HEINEMANN'S Scientific Ifoanbboofcs A KNOWLEDGE of the practical Sciences has now become a necessity to every educated man. The de- mands of life are so manifold, however, that of many things one can acquire but a general and superficial knowledge. Ahn and Ollendorff have been an easy road to languages for many a struggling student ; Hume and Green have taught us history; but little has been done, thus far, to explain to the uninitiated the most important discoveries and practical inventions of the present day. Is it not important that we should know how the precious metals can be tested as to their value ; how the burning powers of fuel can be ascer- tained ; what wonderful physical properties the various gases possess ; and to what curious and powerful pur- poses heat can be adapted 1 Ought we not to know more of the practical application and the working of that almost unfathomable mystery — electricity ? Should we not know how the relations of the Poles to the magnet-needle are tested ; how we can ascer- tain by special analysis what produce will grow in particular soils, and what will not, and what artificial means can be used to improve the produce ] In this Series of ' Scientific Handbooks ' these and kindred subjects will be dealt with, and so dealt with as to be intelligible to all who seek knowledge — to all who take an interest in the scientific problems and discoveries of the day, and are desirous of following their course. It is intended to give in a compact form, and in an attractive style, the progress made in the various departments of science, to explain novel processes and methods, and to show how so many wonderful results have been obtained. The treatment of each subject by thoroughly competent writers will ensure perfect scientific accuracy ; at the same time, it is not intended for technical students alone. Being written in a popular style, it is hoped that the volumes will also appeal to that large class of readers who, not being professional men, are yet in sympathy with the progress of science generally, and take an interest in it. The Series will therefore aim to be of general interest, thoroughly accurate, and quite abreast of current scientific literature, and, wherever necessary, well illustrated. Any one who masters the details of each subject treated will possess no mean knowledge of that subject ; and the student who has gone through one of these volumes will be able to pursue his studies with greater facility and clearer comprehension in larger manuals and special treatises. The first volume is a Manual of the Art of Assaying Precious Metals, and will be found valuable not only to the amateur, but also to the assayer, metallurgist, chemist, and miner. It has proved itself a desirable addition to the libraries of Mining Companies, engi- iieers, bankers, and bullion brokers, as well as to experts in the Art of Assaying. The press has com- mented upon it most favourably, and it has been acknowledged to be by far the best work on the subject suitable alike for the practical assayer and the general inquirer. The second volume of the Series is written by Pro- fessor Kimball, and deals with the physical properties of Gases. He has taken into account all the most recent works on ' the third state of matter,' including Crooke's recent researches on ' radiant matter.' There is a chapter also on Avogadro's law and the Kinetic theory, which chemical as well as physical students will read with interest. In the third volume Dr. Thurston treats, in a popular way, on ' Heat as a Form of Energy ; ' and his book will be found a capital introduction to the more exhaustive works of Maxwell, Carnot, Tyndall, and others. The fourth volume contains the only popular account extant of the science of Geodesy, written by Professor J. Howard Gore, of the Columbian University. The fifth volume, 'A Manual of Bacteriology/ by Dr. A. B. Griffiths, is, as its title implies, a treatise on the science of microbes, a knowledge of which is essential to professional men engaged in checking the spread of disease, and an advantage to all who value health and sanitation. Other volumes will follow, written, like these, by thoroughly competent writers in their own depart- ments ; and each volume will be complete in itself. Ibeinemarm's Scientific fmn&boofcs. i. MANUAL OF ASSAYING