contained in the discharge from the nasal mucous membrane of a horse, will produce nothing but localized symptoms. (This element of quantity can never be ignored in making experiments with bacteria of any kind.) Field mice are extraordinarily susceptible to the glanders poison, whilst white mice and house mice are quite exempt. The pigeon appears to be the only bird that is at all susceptible to the disease, GLANDERS. 269 We have already spoken of bodies that looked like spores in these bacilli, but from the fact that the glanders virus, both in fluids and in tissues, loses its vitality after fifteen days’ drying, it must be assumed that the organism does not form endospores similar to those that are found in bacillus subtilis, for example. The bacillus grows best at a temperature of 37° C., it will not grow at 20° C., nor at 45° C. at the other extreme. At 22° C. it commences to grow slowly, whilst at 25° C. it flourishes most luxuriantly, although it rapidly loses its virulence when cultivated for several genera- tions outside the body. Commenting on this fact, Léffler points out that glanders is essentially a disease of hot countries, where the comparatively high temperature appears to be extremely favourable to the development of the bacillus outside the body, especially in such materials as fodder, manure, and stable refuse generally. We have interesting evidence of this in statistics collected by Krabbe, who gives the following proportion of horses affected with disease per annum per 100,000 horses in the following countries: Norway, 6; Den- mark, 8.5; Great Britain, 14; Sweden, 57; Wurtemburg, 77; Russia, 78 ; Servia, 95 Belgium, 138; the French Army, 1,130; the Algerian Army, 1,548. , As already mentioned, desiccation for twenty-one days is usually quite sufficient to prevent the multiplication of the bacillus when placed in nutrient media. Consequently it may be possible, by proper ventilation, to diminish the mor- tality from this disease even in the warmest countries." Another agent which helps greatly in preventing the mul- tiplication of this bacillus is putrefaction, as the organisms or products developed during that process appear to interfere very markedly with the growth and multiplication of the glanders bacillus. The most satisfactory of all disinfectants, however, is heat, and it has been proved experimentally that a temperature of 55° C., continued for ten minutes only, is quite sufficient to destroy the bacillus, and with it the infective power of the virus, from the fact that no spores, probably, are formed to perpetuate the species. A spray of steam would therefore, in all probability, be the most t Léffler, however, was able to kill with a virus that had been dried on silk threads for eighty-nine days, and Fraenkel states that the organisms or their spores withstand drying. 270 BACTERIA, serviceable and the most available of all disinfecting agents, or perhaps, better still, a thorough washing out of the in- fected stalls with boiling water. Chlorine and carbolic acid are both capital disinfectants. A 2 per cent. solution of carbolic acid applied for twenty-four hours kills the glanders bacillus, and a solution of 4 per cent. applied to the nasal: discharge for a single minute renders it perfectly innocuous. AI per cent. solution of permanganate of potash,'0.23 or even a 0.16 solution of chlorine water, or one-fifth per mille solu- tion of corrosive sublimate, will also render the bacillus perfectly innocuous within a couple of minutes. By means of any of these the process of disinfection may be carried on in stables in which glanders has occurred, with the greatest ease and with absolute certainty. LITERATURE. The following works may be consulted : -BenpaL, Howarp.—Graduation Thesis Univ. of Edin. 1882. BoucHarD, CAPITAN AND CHARRIN.—Bull. de l’Acad. and Rev. de Méd. Francaise. Dec. 30, 1882. CHavuvEAu.—Coniptes rendus, t. yxvut., No. 14, 1869. FLEMING.—Manual of Vet. Sanitary, Science and Police, vol. L, p. 509, 1875. or : GrUNwALp.—Oesterr. Monatsschr. f. Thierheilkunde, No. 4, 1884. IsraEL.—Berl. Klin. Woch., No. 11, 1883. KrasBe.—Deutsch, Zeitschr. f. Thiermed, Bd. 1, Heft. 4, p. 286, 1875. *LOrFLER.—Arbeit a. d. k. Gesundheitsamt, Bd. 1, p. 141, 1886. LOFFLER AND ScHiirz.—Deutsch. Med. Woch. Dec., 1882. *M’FapvYEAN AND WoopHEaD.—Reports National Vet. Assoc. 1888. ; : Scutirz.—Journ. Comp. Med. and Surg., vol. vir., No. 1, p. 196, 1886. VuLPIAN AND Boutry.—Bull. de ’Acad. de Med. 1883. Wassitierr.—Deutsch. Med. Woch., Bd. 1x., No. 11, p. 135, 1883. WEICHSELBAUM.—Wien. Med. Woch., No. 24, p. 754, 1884. * Full references given. CHAPTER XV. ANTHRAX. The Bacillus Anthracis—Early Observations—Pollender—Davaine—Koch — Pasteur—Methods of Examination—Appearances of Bacillus under Different Conditions—Spore Formation—Non-spore Bearing Bacilli— The Vitality of the Bacillus and of the Spores—Cultivation Experi- ments—Cover-glass Preparations—Inoculations into Animals—Methods of Infection—Anatomical Characters of Malignant Pustule—Animals Affected—Spores not formed in the Living Body—The Disposal of Anthrax Carcases—Various Disinfectants—Pathogenic and Sapro- phytic Anthrax—Buchner’s Experiments on Anthrax Bacillus and Bacillus Subtilis—Hueppe and Wood’s Experiments. ANTHRAX, or splenic fever, is perhaps the best known of all the specific infective bacillary diseases. The Bacillus An- thracis, compared with other pathogenic organisms, is of very considerable size; it is from 5 to 20p long, and 1 tol. 5p broad. It multiplies with very great rapidity in the blood of certain animals, and may be very easily cultivated outside the body ; in consequence of these features it was the first organism that was proved definitely to be associated with a specific disease, and it was certainly one of the first to be recognized as occurring in both animals and in man. In 1849, Pollender, and in 1850, Rayer and Davaine described these organisms as occurring in the blood of animals that had succumbed to splenic fever; then again, in 1857, Brauell, examining the blood of a man affected with anthrax, found this same bacillus. Later, as already described, Pasteur’s wonderful experiments on fermentation were published, and these led Davaine, in 1863, to commence a series of observations on anthrax, which, carried on until 1873, gave everything but absolute proof that the anthrax bacillus was the actual exciting cause of this malig- nant disease. This proof, however, was not supplied until 1876, when Koch, who had then been working at the subject for 272 BACTERIA, some time, furnished most rigorous proof of Davaine’s hypo- thesis. At the same time he was able to give much additional information as to the development, mode of production, and general life-history of the organism—information that could only have been obtained by a long continued and careful study of the organism outside the body. : The following year Pasteur also succeeded in cultivating Photo-micrograph of Anthrax bacilli in a preparation of the fresh spleen pulp of a cow that had died from splenic fever. x 1000. . this organism as a saprophyte and from that time up to the present new facts have constantly been garnered, and our knowledge of the biological history of this organism has been greatly extended. To observe the organism, a drop of blood is taken from the spleen of a cow (or of any other animal that has died of anthrax), and spread out between two cover glasses ; these are ANTHRAX. 273 then separated, and one of them is at once lowered on to a drop of three-quarter per cent. salt solution, the other being set aside to dry, after which it may be gently heated in the flame of a spirit lamp and then stained in a water solution of methylene blue, well washed in water and alcohol, and mounted in a drop of water or glycerine. In the unstained specimen there will be found lying between the red blood cor- puscles a number of short rods of the size above mentioned, each of which has slightly rounded ends; sometimes also there may be seen a delicate transverse mark running across the middle, this being especially well marked when the rods are longer than usual. The centre of each rod in the stained specimen appears to be quite homogeneous, and is usually deeply stained ; around this deeply stained portion is a kind of sheath which remains unstained, or is only slightly tinged by the colouring reagent. In some cases there is also at the point of junction, on each side of the transverse line, a some- what oval area slightly stained, so that when a number of these rods are placed end to end without being separated they have very much the appearance of a finger with the joints slightly enlarged, or of a bamboo cane with its charac- teristic thickenings placed at almost regular intervals. Both rods and threads are perfectly motionless. In other cases, in place of a mere transverse line, there is the appearance pre- sented of two bacilli that have only recently become separated from one another, still close together, however, and often so dis- posed that they enclose an angle. In the coloured prepara- tion the ‘same thing is observed. It is now seen that where the rods obtain any considerable length they are distinctly segmented, each chain being divided into a number of short rods, and at regular intervals at the points of segmenta- tion there is usually a slight swelling, although as yet there is no trace or evidence of any spore formation. In cases of wool-sorters’ disease, which is very frequently accompanied by pleurisy, these organisms may be found in the fluid that accumulates in the chest as long threads, which may be grouped together in a kind of network or felt, the individual threads of the bundles often reaching an enormous length without there being any appearance otf segmentation. A similar growth of long threads has also been obtained by cultivating the bacilli, from the blood of the guinea-pig 19 274 BACTERIA. affected with anthrax, for two or three hours in aqueous humour, the threads then become elongated, the marks of division are not nearly so distinct, and the threads remain homogeneous. It is possible, therefore, that the appearances met with in the fluid in the chest are due to the fact that the organisms have been allowed to remain at rest in the exudation, and that a process of cultivation has been going Photo-micrograph—Modified Anthrax bacillus—longer threads leptothrix form. x 1000, | on. In preparations made from the blood it is found that the corpuscles are somewhat irregular in shape, that they are running together in little irregular masses, and that the bacilli are much more numerous than the blood corpuscles, As regards the size of the bacilli it may be observed that the rods vary not only in length (in the same animals), but that they are of different breadths according as they develop in ANTHRAX. 