GOLD, SILVER, COPPER, AND LEAD ORES. By WALTER LEE BROWN, B.Sc. Revised, corrected, and considerably enlarged, with a chapter on THE ASSAYING OF FUEL, etc., by A. B. GRIFFITHS, Ph.D., F.R.S. (Edin.), F.C.S. In One Volume, small crown 8vo. Illustrated, 7s. 6d. n. THE PHYSICAL PROPERTIES OF GASES. By ARTHUR L. KIMBALL, of the Johns Hopkins University. In One Volume, small crown 8vo. Illustrated, 5s. CONTENTS. Introduction. Pressure and Buoyancy. Elasticity and Expansion with heat. Gases and Vapours. Air-Pumps and High Vacua. Diffusion and Occlusion. Thermodynamics of Gases. Avogadro's Law and the Kinetic Theory. Geissler Tubes and Radiant Mat- ter. Conclusion. III. HEAT AS A FORM OF ENERGY. By Professor R. H. THURSTON, of Cornell University. In One Volume, small crown 8vo. Illustrated, 5s. CONTENTS., The Philosophers' Idea of Heat. The Science of Thermodynamics. Heat Transfer and the World's Industries. Air and Gas Engines, their Work and their Promise. The Development of the Steam Engine. Summary and Conclusion. IV. GEODESY. By J. HOWARD GORE. Crown 8vo, cloth. Illustrated, 5s. Science Gossip.— 'It is the best we could recommend to all geodetic students. It is full and clear, thoroughly accurate, and up to date in all matters of earth-measurements.' V. MANUAL OF BACTERIOLOGY. ByA.B. GRIFFITHS, Ph.D., F.R.S. E., F.C.S. Small crown 8vo. Illustrated, 7s. 6d. LONDON: WM. HEINEMANN, 21 BEDFORD STREET, W.C. ^einemann'0 Scientific A MANUAL OF BACTERIOLOGY GRIFFITHS T71TI7ERSIT7 Ibeinemann's Scientific 1ban£>booh0. A MANUAL OF BACTERIOLOGY BY A. B. GRIFFITHS, Ph.D., F.R.S.E., F.C.S. IV TJIIVBRSITT LONDON WILLIAM HEINEMANN 1893 [All rights reserved. \ BIOLOGY UBRAKY 46709 TO ERNEST A. GRIFFITHS, ESQ. OF HER MAJESTY'S CONSULAR SERVICE IN JAPAN, MEMBER OF THE ASIATIC SOCIETY OF JAPAN, ETC. ETC. ETC. THIS WORK IS AFFECTIONATELY DEDICATED BY HIS BROTHER UHI7BRSIT7 PREFACE IN preparing this volume I have endeavoured to meet the requirements of those who are desirous of obtaining a knowledge of the nature and doings, for good or for evil, of those minute beings which are termed microbes or bacteria. The mystic words ' microbes ' and ' bacteria ' have been hurled at the popular head with so much emphasis and so little explanation that it would not be surprising to find many people living under the misapprehension that they are minute ' fiery serpents/ which are always on the look-out for victims, and crawl about them day and night. Not a few people feel comforted by the knowledge that microbes, harmless or harmful, belong to the vegetal rather than to the animal kingdom. Such knowledge takes away the element of repulsiveness arising from the notion of microbes being internal animal parasites or eutozoa. Although microbes are minute plants, they are capable of giving rise to some of the most deadly xii PREFACE diseases to which human flesh is heir. Conse- quently, a knowledge of the science of microbes, or bacteriology, is now incumbent on all medical men, sanitary engineers, chemists, physiologists, and biologists ; and even intelligent householders would be all the better if they had a general knowledge of the subject detailed in the following pages. My sincere thanks are due, and are here most gratefully tendered, to Dr. E. Klein, F.RS. ; Prof. P. F. Frankland, F.E.S. ; Prof. A. Gautier (of Paris) ; Prof. L. Brieger (of the University of Berlin); Prof. C. Tommasi-Crudeli (of the University of Eome) ; Prof. I. Giglioli (of Portici, near Naples) ; Dr. Eoux (of the Pasteur Institute) ; Dr. P. Miquel (of Paris); Dr. T. Lauder Brunton, F.RS.; Mr. W. Watson Cheyne, F.E.C.S. ; Dr. G. Sims Woodhead, F.E.S.K ; Dr. C. Zeiss (of Jena) ; and Messrs. F. E. Becker & Co. (of London) for valuable aid in various parts of the book. In conclusion, it is hoped that this volume may contribute something towards a proper understand- ing and an intelligent appreciation of the important and far-reaching subject of bacteriology. A. B. GEIFFITHS. EDGBASTON, January 1893. CONTENTS CHAPTER I INTRODUCTION : Koch's Canons — Vivisection— General Properties of Microbes — Products of Microbian Activ- ities— Sizes, Weights, and Reproductive Powers of Microbes, etc., ...... CHAPTER II BACTERIOLOGICAL LABORATORY AND ITS FITTINGS : The Edinburgh Laboratory — The Pasteur Institute— The Microscope — Microphotographic Apparatus — Dissecting Instruments — Microtomes — Sterilisers — Incubators — Cultivation Tubes, etc. , . CHAPTER III METHODS OF CULTIVATING, STAINING, AND MOUNTING MICROBES, ETC. : Cultivation Media — Cultivation Methods — Staining Preparation sand Tissues — Harden- ing, Imbedding, Cutting, and Mounting Preparations — Methods of introducing Microbes into Living Animals— The Unit of Microscopical Measurements, etc., ...... 49 CHAPTER IV THE ORIGIN, CLASSIFICATION, AND IDENTIFICATION OF MICROBES : Pleomorphism— Modes of Reproduction— The Classifications of Cohn, Zopf, Baumgarten, Hueppe, and De Bary, etc., ..... 98 CONTENTS CHAPTER V THE BIOLOGY OF MICROBES, ETC. : Micrococci — Bacteria — Bacilli— Spirilla — Spirochaetae— Yeast-Fungi, etc. , . CHAPTEE VI INFECTIOUS DISEASES AND MICROBES : Yellow Fever — Hydrophobia— Erysipelas — Puerperal Fever — Influ- enza — Pneumonia — Scarlatina — Leprosy — Syphilis — Tetanus — Malaria — Typhoid Fever — Cholera — Glanders — Diphtheria — Tuberculosis — Anthrax — Actinomycosis— Thrush, etc., . . . . CHAPTER VII MICROBES OF THE AIR : Examination of Air — Number of Dust Particles in Air — Air of Lincoln, Paris, London, etc. — Air of Country Places, etc., CHAPTER VIII MICROBES OF THE SOIL : Examination of Soils— Soils of Lincoln, Manchester, London, Paris, Dieppe, New Zealand, New York — Microbes and Leguminous Plants— Nitrification, etc., . . 276 CHAPTER IX MICROBES OF WATER : Examination of Waters — Water from Rivers Witham, Irwell, Thames, Lea, Seine, Marne, Isar, Spree— Self-purification of Rivers— Sand Filtration— Sterilisation of Water by Electricity, Heat, and Filtration through Porous Porcelain — Classification of Waters, etc., . . 286 CONTENTS xv CHAPTER X PAOE PTOMAINES AND SOLUBLE FERMENTS : Properties of the Ptomaines— Extraction of the Ptomaines — The Non- oxygenous Ptomaines — The Oxygenous Ptomaines — The Leucomaines — Albumoses, etc., . . . 305 CHAPTER XI GERMICIDES AND ANTISEPTICS : Metallic Salts— Halogen Elements — Aromatic Compounds — Oxidising Com- pounds— Miscellaneous Germicides — Concluding Re- marks, ....... 325 APPENDIX, ...... 332 INDEX, . 343 UNIVERSITY CHAPTEK I INTRODUCTION DURING the past ten years or so there is hardly a subject which has received so much attention as the Science of Bacteriology — the Study of Microbes. No one need wonder that the scientific world has been so busy in such a fruitful field of research, for it has not only been demonstrated that microbes play important parts in the processes of fermenta- tion, putrefaction, nitrification, etc., but that many of these lowly beings are intimately connected with infectious diseases. Phthisis, diphtheria, cholera, malaria, glanders, scarlatina, etc., have been proved to be the result? of the action of certain microbes on the blood an* tissues. Infectious diseases being due to the action of cer- tain microbes, it is necessary to isolate the microbes and to study them apart from the body. Hence the necessity of obtaining a pure culture of any particular microbe (i.e. its freedom from other microbes, etc.) before we can accurately study its 2 A MANUAL OF BACTERIOLOGY mode of growth, multiplication, and the products it may give rise to. In fact, Dr. E. Koch l has laid down the following canons _to ascertain whether a microbe is, directly or indirectly, the causa causans of a particular disease: — (1.) The microbe in question must have been found either in the blood, lymph, or tissues of the man or animal which is suffering from, or who has died of, the disease. (2.) The microbe taken from this medium (blood, tissues, etc.), and artifically cultivated in certain media, 'must be transferred from culture to culture for several successive generations, taking the pre- cautions necessary to prevent the introduction of any other microbe into these cultures, so as to obtain the specific microbe, pure from every kind of matter proceeding from the body of the animal whence it originally came. (3.) The microbe, thus purified by successive cul- tures, and reintroduced into the body of a healthy animal capable of taking the disease, ought to re- produce the disease, in the animal, with its char- acteristic symptoms and lesions. (4.) Finally, it must be ascertained that the microbe in question has multiplied in the system of the animal thus inoculated, and that it exists