275 one species or in another, those found in a guinea-pig being thicker than those seen in the mouse or sheep, whilst those in the rabbit are thinner than any of those above mentioned. If one or two of these bacilli be placed in a moist cell, in what is called a hanging drop of nutrient broth, and the temperature be kept at about 37° C., the organism may be kept under observation under the microscope during the whole course of its development. First, the short rods so increase in length that they may ultimately cover a whole field of the microscope, the protoplasm at the same time becoming granular ; then a large number of very minute points make their appearance, the hyaline appearance is gradually lost, and the threads become quite opaque. A little later, if the observation be continued, it will be found that instead of a single thread, little bundles of the same long threads make their appearance, in which there are found, at pretty regular intervals, the highly refractile bodies with well-defined margins and sheaths which have already been described as spores. When the spores occur in long threads there is usually a slight enlargement at the point at which the spore is situated, and this, with the bright shining point, gives to these spore-bearing threads an appearance that is described as being like a chain of pearls. In many cases the spores make their appearance in the shorter rods.t_ In place of the regular forms above described there are others, or involution forms, which are found to present themselves when the organism is grown under unfavourable conditions ; for ex- ample, irregular moniliform threads are usually met with where the temperature is too high or too low, the soil is exhausted and so on. It has been found by Lehmann, Hime, Buchner, Behring, and Roux that anthrax bacilli may be obtained that do not give rise to spores. This appears really to be due to an interference with the vitality of the protoplasm, as asporo- genous organisms are best obtained by acting upon them with antiseptics, be these chemical or physical. Roux, for example, was able to obtain asporogenous bacilli, and bacilli that remained asporogenous for several generations, simply t The development of the spore has already been described, as most of the descriptions of the setting free and development of the spores are taken from observations made on this species and described by Koch, Buchner, Prazmowski and others. 276 BACTERIA. by immersing them for some time in 1,000 parts of nutrient fluid, to which one part of carbolic acid had been added. Under these circumstances the growth of the organism is not completely prevented, but no spores appear to be de- veloped. It cannot multiply at a temperature below 16° C. nor above 45° C., 30° to 37° C. being the optimum tempera- ture. It is distinctly zrobic in its growth, and cannot develop unless it can obtain a pretty free supply of oxygen. It has already been stated that the organisms which occur in the blood are homogeneous, and it is a well-known fact that spores are never developed in the bacillus that grows in the blood, the multiplication there being entirely vegetative in character, and being due to fission or division of the rods as they increase in length. The same thing holds good even in the dead body so long as it remains intact ; when once, how- ever, the blood is allowed to come to the surface and in con- tact with oxygen, spores are very rapidly formed within the bacilli. This spore formation will not take place below from 24° to 26° C. (Koch says 18° C.), and then only in the presence of oxygen, so that they can best be seen in those bacilli that are cultivated on the surface of such nutrient substrata as agar-agar, solidified blood serum, and potato, or in fluid media through which a pretty constant stream of oxygen is allowed to pass. In this respect the spore formation of anthrax bacilli ap- pears to agree with the sporulation of yeasts, which, it will be remembered, takes place best on a surface of plaster of Paris that is constantly kept moist and well supplied with air. Anthrax bacilli as distinguished from the spores are very readily killed. The ordinary putrefactive processes that are undergone in the decomposition of carcases in which these organisms have been present during life, especially if the air be excluded, cause the death of these bacilli in about a week, The temperature of boiling water maintained for a few seconds kills the bacilli, but, according to Klein, boiling for ten minutes is not to be relied upon to kill the spores although Koch states that at 100° C. the spores are killed in five minutes. The bacilli are killed by a two minutes’ ex- posure to a one per cent. solution of carbolic acid in water, whilst the spores may remain alive for more than a week in a similar solution. ‘They must be kept for nearly a week in a three per cent. solution, and twenty-four days in a five per ANTHRAX, 277 cent. solution if these reagents are to have any lethal effect on them. Carbolic acid in oil—five per cent. solution —had little more, if any, effect than pure olive oil. The spores remain alive, for an indefinite period almost, ina five per cent. solution of olive oil. Sulphurous acid vapour in the propor- tion of one to one hundred of air kills the bacilli in half an hour, but the spores resist the same antiseptic for seventy-two hours. Corrosive sublimate in one per mille of water suffices to kill spores by simply wetting them. ‘These tests of the vitality of the spores, however, were made by inoculating them into gelatine after they had been treated with the antiseptic. If, in place of inoculating into a nutrient medium of this nature, the spores are introduced into the circulating blood of a living animal, it has been found by Klein that it requires much longer exposure and much stronger solutions to hinder the development of the spores into bacilli and prevent the production of anthrax. We have already stated that the organism cannot con- tinue its growth at any temperature above 45° C., but it may still remain alive up to about 60° C. if this temperature be not continued for too long a period, the spore-bearing bacilli, though themselves killed at this temperature, leave their spores, which will withstand a temperature of 100° C. if continued only for a short time, and start into life and into active vegetative growth when again placed under suitable conditions. The only other physical condition that appears to be fatal, or at any rate injurious, to anthrax spores is strong sunlight ; this appears to deprive them in whole or in part of their powers of further development in a most remarkable manner, always causing distinct attenuation of their pathogenic virulence before completely destroying them. The best way of maintaining cultures of anthrax bacillus is to take a drop of anthrax blood, sow it on a potato or agar-agar, . allow it to grow there for several days at about 30° C. until spores are well developed, triturate a small quantity of the growth with some distilled water, place a number of silk threads which have previously been sterilized by heat in this mixture, and then dry them carefully, cut into short lengths, and keep them ina stoppered bottle or in a plugged test tube. From these threads cultivations may be made on almost any artificial nutrient medium. In addition to the media already 278 BACTERIA. mentioned, starchy materials, vegetable infusions, hay and meat infusions, and even sterilized alkaline urine, may be used, the only thing that appears to interfere with their growth being an acid reaction. Even this, however, may be present to a slight degree if other conditions are favourable; for example, the anthrax bacillus grows readily on potato, which gives a slightly acid reaction. On gelatine plates colonies grow and are visible to the naked eye, on the second or third day, as small white points which gradually spread outwards, and as they come to the surface cause a slight liquefaction of the surrounding gelatine; théy are then seen as small white masses with wavy margins lying in a clear space, formed by the liquid gelatine. When examined under the low power they appear as round dark-green points with an irregular outline ; on the second or third day this irregularity becomes much more marked and, as Fliigge describes it, when it reaches the surface of the gelatine “the dark remnant of the deeply-placed colony can only be seen in the middle. Around this centre, however, there is a light- brown or light-grey shimmering mass, which consists of numerous wavy, curling bundles, recalling the appearance of locks of hair or snakes on the head of a Medusa. Ultimately individual threads, or bundles of threads, branch off from the irregular periphery, and grow over the gelatine in various directions. At the same time the gelatine is liquefied over a small area; the colonies, which have now a diameter of 2 to 4 mm., begin to float and break down at their margins under the action of the fluid formed.” An exceedingly good method of obtaining a permanent record of the appearance of these colonies, is to lower a cover glass on to one of them and then to raise it carefully without sliding it over the surface, so as to take an imprint of the colony upon the glass; the surface of the colony adheres to the glass and a thin layer is removed. This, when stained with fuchsine or methyl blue and mounted in Canada balsam, gives an exceedingly faithful ‘‘impression” of the appearance of one of these masses. The puncture cultivations of the bacillus anthracis pre- sent most characteristic appearances. Along the track of the inoculating needle there appear delicate feathery rays, which pass for some distance into the gelatine ; small lateral rays are given off from these, and the whole of the young growth has a peculiar feathery appearance. These rays are always longer near the surface, and gradually become shorter ANTHRAX. 