in greater number than in the inoculating medium. Microbes are everywhere present — in the air, in the earth, and in waters ; in and on food, clothes, etc. ; consequently they gain admittance into the bodies of man and animals. These microbes do not 1 Die Milzbrand-impfung, 1883. INTRODUCTION 3 necessarily give rise to disease, for many are harm- less, although they may be present in the blood and tissues. Not even in the case of an infectious disease, where a certain microbe is present, can one say that it is the cause of that disease. Not until Koch's canons are fulfilled, is the experimenter justified in saying that any particular microbe is pathogenic or disease-producing. From what has been said, it will be seen that bacteriology, as applied to disease, is dependent upon observation of, and experiments upon, living matter. Among phenomena of so complex a char- acter as infectious diseases, simple observation goes but a very little way, and our knowledge of all the most important truths of bacteriology, as applied to these diseases, has been obtained by experimentation upon living animals. Vivisection is necessary for a proper interpreta- tion of the phenomena. But ' every now and again a loud outcry is raised against this method, partly from ignorance and partly from prejudice. Many — probably most — of the opponents of experiments on animals are good, honest, kind-hearted people, who mean well, but either forget that man has rights against animals as well as animals against man, or are misled by the false statements of the other class. These are persons who, blinded by prejudice, regard human life and human suffering as of small import- ance compared with those of animals, who deny that a man is better than many sparrows, and who, to the question that was put of old, " How much, then, is a man better than a sheep?" would return the reply, 4 A MANUAL OF BACTERIOLOGY " He is no better at all." Such people bring un- founded charges of cruelty against those who are striving, to the best of their ability, to lessen the pains of disease both in man and also in animals, for they, like us, are liable to disease, and, like us, they suffer from it.' 1 Without vivisection, the important researches of Pasteur, Koch, Klein, and others could not have been conducted; in fact, vivisection is absolutely necessary to ascertain the pathogenic nature of any microbe. We now proceed to detail the general properties, etc., of microbes. All microbes contain two principal parts — a cell-wall or limiting membrane and a semi-fluid contents — the protoplasm. The cell-wall is composed of cellulose — a carbohydrate having the empirical formula C6 H10 05. The protoplasm ap- pears to vary somewhat in its chemical composition; for, in some microbes, this complex substance is devoid of sulphur and phosphorus, whereas in others, both of these elements are present. The protoplasm which is devoid of sulphur and phos- phorus, has been termed mycoprotein by Nencki, and has entirely different reactions from the proto- plasm containing sulphur and phosphorus. Microbes are capable of giving rise to various products, such as acids, alkaloids, colours, enzymes, albumoses, etc. This property depends upon the present potentialities of the protoplasm (in each case), and the inter-relation of its various functions, and these again result from, or are modified by, the 1 Dr. Lauder Brunton in Nature, vol. xliv. p. 331. INTRODUCTION 5 adjustment which takes place between an organism and its environment. For instance, the cholera bacillus grown on albumin produces toxines or alkaloids, and is pathogenic; on potatoes, it gives rise to a brown pigment and is chromogeuic ; while on sugar it produces butyric acid, and is con- sequently zymogenic or fermentive (Hueppe). The action of gases, heat, light, electricity, and various antiseptics have the power of altering the common properties of a microbe ; but in every case the usual products, etc., are formed when the microbe is once more transferred to its natural mode of life. ' Every organism has more potentialities or modes of action than those which are actually in operation at any given time, and when the environment is changed one or other of these potentialities may come into action, replacing, more or less completely, a former one.' The extent of the powers of