279 and fade off as the lower part of the track is reached. After a time the gelatine begins to liquefy, and slowly the feather- like mass sinks to the bottom of the liquefied medium, eventually forming an opaque white layer, which is found to consist of bacilli in different stages of degeneration. The upper layer of the gelatine, though perfectly clear, is now quite fluid. This process of liquefaction comes on first along the track of the needle, near the surface, and gradually extends downwards until the whole needle track has dis- appeared. We have then in the tube, the upper liquefied gelatine, then the opaque layer of bacilli, and lastly the clear solid gelatine below. If agar-agar be used in place of gelatine, much the same appearances are presented ; there is a smooth glistening greyish-white surface growth, and the feathery rays appear to get more and more solid as the growth continues, but no liquefaction takes place. On solidified blood serum the colonies grow as greyish-white layers on the surface, and the medium is very slowly liquefied. On sterilized cooked potatoes the bacillus grows asa creamy- white somewhat parchment-like granular mass, which rises above the surface for some little distance but never extends far in a lateral direction ; it has a peculiar dry appearance. The growths on potatoes, as on other media, take place at the ordinary temperature of the room. This organism can be very readily inoculated into certain animals, with results that may be looked upon as very definite ; for example, if with a needle the point of which has been dipped into the spleen of a cow that has died from anthrax a mouse be pricked at the root of the tail, it will die in from seventeen to eighteen hours, enormous numbers of bacilli being found in its blood. It has been observed that in the case of some- what larger animals, such as the guinea-pig, death does not take place till a rather later period ; for instance, a guinea- pig setoned with a silk thread containing spores of anthrax bacillus will die on about the second or third day. If the animal be examined after death, it will be found that near the seat of inoculation there is usually very little to indicate that this was the point at which the infective material was introduced. Should, however, the inoculation be made into the abdominal wall, there is usually marked cedematous swelling of the peritoneum; there may be small hemor- rhages into the subcutaneous tissue, and there is usually 280 BACTERIA, some emphysema ; the abdominal muscles are pale, moist, and are evidently in a condition of cloudy swelling, or in some cases there may be in the muscles, near the point of inoculation, hyaline degeneration. The spleen is enlarged, soft, and pulpy, and appears to contain a very large quantity of blood ; itis dark in colour. The liver also is changed ; it has a half-boiled look, and contains a considerable quantity of blood. The lungs are bright red and the cavities of the heart are distended. As already stated, the blood and lymph from the tissues contain the bacilli in very considerable numbers. If sections of various organs and tissues are afterwards made, it will be found that the bacilli are most numerous near the capillary vessels or in those small venules that arise from the capillaries. They are found in all parts of the spleen and in the small venules of the liver. They may also be found in the small capillaries of the glomeruli of the kidney. Another method of infection, especially met with in wool- sorters’ disease, is by the air passages, a condition that was most carefully described by Greenfield in. this country, and has since been made the subject of careful observations by Buchner in Germany. It appears that the spores are inhaled along with dust from hair, wool, &c. ; they develop in the air passages of the lung, make their way through the alveolar membrane or through the walls of the bronchi, and so into the lymph channels and blood vessels of the lung. In some cases, however, they appear to " operate” from the air channels themselves,-and, multiplying in these positions, give rise to a kind of pneumonia. The air vessels become filled with sero-fibrinous exudation, in which the bacilli may develop most rapidly, the walls of the alveoli become cedematous ; the bacilli make their way from these points into the circulation, and general anthrax is set up. Pleurisy, with effusion into the thoracic cavity, as already mentioned, may also’ develop in these cases, and the walls of the bronchi appear to be invaded by the bacillus. It has been found that if the bacillus is taken into the alimentary canal the acid contained in the stomach usually destroys it.. If, however, resistant spores make their way into the alimen- tary canal, they may pass untouched through the stomach and so into the alkaline contents of- the intestine. At the body temperature they are then under very suitable conditions ANTHRAX, 281 for development into the vegetative forms ; and as they are developed. they make their way, especially through damaged epithelial cells which they gradually push to one side, into the deeper layers of the intestinal wall, into the lymph channels, or directly into the blood vessels, so giving rise, in many cases, to a general infection. Susceptibility of animals to infection through the intes- Photo-micrograph of Anthrax bacilli in the blood vessels of the mesentery of a mouse, At one or two points the bacilli may be seen lying in the substance of some of the white blood cells (phagocytes). x 375. tinal canal varies very greatly, the guinea-pig, which is very susceptible to the disease conveyed by inoculation, withstand- ing a very large dose. of the spores when given by the stomach, whilst sheep and cattle, both of which are more resistant as regards inoculation, are comparatively easily in- fected through the alimentary canal. I have stated that at — the point of inoculation in animals there is usually no evi- 282 BACTERIA, dence at all that it has been the point of entrance of the bacilli, but in the human subject there very frequently occurs at the point where the bacilli enter a wound, a marked local reaction, the result apparently of an effort on the part of the tissues to prevent the further advance of the bacilli. There is first irritation at the point of in- oculation, this usually occurring in from one to three days after the inoculation ; about a day later a minute vesicle surrounded by a zone of inflammatory redness and swelling makes its appearance. The serum in this vesicle becomes brown, and gradually a- kind of mortification is set up; other vesicles, forming a ring, appear around the original point ;- these in turn become brown or black, until gradually an extensive black scar is formed. If these little pustules, with the surrounding tissue, be freely excised and the wound well cauterized with strong carbolic acid, there may be no return of the disease ; in fact, if the removal be free enough, this result is most certainly ob- tained ; but if the inflammation be allowed to go on unchecked, there is gradual extension of the vesication, sloughing, and blackening, until a very considerable area is affected, the bacilli sooner or later making their way into the blood vessels and giving rise to general anthrax. In the vesicles, and also in the tissues around the point of inocula- tion, anthrax bacilli can usually be found in considerable numbers, the tissues are somewhat ‘cedematous, and there is an increase of leucocytes in the inflamed area. When the blackening commences there are usually found along with the bacilli, or in some cases in the centre of the ulcer. re- placing them, chains of micrococci ; these are seldom seen in the very early stages, but in the later stages they are almost invariably present. The animals most susceptible to this disease are sheep, mice, rabbits, guinea-pigs, and, according to Fliigge, horses, hedgehogs and sparrows, all of which die or are very ill when the organism is inoculated directly into the sub- cutaneous tissue. Most birds are not readily inoculated, and for a long time it was found impossible to kill fowls by means of anthrax. White rats, old dogs, and amphibians are ex- ceedingly resistant to the disease in almost any form ; cattle, too, which are readily infected through the alimentary canal, are but slightly susceptible to anthrax introduced by direct ANTHRAX. 