adaptation of an organism depend on its potentialities and their capacity of extension, and these vary, in each case, enormously, a view in perfect consonance with the results which experiments have already yielded. As microbes differ in their actions, they likewise differ in their dimensions ; and, as a general rule, they vary from about 0*0005 mm. to 0'05 mm. in length or diameter, as the case may be. Dr. F. Cohn calculated that one bacterium (Bacterium termo) weighs 0*000,000,001,571 milligramme, or that six hundred and thirty-six millards of bacteria would weigh one gramme, or six hundred and thirty-six thousand milliards a kilogramme; and the late Professor J. Clerk Maxwell stated that the 6 A MANUAL OF BACTERIOLOGY smallest organised particle visible under the micro- scope contains about two million molecules of organic matter. The reproductive power of microbes is most prolific; and Cohn has calculated that a single microbe at the end of three days would have in- creased to nearly forty-eight billions, a mass which would weigh no less than 7500 tons. But this astounding rate of reproduction is kept in check by the limited supply of food, as well as by various circumstances which make the environment unsuit- able for such a rapid rate of increase. ' As a con- sequence of their enormous fecundity, it will be readily understood that they are ubiquitous. Every surface teems with them; all natural waters are infested by them ; even the skin of the most washed of mankind ; even the moisture of the sweetest mouth harbours them by the million ! One thing, however, they cannot stand, and that is boiling. Boil them or the stuff in which they are nourishing, and they cease to live — or, in other words, the liquid or solid substance so treated is sterilised. By means of sterilised nutriment we can test any object for the presence of microbes or bacteria, as they are sometimes called. We prepare a broth suitable for their nourishment, and sterilise it. If kept her- metically sealed (as are preserved vegetables and tinned meats), no microbes will appear in the broth. Touch the broth with any stick or stone, or add to it a drop of purest spring water, and it will, after a few hours, swarm with microbes and putrefy. This was the discovery of Theodore Schwann, also cele- INTRODUCTION 7 brated for his cell-theory. He showed fifty years ago that what we call 'putrefaction' is not the result of death, but of life. The unpleasant smell and the disintegration of dead bodies, whether of plants or animals, is entirely due to microbes — it is the accompaniment of their digestion. If you destroy all the microbes present by means of boiling heat, and then prevent the access of new microbes (which are blown about in the dust of the air), dead bodies never putrefy. Supposing that by the fiat of an omnipotent Being all microbes could be annihi- lated, the earth's surface would soon be covered with dead bodies remaining unchanged year after year, century after century. The seas and lakes would be choked with them, and we should have to use them for paving our roadways and building our houses. But worse than that, all the carbon and nitrogen which living things use in turn in their successive occupation of the earth's surface from generation to generation, would soon be tied up. There would be no food for the green plants : herbi- vorous creatures would cease to exist. The con- templation of these imaginable horrors gives us some notion of the part played by microbes in the order of nature.' CHAPTEK II THE BACTERIOLOGICAL LABORATORY AND ITS FITTINGS BEFORE describing the necessary apparatus, etc., for the proper investigation of bacteriological problems, we give a general account of the laboratories of the Koyal College of Physicians, Edinburgh, and those of the Pasteur Institute, Paris : these being chosen as typical examples of bacteriological laboratories. The Edinburgh Laboratory. — The ground floor of this laboratory, which is situated in Lauriston Lane, contains a workshop, stores, and a room set apart for experimental physiology. The latter is 32 feet long, 18 feet wide, and 14 feet high: it is fitted with tables for microscopical work ; a respira- tion apparatus driven by water-power ; recording apparatus ; galvanometer and other electrical ap- pliances ; and sink and draining apparatus. ' On