283 inoculation into the tissue. It has been found, however, that by reducing the temperature of the fowl and by raising that of a frog, these animals may be infected by inoculation, and they have even under certain conditions been killed. The usual method of infection in man, of course, is by the skin, and it is found that anthrax usually affects those who attend on or slaughter animals that are subject to this disease, so that those usually affected are butchets, grooms, and others engaged in similar employments. During the time that I was acting as physician to the Western Dispensary in Edinburgh, which. was situated near the public abattoir, several cases of localized poisoning and two of general anthrax poisoning came under my charge within a comparatively short time. ‘These were all cases of butchers, who having slight scratches on their hands or arms, had been infected by the blood of animals that they had killed which were suffering from splenic fever. T'anners, skin-dressers, and wool-sorters are also specially liable to this form of the disease, though the latter class are also very frequently affected through the lungs. In certain regions where the disease seems to be almost endemic the spores of the bacillus may make their way into the alimentary canal of the human and other subjects, through the medium of food or water. It has already been stated that the anthrax bacillus within the body of an animal is incapable of forming spores, but in those cases where the bacilli can make their way from the lungs into the saliva, from the mouth, or from the lower part of the alimentary canal, whence they are discharged with the secretions, spores may be readily enough formed. Consequently an affected animal must always remain a centre of infection, and even the dung from a diseased animal may contain a large number of spores which, when scattered over a field, are sufficient to infect whole herds or flocks. The only way in which to get rid of the infection in such a case is to burn the animal at once, or to bury it deep down in the earth. This latter method of disposing of the animal should only be resorted to where it is possible to bury it six or seven feet deep. It is a well-known fact that the organism cannot form spores where oxygen is absent, and at any tempera- ture below-12° C., and below this temperature the bacilli are incapable of existing for any length of time. At a distance of six or seven feet from the surface the temperature of the 284 BACTERIA, ground is usually below 12° C., so that animals put below this depth, even if swarming with bacilli, cannot be looked ‘upon as centres of infection; the organisms, no longer able to form spores, soon lose their virulence and die out altogether. In order to get rid of any spore- bearing bacilli, all discharges or blood that may have escaped at the place where the animal lay after death should be carefully disinfected with 5 per cent. carbolic acid. It was for long thought that by cultivating the anthrax bacillus through a great number of generations on ordinary nutrient media it could gradually be converted into the hay bacillus or the bacillus subtilis — a very similar organism, but one that has no pathogenic properties—and Buchner, in a published series of experiments, claimed that he had obtained this conversion, and that in the same way he could again turn back the wheel from the hay bacillus to the pathogenic form. These results, however, have not been repeated, although many have tried similar experiments ; but Hueppe and Wood, by using a species of earth bacillus, which, in its morphological characters and method of growth on nutrient media, appears to be almost identical with anthrax bacillus (but possesses no pathogenic properties), have found that it must be very nearly related phylogene- tically, from the fact that when inoculated into animals it acts as a kind of vaccine, and renders the animal immune to an attack of anthrax. It will be well, however, to take up this and other ques- tions relating to anthrax in connection with vaccination and immunity. LITERATURE. The following works may be consulted: Bot.incer.—Centralbl. f. d. Med. Wiss., No. 27, p. 417, 1872. BraAveELt.—Virch. Arch., Bd. xt, Heft. 2, p. 121, 1857, and Bd. xiv., Heft. 5, 6, p. 432, 1858. Bucuner.—Nageli’s Untersuch. ti Nied. Pilze ; Eine neue Theorie i. Erziel d. Immunitaét gegen Infectionskran- kheiten Minchen, 1883; Virch. Arch., Bd. xc1., Heft, 3, Pp. 410, 1883. CHAuVEAU.—Comptes rendus. 1880-1885. Davaine.—Comptes rendus, 1863-1866. ANTHRAX. 285 GREENFIELD.—Proc. Roy. Soc., vol. Xxx., p. 557, 1880. JouBert.—Comptes rendus, t. XCVI1., p. 641, 1883. Kien.—Rep. Med. Off. Local Gov. Board, p. 207, 1882. Kocu.—Beitr. zur. Biol. der. Pflanzen, Bd. 1, p. 277, 1876; Mitth. a. d. k. Gesundheitsamt, Bd. 1, p. 49, 1881. *M’FaDYEAN AND WoopDHEAD.—Reports Nat. Vet. Assoc., 1888, PasTEur.—Comptes rendus, t. LXXXIV., p. 90, 1877, t. XCVI., P- 979, 1883 ; Rev. Scientifique, Jan. 20, 1883. PoLLENDER.—Casper’s Vierteljahrsschrift, 1875. PrRazMowskI.— Biol. Centralbl., Bd. rv., No. 13, p. 393, 1884. Rocer et Davaine.—Bull. de la Soc. de Biol. de Paris, 1850. Toussaint.—Comptes rendus, t. LXXxIV., p. 503, 1877. See also various papers in Centralbl. f. Bakt. u. Parasitenk ; Annales de l'Institut Pasteur ; Corni and Bases Les Bac- téries ; HuEpPE Joc. cz#. and LérFLer, Vorlesungen tiber Die Geschichtliche Entwickelung der: Lehre von den Bacterien, Leipzig, 1887. : * Full references given. CHAPTER XVI. TETANUS. Tetanus a Specific Infective Disease—A Wound Fever—Organism found by Nicolaier in the Soil taken from Streets and Fields—Experiments on Animals—Symptoms of Disease—Pure’ Cultivations Obtained— Description of Organisms-—Characteristic Shape—Spore Formation— Organism Anzrobic—Cultivations—Kitasato’s Method of Cultivating the Organism—The Bacillus found only at the Seat of Inoculation— Wide Distribution of Spores—Bossano’s Examination of Earth— Vaillard and Vincent’s Observations—Tetnaus Bacillus a Facultative a under which Tetanus is Contracted —Poisoned Trows. TRAUMATIC tetanus, or convulsions resulting from poison- ing associated with an open wound, was for long sus- pected to be the result of some condition similar to septic or hospital fever; that it was an infective disease was recognized by many of the older surgeons, and attempts were made at a very early date to treat it as a traumatic infective disease. That this poisoning was the result of the activity of an organism, which made its way to a wound and there flourished and gave rise to the characteristic pro- ducts and symptoms, was not the result of direct experiment made with the object of finding out such an organism, although attempts were not wanting to demonstrate its presence in the wounds and along the course of the nerves in cases of tetanus. All efforts, however, proved un- successful until after an organism obtained from other sources had been obtained and described, and an artificial tetanus had been produced. In 1884 Nicolaier, working with soils obtained from the streets and from the fields, found that these when inoculated into certain animals produced effects different from those produced by soils taken from cultivated gardens and from woods. ‘The former he found, when a small particle was placed in a little pocket under the skin of mice, rabbits, TETANUS. 287 and guinea-pigs, gave rise to symptoms which he described as tetanic in character. In from two to four days after the inoculation the hind-quarters of the animal became paralysed, first the one near the seat of inoculation, then the other ; then rigidity came on followed by loss of motion ; the forelegs were in turn affected, then the neck, and at length the whole:of the body became rigid, and sometimes curved as in tetanic convulsions occurring in the human subject. On examination after death there was found, at the point of in- oculation, a small abscess, in the pus of which were several species of micro-organisms. One of these, when obtained pure or practically pure, if inoculated into another animal produced exactly the same symptoms. Nicolaier was not able to obtain any perfectly pure cultivations, but he described an organism which was afterwards separated by Kitasato and by Tizzoni and Mdlle. Cattani independently. This organism, although never directly demonstrated in the earth that produced the disease, is constantly found in the pus of the abscess, in the walls of this abscess, and even in the immediately surrounding tissues. It occurs as long delicate threads scarcely thicker than the bacilli of mouse septicemia with slightly rounded ends; like some other organisms, especially those met with in putrefactive pro- cesses, they give rise to spores which are usually developed at the end of the shorter rods into which the long threads break up ; this spore, forming the head of what is called the drum-stick-shaped bacillus, is usually large and may be seen as a clear mass causing enlargement of one end of the bacillus. The spore develops best at the temperature of the blood, and under favourable conditions is completely formed about thirty hours after multiplication has commenced ; at the temperature of the room this does not occur for about aweek, although the organism itself developes readily enough at thistemperature. The rods are motile. Owing to the fact that the organism is anzrobic and that the presence of oxygen interferes very greatly with its development (it is said that oxygen kills it altogether), it proved a somewhat difficult matter to obtain perfectly pure cultivations, although when once it had been recognized that the organism was anerobic, plate cultivations were readily enough obtained For further description of the disease see ‘‘ Micro-Organisms,” Dr. C. Fliigge, New Sydenham Society, 1890, 288 BACTERIA. by keeping the plates on which the nutrient medium was spread in an atmosphere of hydrogen. When puncture inoculations are made in tubes of gelatine to which grape sugar has been added, there is no growth along the part of the track of the needle near the surface, but in the deeper part away from the air, there is a moderately luxuriant growth which appears in the form of a central Specimen from pure culcure of the Tetanus Bacillus, with enlarged spore-bearing “drum-stick” ends. x 1000. streak, from which numerous spikes pass laterally into the surrounding medium. The culture at this stage has very much the appearance of a spruce-fir, the lower branches (z.e., those in the deeper part of the gelatine where the access of oxygen to the organism is entirely cut off) being longer and more distinctly marked than those nearer the surface, Later this characteristic appearance is lost, the organism invades the whole of the nutrient medium, TETANUS. 289 forming a kind of cloud; the gelatine becomes softened, and there is emitted a peculiar fusty smell which appears to be almost characteristic of this organism. Most of the earlier experiments were somewhat unsatis- factory, and considerable doubt was cast on this organism as a producer of tetanus from the fact that in many cases pus that was known to contain this specific bacillus, and even old pure cultivations of the organism, failed to set up the characteristic symptoms when inoculated in the usual manner. More recent observers, however, have pointed out that the tetanus bacillus, like many others of the septicaemia group, is virulent only so long as it is grown under anzrobic conditions. This is especially the case in those organisms in which there has not been time for the spores to develop; so that when grown in the presence of free oxygen (when that is possible), or when exposed to the air after the growth has been com- menced under favourable conditions, and has gone on for some time, but before there has been time for the formation of spores, the organism rapidly loses its virulence, or, as we have seen, dies off altogether. Experiments made by inocu- lating the pus from tetanic patients often gave entirely nega- tive results. Here it was evident that the failure was due, in many cases at any rate, to the fact that the pus with its contained organisms had been exposed to the air for some time, and the bacilli had been compelled to grow under conditions unfavourable to the retention of their specific viru- lence, before they were used for purposes of inoculation. In such organisms, as we should expect, it is found that spores are not seen, or they are very imperfectly developed, and it may be in the case of the older cultures, where these spores are developed into the young bacilli, that these, not having attained their full resisting power, die off very readily in the presence of oxygen. By paying attention to this point it.has gradually been proved, almost beyond doubt, that the tetanus that may be produced in white mice or guinea-pigs by the inoculation of small particles of garden earth, is of the same nature as the tetanus that is produced by the inoculation of the pus from a primary wound which has apparently given rise to tetanus in the human subject. Further, now that the conditions under which the organism exists have been studied, and its anzrobic character recognized, pure cultivations have. been made, and it has 20 290 BACTERIA. been proved that the disease may be produced without fail if certain definite precautions are taken before the inoculation be made. One great difficulty connected with the obtaining of pure cultivations was, of course, that under the conditions favourable to the growth of the tetanus organism, other anzrobic bacteria would also take the opportunity of developing. Some only of these other organisms, however, give rise to the formation of spores, and this occurs at a later date than in the case of the tetanus bacillus. Kitasato, very ingeniously, made this fact the stepping-stone to the cultivation of a pure growth of the spore-bearing tetanus bacillus. As we have already seen, spores of many bacteria can readily withstand a high tem- perature (in some cases of even 100°C., if this is not continued for too long a time, and they will withstand for a con- siderable period the action of a temperature of 80° C.), whilst the vegetative or fully developed forms are killed off very rapidly at a comparatively low temperature. Kitasato’s method of procedure was as follows : From the immediate neighbourhood of the suppurating wound of a patient who had died from tetanus, he took a small fragment of tissue, and placing it under suitable nutrient conditions, z:e., in the specially prepared gelatine at a temperature of little over 30° C., and in an atmosphere of hydrogen, he obtained a very luxuriant growth of anzrobic organisms ; amongst these he observed that the drum-stick-shaped organisms developed their spores at a much earlier period than any of the others that were growing in his cultures. As soon as these spores made their appearance he raised the tem- perature to 80° C., with the result that all those bacteria, in which spores were not already developed, were very rapidly destroyed ; the tetanus dacz/dz were also destroyed (that is, the vegetative forms were destroyed), but the sfores still retained their vitality, and on being transferred to suitable nutrient media, and placed under other suitable conditions, they “‘ hatched ” out into the vegetative form, and a pure cultivation of the tetanus bacillus was obtained. It is a rather curious fact that here, as in the case of diphtheria, the organism seems to be localized at the actual point of inoculation, for although, as we have seen, the bacilli, how- ever numerous in the pus and in the walls of the abscess, and in the infiltrated tissues immediately around the abscess, TETANUS. 291 are confined to a well-defined and localized area, and the most careful researches, conducted with the help of both cultivation and histological. methods, have hitherto failed to enable any observer to demonstrate the presence of these organisms in the internal organs. It would appear, then, that as in the case of septicemia and diphtheria, the poison is manufactured by the organisms at the site at which they are actually introduced, and that from this point it is absorbed into the body, and is carried to the special tissues on which it acts. Brieger, indeed, was able to separate from the limb of a patient who had died from tetanus an exceedingly virulent basic poison or ptomaine which he speaks of as tetanin, whilst he also found a very poisonous proteid substance, tetano-toxin, which he called a toxalbumen, a substance of which we shall have to speak in a later chapter. At first sight it appears somewhat extraordinary that the development of the tetanus bacillus should be so extremely localized, and that the localization should be confined to a position so near the surface—z.e., to the surface of a compara- tively open wound, to which the oxygen of the atmosphere might at first sight appear to have easy enough access. When, however, we come to think more carefully of the conditions that prevail in the wound, this does not seem quite so extraordinary. In the first place, there is, covering it, a layer of pus which, as is well known, has little power of holding oxygen in solution, any small quantity that is there being gradually taken up by the leucocytes that escape to the free surface or into the abscess, and utilized by them for their own purposes. Beneath this, especially in the later stages of infiltration, and before the capillary vessels of the granula- tion tissue have begun to form, or are fully formed, the supply of oxygen must necessarily be comparatively small, and any that is present is promptly taken up by the active wandering and proliferating connective tissue cells. Here then we have the conditions under which the tetanus bacillus is enabled to flourish ; there is a condition of anzrobiosis, or oxygen famine. Beyond this, however, where the infiltration is not so great, and where the vessels are considerably dilated, the red blood corpuscles are constantly bringing up their fresh supplies of oxygen to the tissues, and in consequence the fluids at some little distance from the wound contain more oxygen than there is near the surface of the wound, and the conditions 292 BACTERIA. are consequently much less favourable for the continued existence of the tetanus bacillus. The spores of the tetanus bacillus seem to have a remark- ably wide distribution. Originally they were only cultivated from garden soil, but successful inoculations have since been made with such material as the sweepings of a hay-loft, and the dust that had accumulated on the furniture of horses. The specific bacillus has been found on the grime on a man’s hand, and on imperfectly cleansed surgical instruments. Tetanus is said to be specially associated with the horse, but the more recent observers insist that this is simply because the horse is susceptible to the action of the bacillus and its poison, and because the germs have such a widespread—in fact, an almost universal—distribution. How universal this distribution is may be gathered from the fact that M. Bossano, who was able to obtain the soil from forty-three different regions in various parts of the globe, got positive results with twenty- seven of them. With the soil from these forty-three places he inoculated a number of animals, introducing a small por- tion, about the size of a pea, into a little subcutaneous pocket of a white mouse or a guinea-pig, and with the soil obtained from twenty-seven of these places tetanus was produced in from two to four days. He says of the soil obtained from England, that from Bath produced tetanus in two out of three white. mice inoculated, both of them dying in about two days. Soil from Portsmouth did not contain tetanus bacilli, whilst that from Plymouth and from Manchester caused the death of some of the animals that were inoculated with it, all the characteristic symptoms of tetanus being developed. Speaking colloquially, a worker at the Brown Institute told a friend that they grew the tetanus bacillus in the garden there. From his experiments Bossano concluded that soils which contain much organic matter, almost in- variably contain tetanus bacilli, and that latitude, climate, and special meteorological conditions, have far less influence on its development than defective drainage, imperfect hy- gienic conditions, and the degree of cultivation of the soil. It appears, however, that our’methods of cultivation are not yet perfect, for it is an undoubted fact that failures to pro- duce tetanus with pure cultivations are of very common occurrence, even in the hands of those best fitted to carry on experiments of this kind. So markedly is this the case that TETANUS. 