each microscope table, which is painted black and hard varnished, a white band about four inches broad is painted, four inches from the edge of the tables. Some of the tables, instead of being varnished, are covered with plate glass, painted as above on the under surface, and imbedded in felt. On these glass-covered tables the microscope stands . THE BA CTERIOLOOICA L LA BORA TOR Y 9 on a felt circle, to diminish the risk of breakage when the bell jar is lowered over the microscope/ On the second floor there are five rooms, three of which are occupied by the laboratory assistant. Of the other two rooms, one is used as a library and museum ; while the other is the director's private room. The former room is fitted with an Oertling's balance, a barometer graduated in inches and millimetres, a thermometer with Fahrenheit and centigrade scales, and a large spectroscope. This room is also used for the meetings of the committee. The third floor (counting from the basement) contains six rooms. ' The first of these, a small one, is used as a still-room ; the still is connected with the water-pipe and is self -feeding, so that to obtain a supply of distilled water all that is necessary is to turn on the tap and light the Bunsen burner.' A second room — the chemical room — is fitted with a good supply of gas and water, working benches, evaporating chamber, sandbaths, and the necessary apparatus and reagents for the analysis of water, air, food, and for physiological chemical work. The next room is fitted with a table for histological work, but is chiefly used for blow-pipe work, metal injections (Cathcart's method), imbedding in paraffin and celloidin, and section-cutting by means of the microtome, etc. The same room is also used as a store for some of the glass apparatus. The next room is used as a store for chemical reagents. This is followed by another small histological room ; and finally, a room is set apart for the estimation of urea, albumin, and glucose in urines. 10 A MANUAL OF BACTERIOLOGY The fourth and top story contains three well- lighted rooms (Fig. 1). The south room is the true bacteriological laboratory (Figs. 1 and 2), and is fitted with tables, micro- scopes, sterilisers, incubators, and the apparatus neces- sary for research in the various branch- es of bacteriology. The other two rooms on the top story are fitted for histological work. The original cost for the whole equip- ment of the ' Edin- burgh Laboratory ' CUPBOARD was only fSSO.1 BACTERIOLDG/CAL ffi ^ Of course this la- boratory, being a public one, has been fitted for many workers ; but a good private laboratory, suit- able for one's own ROOK' o /H CUB AT OR STOVl *'- — ,*-»—• 27 fT . — - . i. PLAN OF BACTERIOLOGICAL LABORATORY, ETC. '-? fitted at less than a fourth of the above-mentioned amount. 1 See Dr. G-. Sims Woodhead's paper in Proc. Roy. Physical Soc., Edinburgh, vol. ix. p. 521. 12 A MANUAL OF BACTERIOLOGY The Pasteur Institute. — This celebrated institu- tion (see frontispiece) is not simply an hospital for the treatment of persons suffering from hydrophobia or rabies, but is a building set apart for the study of micro-biology in all its branches. The Pasteur Institute is situated in the Eue Dutot — not far from the Cimetiere Montparnasse — on the south side of Paris. It is the most perfect building of its kind in the world ; the cost of erection, fitting, and endowment being £100,000. The anti-rabic de- partment forms a relatively small portion, there being in addition an important department, in which are prepared vaccines for the prevention of several of the infectious diseases of cattle — rouget de pore (swine fever), anthrax, etc, — as well as laboratories, lecture-rooms, and a large library. In the same building is the residence of M, Pasteur, who naturally takes the greatest interest in the work of the institute. The Pasteur Institute covers an area of 11,000 square metres, and consists of two blocks, running parallel, one behind the other. These blocks are united by a long corridor. On the first floor of the front block is a room used as a library and council chamber ; and the second floor of the same block is entirely occupied by the attendants and servants of the establishment. On the right of this block is M. Pasteur's residence. The block in the background is divided into