293 Chantemesse and Widal at one time thought that the pre- sence of other organisms was necessary in order that the tetanus bacillus might act, and it has been suggested that zrobic organisms which have great avidity for free oxygen must also be present in order to allow of the development of the full virulence of the anzrobic tetanus bacillus. It is a fact, whatever may be the explanation, that the tetanic organism soon loses its virulence under cultivation, especially when it is grown “ pure.” ; Recently, however, Vaillard and Vincent, as the result of a series of most careful observations, have arrived at the conclusion that it is usually the tetanus poison — which they compare to snake poison—and not the organism itself that gives rise to the tetanic symptoms in animals that are infected experimentally. They also find that until the organism has grown in artificial culture media for some time it has not the power of setting up disease, a fact that was accounted for when they found that no poison was developed until some time after the organism had begun to grow, the production of the poison appearing to go on simultaneously with the formation of a peptonizing enzyme. Even spores, when injected alone, could not set up tetanic symptoms, but when these were injected along with other organisms such as lactic acid bacillus, or even with lactic acid itself, with bacillus prodigiosus, or into a bruised wound, or where they were injected along with a quantity of their own poison, tetanus was invariably set up. The tetanus organisms form their poison slowly, and in healthy tissues they are rapidly destroyed by the tissue cells long before they have time to form sufficient poison to produce the nervous symptoms of tetanus ; whilst in the cases above mentioned the tissue cells are so engaged in removing the other foreign matter, or are so paralyzed by the action of the lactic acid, or the small portions of tetanus poison, that they are not able to contend on equal terms with the tetanus bacilli, which being under favourable conditions grow rapidly, give rise to the formation of the special poison, and the patient succumbs. This remarkable poison in doses of 25 millegrammes is quite sufficient to kill a rabbit, or the 25th of a millegramme to kill a mouse. The tetanus bacillus is a facultative saprophyte, the nature of the wounds through which tetanus is inoculated bearing, 294 BACTERIA. out in a most remarkable fashion the experimental proofs that have been already adduced. A horse which, in the stable and in the field, always collects a certain quantity of earth on his skin and in his hoofs, may be easily. inoculated ; he, in turn, may readily inoculate a man or another animal by a kick with the sharp iron of his dirty shoe. Gardeners, agri- cultural labourers, and all who work with horses or in the soil, bear on their hands a virus which only needs a bruise or a cut, but especially the former, to allow of its setting up the characteristic symptoms of tetanus. Soldiers, during a cam- paign, when their garments and equipments are soiled from contact with the ground during their camping and bivouack- ing are always more liable to the disease than when they are hurt accidentally during times of peace. It is also pointed out that in warm countries where people are in the habit of sleep- ing out in the open air and on the ground, tetanus is very frequently met with as the result of comparatively slight wounds, whilst amongst children during the years they crawl: on the ground, or play in gardens or in fields, tetanus is always more common than in later life, when: the parts that come in contact with the ground are usually pro- tected by shoes and gaiters. Many of these facts are well known to savage tribes, whose powers of observation and opportunities for experimentation are of a very high and extended order, and we find that Dr. Ledantec, in an interest- ing account of the poisonous arrows used by the inhabitants of Santa Cruz, of the Solomon Isles, and of the New Hebrides, speaking of the deaths from this cause of Bishop Patteson and Commodore Goodenough, with their companions, refers to the fact that they were all attacked by tetanus, or lockjaw. He gives a short description of these poisoned arrows. They are about three feet in length ; the shaft is made of a reed, then comes a middle portion composed of hard wood, and lastly a point which is usually composed of a fragment of human bone, which is carefully sharpened to a very fine point, and is so fixed that it readily snaps off on the slightest shock. Witha sticky substance obtained from an incision made in the bark of a tree, the point composed of the fragment of bone is smeared. This fluid, on exposure to the air, becomes thicker, and of a more viscid consistence. Thread is then wound in a spiral direction round and round the sticky point. A quantity of soil from the edge of a mangrove TETANUS. 295 swamp is taken in a cocoanut shell, or some similar vessel, and into this the arrow-head is plunged. It isthen carefully dried in the sun, after which the thread is removed, when a roughened point covered with a film of dry mud and dust is left. In this mud there are probably both septic vibrios and tetanus bacilli, the former, however, are rapidly killed by exposure to the sun, whilst the tetanus bacillus of Nicolaier, which, as we have seen, developes a well-formed spore at one extremity, may remain active for months and even years, although, as the savages well know, the poison gradually becomes more and more attenuated, until old arrows are known to become entirely inoffensive, except as mere mechanical weapons of warfare or hunting. Stanley, speaking of the hunting appliances and offensive weapons of the pigmies whom he encountered during his African wanderings, makes a similar interesting observation, to the effect that these small specimens of humanity use arrows so poisoned that the slightest scratch with one of them pro- duced one of two things, tetanus (or convulsions), which is evidently due to the action of a poison similar to that above described, or death, accompanied by peculiar gangrenous sloughs at the seat of the wound, which is very probably associated, so far as we can see, with malignant cedema or “black quarter,” a condition that was also described by Nicolaier as resulting from the inoculation of soil collected from gardens and forests. These pigmies are described as dwellers in the dense forests of that part of Central Africa through which Stanley travelled. LITERATURE. The following works may be consulted : ARLOING ET Lifton Triprer.—Arch. de Physiol. Normale et Path., t. IIL, p. 235, 1870. _ BeELFANTI u PescaRoLo.—Centralbl. f. Bakteriol. u. Parasi- tenk, Bd. v., No. 20, p. 680, and No. 21, p. 710; Bd. vi., No. 10, p. 283, 1889. Bonome.—Fortschr. der Med., Bd. v., No. 21., p. 690, 1887. Bossano.—Comptes rendus, t. Cvil., p. 1172, 1888; Re- cherches Expérimentales sur l’Origine Microbienne du Tétanos, 1890. BRIEGER.—Bericht. d. deutsch. Chem. Gesellsch., Bd. xrx. 296 BACTERIA BRIEGER UND FRAENKEL.—Berlin Klin. Woch., pp. 241, 248, 1890. CarRLE E Ratrone.—Giornale della R. Acad. de Med. di _ Torino. March, 1884. CLarkE.—Trans. Roy. Irish Acad. 1789. CHANTEMESSE ET W1DAL.—Les Bactéries, Cornil and Babes, t. 1, p. 567, et seq., 1890. Faser (Knup).—Om Tetanus som Infektionssygdom. Copenhagen, 1890. Fiiigce.—Die Micro-Organismen, Leipzig, 1886, Tran. W. Watson Cheyne, New. Syden. Soc., 1890. Kitasato.—Kongress f. Chirurgie, 1889. Kirasato unD WEyYL.—Zeitschr. f. Hygiene, Bd. vu, pp. 41, 404, 1890. Kocu.—Mitt. aus d. k. Gesundheitsamte, Bd. 1., 1881. KussmauL.—Deutsch. Arch. f. Klin. Med., Bd. 11, Heft. 1, p. 1, 1873. : Lepantec.—Annal de l'Institut Pasteur, t. 1v., Nov. 25, 1890. MarsHat Hatt.—On the Diseases and Derangements of the Nervous System. London, 1841. Nicotarer.—Beitr. zur. Aetiol. des Wundstarrkrampfes. Dissert. Géttingen, 1885. ‘ Nocarp.—Recueil. de Méd, Vét., t. 1v., No. 18, p. 617, 1887 ; Arch. Vét., 1882. Rietscu.—Comptes rendus de l’Acad. d. Sc., t. CVIL, p. 400, 1888. . Rose.—Ueber d. Starrkrampf., 1870. ; SANCHEZ-TOLEDO AND VeEILLON.—Arch. de Med. Expérim. Bd. 11, p. 709, 1890. ‘ SaxTorpH.—Clinisk Chir. 3dje. Del. Copenhagen, 1878. Simpson, Sir J. Y.—Works, vol. 1., 1871. Tizzoni AND CaTTANI.—Riforma Medica. April, 1889. VAILLARD ET VincenT.—Annal. de l'Institut Pasteur. Jan., 1891. VERHOOGEN ET, BaerT.—Premiéres Recherches sur la Nature et l’Etiologie du Tetanos. Bruxelles, 1890. VERNEUIL.—Soc. de Chir., t. x1., pp. 327, 438, 1885. WIeDERMANN.—Zeitschr. f. Hygiene, Bd. v., p. 522, 1889. CHAPTER XVII. DIPHTHERIA. Diphtheria an Infective Disease—The Organism found in the False Mem- brane in its Deeper Parts—Method of Staining the Bacillus—Characters of the Bacillus—Involution Forms—Cultivation Methods—Appearance of Colonies—Nutrient Media—Results of Inoculation Experiments— Klein’s Bacilli differ somewhat from Léffler’s—Streptococci found in Diphtheria Poison—Extreme Virulence—Resemblance to snake-bite poison—Toxicity—Predisposing Conditions—Conditions fatal to the Bacillus—Roux and Yersin’s Observations—Fraenkel’s Observations —Attenuated Diphtheria Virus—Increase of Virulence. ALTHOUGH it has long been known that diphtheria was an extremely infectious disease, it is only within compara- tively recent years that any reliable information as to the nature of the specific infective poison has been forth- coming. Even when the organic nature of other specific infective poisons had been practically proved many difficulties still remained to be overcome; in most other cases where the disease could be proved to be the result of the vital activity of a micro-organism, such organism could usually be found in a pure condition, or greatly predominating over all others, in the blood, in the internal organs, or in some special fluid in the body. This was found not to be the case in diphtheria. Most careful and elaborate researches were entered upon, but it was found impos- sible to demonstrate any special organism as occurring in the blood, in the lymph, or in any of the organs of the body. The only position in which any could be found was in the false membranes (composed of fibrinous lymph and _altered epithelial cells) that are found in the throat, and it was at once surmised—a surmise that was afterwards found to be correct—that the poison which gives rise to the constitu- tional symptoms must be formed at the point at which the micro-organisms are found; and that, being of an exceedingly diffusible nature, it is thence absorbed and carried to various 298 BACTERIA. parts of the body, giving rise to heart failure, or to certain forms of paralysis, partly through its action on the nervous system and partly owing to its interference with the nutrition of the various tissues of the body. Here again, however, those who were studying the subject were con- fronted with another difficulty ; there were so many forms of ‘micro-organisms that found in this false membrane a suit- able subsoil on which to grow, it was almost impossible with the methods then at command to separate them and so to obtain pure cultures, in order that the specific and bio- logical characters of each might be investigated. In 1875 Klebs found in the false membranes a small bacillus with rounded ends, and with, here and there, small clear spaces in its substance, a bacillus that was not readily stained, that grew luxuriantly in broth, and which, inoculated into animals, gave rise to a peculiar dirty fibrinous-looking slough at the seat of inoculation. He found, however, that in certain cases this bacillus was absent, the predominating organism then seeming to be a micrococcus arranged in masses or in short chains. This, when cultivated in broth, gave rise to the formation of chains of considerable length. As a result of thése observations he described diphtheria as occurring in two forms, one form resulting from the action of one organ- ism, the second being caused by the other. These researches were continued by other workers, and Formad, in America, came to the conclusion that the rod- shaped bacillus had little to do with the disease, but that the streptococcus, or chain-forming micrococcus, was the real exciting cause. Matters remained at this stage for some time—in fact, until Léffler took up the subject. After examining a number of cases of diphtheria, he found that, although there are numerous organisms in the false membranes or diphtheritic patches, these were mostly near the surface, and many of them were simply the organisms that were usually found in the mouth now growing under more favourable conditions of nutrition. He found, however, that in the deeper layers, or at the inner margin of the layer of exudation, the Klebs bacillus might almost invariably be found. It was more deeply situated than any of the others, and was always most numerous in the oldest part of the membrane. This was in cases of pure diphtheria, In the so-called diphtheritic sore throats met with in other diseases, DIPHTHERIA. 299 especially in scarlet fever, the streptococcus appeared to be the predominant and characteristic organism. These rods described by Klebs and Léffler vary much in length, but they average from 3 to 6; they are straight or slightly bent, one end or both sometimes being a little swollen. There may be deeply stained or bright glistening points in the protoplasm, though these are not usually met with. Babes indeed ‘describes spores as occurring in the diphtheria bacillus, but, as is mentioned later, these are not real endospores but consist merely of altered protoplasm. In order to stain the bacillus it is only necessary to remove a small fragment of the false membrane by means of a piece of absorbent cotton wool tied firmly to a pair of forceps or to a pen holder; from this it is trans- ferred to a scrap of blotting-paper, and thence to a cover glass, where it is broken down as finely as possible, heated over a flame in the ordinary fashion, and stained with Loffler’s alkaline methylene blue, or by Gram’s gentian violet method (washing thoroughly with water before attempting to ere): or bya method adopted by Roux and Yersin, who use a blue, com- posed of equal parts of aqueous solution of violet dahlia and methyl green, with water added until a clear, but not too deep, blue is obtained. A drop of this is placed on the slide, the cover glass on which the fragments are dried is inverted and lowered on to it, the superfluous fluid is removed with a piece of blotting-paper, and the organism is examined at once. The vital characteristics of the organism may be used in separating it from false membranes, even where contamination from the organisms of the mouth and pharynx has occurred ; and it is recommended that in order, wherever there is any doubt, to be absolutely certain of the diagnosis of diphtheria, cultivations on blood serum should be made. The specific diphtheria bacilli appear to be stained more readily and more deeply than any of the organisms that usually accompany them. They occur in small groups, as short, straight, or curved rods, with ends sometimes pointed, sometimes curved ; they are never absent from cases of true diphtheria in the early stages, and in some cases the membrane consists of an almost. pure cultivation of the bacillus. In older cases, however, the organisms do not stain so equally ; many pear-shaped and club-shaped bacilli are present, and, in some very old membranes, it is difficult or impossible to distinguish any characteristic bacilli, the ac- companying organisms becoming more numerous, especially as the surface becomes fcetid and softened. In such cases the specific organisms can only be found entangled in the deeper fibrinous net-work. Roux and Yersin hold that microscopic examination gives the most precise 390 BACTERIA. information, even in the case of dried false membranes sent from a distance wrapped in linen or blotting-paper. They also hold that where improve- ment is taking place, the specific bacilli become less numerous, the other microbes increasing in number, and that this may be followed day by day with a microscope, the course and prognosis of the disease being indicated by the changes that are met with. They also hold that at the beginning of a case of diphtheria it is possible to predict a favourable issue from thé presence of a small number only of the specific bacilli, and a large number of other forms, and they also believe that some of the micro-organisms met with under these conditions interfere with the growth and activity of the specific bacillus. > The Klebs-Léffler bacillus cannot be cultivated outside the body on peptone meat gelatine, as it will not grow sufficiently rapidly at the room temperature to outpace the putrefactive organisms which accompany it ; it was, therefore, found impossible to make plate cultivations in order that the different forms might be separated. By mixing the membrane with boiled distilled water and allowing drops of the mixture to trickle over the surface of solidified blood serum, Léffler succeeded in obtaining pure cultivations. Weyssokowicz, using agar instead of gelatine for making plate cultivations, and incubating at 35° C., was able, on a small percentage of his plates, to obtain pure cultivations of the characteristic diphtheria bacillus ; most - of his plates, however, were overgrown with putrefactive and other non-pathogenic organisms. To obtain a somewhat purer inoculating material, put a scrap of mem- brane between blotting-paper, or on a cover glass in a test tube, allow it to dry, and then heat toa temperature of 98° C. for a whole hour—a tempera- ture which few ordinary organisms can withstand for even a less time than this—so that, when the dried material from a cover glass so treated is inoculated on solidified blood serum, a very pure cultivation is usually obtained, Roux and Yersin adopted a simpler method. They used a platinum needle beaten out at the end to form a kind of spa- tula ; on this they took a particle of the false membrane from a case of diphtheria and then made stroke cultivations on the surface of the solidified blood serum, using the same needle without re-charging for some half-dozen tubes. When these are incubated at from 33° to 35° C. the bacilli rapidly make their appearance ; they are visible at the end of twenty hours and have a characteristic appearance in forty-eight hours. This rapid growth is very characteristic of the diphtheria DIPHTHERIA. 301 bacillus, which appears to be the only organism of all those found in the membrane that can form colonies visible to the naked eye in twenty hours. Such colonies grow as small rounded greyish white points, the centre of each of which is more opaque than the periphery; they spread rapidly, form greyish rounded discs, and continue to develop so quickly that they are very evident before the other organisms have begun to form a colony at all visible to the naked eye. From these points, inoculations on to blood serum may be made. Probably the best nutrient material for the diphtheria organism is that recommended by Léffler ; it is composed of three parts of blood serum, one part of neutralized broth, to which has been added 1 per cent. of pep- tone, .5 per cent. of common salt, and 1 per cent. of grape sugar. Ordinary blood serum comes next, and then agar-agar jelly. On agar plates the colonies situated in the substance are coarsely granular, dark brown, and somewhat rounded or oval; although where several colonies have run to- gether they may give rise to somewhat irregular outlines. The superficial colonies are much lighter in colour, are not so dense, and have an irregular scalloped border. These cultivations are found to be made up of bacilli similar to those described by Klebs ; they are not quite so long as the tubercle bacillus, but are rather thicker, the extremities, which are more deeply stained than the central portion, are often slightly enlarged. In older cultivations the rods are not uniformly coloured, and there may be seen in the substance of the protoplasm bodies which somewhat re- semble spores. It was soon noticed that with this inequality of staining, certain other changes in the organism might be met with ; so-called involution forms make their appearance. In these the bacilli are cut up into small rounded masses of protoplasm, some of which have a less diameter than that of the bacillus, whilst cthers may be considerably larger and are oval in shape. These involution or degenerate forms soon make their appearance where the conditions for the growth and development of the bacillus are unsatisfactory, and it is supposed that many of the failures to find the typical rod- shaped bacilli in old diphtheritic membranes are due, in some measure, at any rate, to the occurrence of these involution forms in the later stages of the disease, where the membrane is invaded by putrefactive and other organisms which inter- fere with the growth of the specific bacillus. It is also 302 BACTERIA, found that the diphtheria bacillus has a tendency to lose its virulence on cultivation. It was at first concluded that the bright, strongly refrac- tile particles and deeply stained granules were more or less perfectly developed endospores, and that some of the involution forms might possibly be arthrospores. Although, however, the organisms remain alive and potentially active after being subjected to drying for a considerable time, a moist temperature of 58° C. is quite sufficient to kill them off, which would scarcely be the case were these bodies true spores. The bacillus, as we have seen, grows freely on meat fluids that have an alkaline reaction, but as it grows it first renders these media slightly acid, but later, if there is free access of air, the fluid again becomes alkaline. The acid reaction is always most marked in those cases in which there is glycerine in the cultivation medium. The organism grows ‘in vacuo,” but more slowly than in air; the exclusion of air, however, interferes with the formation of acid, so that under these conditions the bacillus may retain its vitality for a period of six months, or even longer. Examined in a fluid medium it is found to be quite motionless, Although Léffler was able to produce some of the symp- toms of diphtheria by the inoculation of this bacillus on an excoriated mucous membrane, he was not satisfied that this was the real causa causans of the disease, and it was left for Roux and Yersin to demonstrate the intimate causal relation that actually exists between this organism and true diphtheria. They repeated Loffler’s experiments of inoculating the bacilli on the damaged mucous membrane of rabbits, guinea-pigs, and pigeons, and in all cases they found that characteristic diphtheritic patches were produced. Injected under the skin, the bacillus causes swelling at the point of inoculation, and the animal dies with symptoms of acute poisoning. In some cases there is found congestion and effusion into the serous cavities ; in others there is evidence of fatty degeneration of the liver, similar to, but more acute than that met with in cases of diphtheria in the human subject. A very important point determined by them was, that if death did not take place too rapidyy characteristic diphtheritic paralysis usually supervened. The bacillus in these cases was found only at the point of inoculation, and even in the most congested organs it could not be demonstrated either in the blood channels or in the lymph spaces, whilst it often disappeared DIPHTHERIA. 303 even from the seat of inoculation during the latter stage of the disease ;—another fact that helps to explain Loffler’s inability to find the organism in certain of his cases. From all this it is concluded that the local symptoms of diphtheria are due to the action of a specific bacillus on a weakened mucous membrane or on a wounded surface ; that once having gained a footing it gives rise to an acute in- flammatory process, probably by the direct action of the poisonous material that it forms on the cells and on the blood vessels in the immediate neighbourhood ; this caustic action is so intense that the epithelial cells undergo de- generation,—the fibrinous lymph and leucocytes which are exuded also become more or less rapidly degenerated—and give rise to the grey false membranous patches that are so characteristic of true diphtheria, When the growth of the organism is rapid, and where the area of surface attacked is extensive, the amount of poison developed may be very great indeed, and where this latter is greater than can be dealt with in the inflammatory area, owing to the rapidity with which it is produced by a large number of organisms, especially when they are situated deep down in the tissues, there is rapid absorption of the poison, but not of the bacilli, into the system, and the characteristic constitu- tional symptoms of the disease are set up. We must thus distinguish carefully between the local action of the bacillus and its products, and the toxic constitutional effects of these products. Additional proof that these products are the active agents in the causation of the disease was found in the fact that from pure cultures of the diphtheria bacillus there may be separated, by means of Chamberland’s porcelain filter, a special chemical substance, or series of substances, which, after being proved quite free from bacilli and injected under the skin of animals, gives rise to all the constitutional symptoms and lesions of the disease that follow the inoculation of the bacillus itself, the only feature wanting being the false membrane, which usually does not make its appearance. Animals into which small doses are injected are frequently attacked by diph- theritit paralysis. Of course it has been objected that the diphtheria pro- duced in animals is not necessarily the same thing, nor is it necessarily due to the same organism, as the diphtheria of 304 BACTERIA, man. Léffler himself has examined the diphtheria of a calf, and he acknowledges that it is not even related to the diphtheria of the human being. Klein considers that cats are especially susceptible to diphtheria, and that they may act as the intermediate hosts for the development of the diphtheria bacillus between two human patients, whilst he also holds that cows may be inoculated, especially on the udders, with diphtheria bacillus, the disease manifesting itself in the form of small pustules. He considers that milk from such cows may readily become the agent by means of which the disease is spread. Eminent veterinarians, however, are strongly opposed to this view, and it can yet scarcely be maintained that Klein has fully proved his point, although he seems to have obtained strong evidence in support of his position. As he is now working most carefully at the subject, however, it may be well to suspend judgment until he publishes a full report of his observations and experiments. Klein’s bacilli, however, undoubtedly differ in certain ‘ essential points from the bacillus described by Loffler ; most important of all in the fact that whilst Loffler never suc- ceeded in obtaining growths of his bacillus at the ordinary temperature of the room, and was not able to grow it in gelatine, those that Klein describes as occurring in the pustules on the ulcerated udder of the cow grow luxuriantly- in gelatine at the ordinary temperature of the room. Klein describes a second bacillus as growing slowly upon gelatine, but this he does not consider to be so important as the more rapidly growing one. Young rabbits and guinea-pigs are the animals with which most experiments have been made. It has been found that rats and mice enjoy almost perfect immunity from the disease, a fact which has recently been utilized by Behring in connection with the production of an immunity against diphtheria in rabbits and guinea-pigs. We shall have to wait for further light on these points, but from what we have already seen, young rabbits, guinea- pigs, and young dogs, may undoubtedly contract diphtheria, or something very similar to that disease ; both local and toxic symptoms resulting from the introduction- of the poisonous material. It must be borne in mind that there are usually several kinds of bacteria present, that the compli- DIPHTHERIA. 305 cations of the disease are numerous, and that it undoubtedly occurs as a complication in other diseases. But when all this is taken into consideration the fact must still be accepted that careful clinical observation and experimental investi- gation have been made by many thorough workers, and that these workers have assigned to a special bacillus the power of giving rise to at least one form of diphtheria. It is possible that the streptococcus may play an important part in certain cases of diphtheria, but this has not yet been actually proved ; whilst the evidence in favour of the specific infective Klebs-Loffler bacillus is now almost overwhelming. As regards the streptococci that are found in this disease, it appears that these organisms so fully described by Klebs, Formad, and Prudden and Northrup, and later by Loffler and Babes, when inoculated give rise to local inflammation, and.even to inflammation of joints, &c.; in no case, however, do they appéar to give rise to the formation of a false or a fibrinous membrane. No doubt they play a part in the preparation of the tissues for the diphtheria by weakening them so as to enable them to offer less resistance to the action of. the specific organism. These streptococci may actually make their way into distant organs of the body, and it has been found possible to make pure cultivations of them from such organs. These, then, differ very markedly, as regards their distribution in the body, from the diphtheria bacillus, which grows only at the point of inoculation, manufactures all the poison in that position, the poison only being carried. into the body; the bacilli remaining z# sz#w, multiply during the advance of the disease, and degenerate as soon as the tissues ‘begin to obtain the upper hand, which they do in all cases where recovery occurs, and even in some cases where the patients afterwards die from the effects of the absorption of the specific poison. The diphtheria bacillus, having been obtained pure, was naturally seized upon by Loffler, then by Roux and Yersin, and, lastly, by Brieger and Fraenkel for their experiments on toxalbumens ; the way having been paved somewhat by Hankin’s discovery of albumoses in certain anthrax culti- vations. The diphtheritic poison is always most active when alka- line ; during the acid period already mentioned, the toxic power is very considerably diminished. Moreover, if an acid be added to a virulent alkaline filtered liquid, its poisonous activity is immediately diminished ; but, curiously enough, this property is immediately regained when the fluid is again neutralized by the addition of a fixed alkali. So virulent is the alkaline liquid that one-fifth of a cc. of ’ the filtered fluid, from a cultivation of the diphtheria bacillus 21 306 BACTERIA. that has been allowed to grow for forty-two days, proves fatal to a guinea-pig in about thirty hours. Larger doses, from 4 to 20 cc. injected into dogs of: various sizes kills them in from fourteen to twenty-six hours. In doses of 2 cc. it proves fatal in four to six days. In all cases the symptoms are those of more or less acute poisoning, re- sembling septic poisoning in some respects and phosphorus or metallic poisoning in others.