two wings, each about 25 metres long, and 15 metres from back to front. In the right wing, on the ground floor, are the rooms set apart for the anti-rabic treatment, and a laboratory in THE BACTERIOLOGICAL LABORATORY 13 which the preparation of the virus is carried on. This laboratory is always maintained at a tempera- ture of 23° C. In the left wing, on the ground Hoor, is a lecture-theatre for biological chemistry, a laboratory, and a room set apart for photographing microbes, etc. At the end of the block (i.e. at the back) are two rooms (one on each side of a central corridor) used as aquaria. The remaining portion of this block is occupied as a store-room and a general laboratory; the latter being used for the preparation of the various cultivating media, glass-blowing, etc. The second story is, likewise, divided into two halves ; on the left is the micro- biological department, and on the right that of practical biology. On the same floor there is a large laboratory fitted with sterilisers, incubators, evaporating chambers, etc. : in fact, this room is used for the growth of all kinds of microbes. Joining this room is a smaller laboratory, out of which one steps into the museum. In addition, there is a chemico- biological laboratory and a lavatory. The third story of the rear block com- prises two series of rooms, which are all used for research ; the left wing is occupied by the depart- ment of applied or practical bacteriology, while the right wing is devoted to the study of comparative micro-biology or bacteriology. Each of these de- partments is fitted with incubators, sterilisers, and other bacteriological apparatus. In each of the departments of the institute there is a director's private room and laboratory. Besides the two main blocks there are separate 14 A MANUAL OF BACTERIOLOGY buildings, etc., in the grounds of the Institute. Among these are the cages for the accommodation of animals; a special house for the reception of dogs ; stables, etc., for large animals ; a rabbit- house ; a run, etc., for hens ; and an aviary. All these places are kept in a state of perfect cleanliness. This important establishment would not be com- plete without a crematory ; this consists of two large furnaces, situated in one corner of the grounds, whicli are used for destroying all useless animal matter. The Pasteur Institute accommodates fifty workers, and is open to foreign as well as French scientific and medical men. Besides being an institute for research, it is also used for the instruction of pupils in both general and special methods of bacterio- logical investigation. The above is only a general account of the Pasteur Institute ; but the reader desirous of ob- taining fuller information is referred to the Annales de I'lnstitvt Pasteur, 1889. Having given a description of the Edinburgh and the Paris laboratories, we now proceed to describe the various apparatus and appliances used in the study of bacteriology. The Microscope. — In our experience the best microscopes suitable for the study of 'les infini- -ment petits ' are those made by Carl Zeiss, of Jena (Fig. 3). These instruments are monocular micro- scopes, and consist of the usual parts — the stand, eye-pieces, and objectives. Very few workers in bacteriology use the binocular microscope, because there is a great -loss of light, and the definition of THE BA C TERIOLOGICA L LA BORA TOR Y 15 high-power objectives is impaired when this instru- ment is used. It is believed by some that when the monocular microscope is used continuously the eyes are apt to become fatigued. The student should learn to keep both eyes open when work- ing with the monocular micro- scope. There are several ways by which this may be effected. One is by having a black sheet of paper near to the second eye; an- other plan is to put the hand be- fore the eye. Perseverance is all that is needed. One evening is quite enough to make any one skilful, if he is determined to ' succeed. The student need no more fear seeing things on the table with the second eye than seeing the crown of his head, unless he is^training for drawing objects -on the table by FIG. 3. ZEISS' MICROSCOPE. 16 A MANUAL OF BACTERIOLOGY means of a camera lucida from the instrument, whilst with one eye he looks at the object, and with the other draws the figure. A very little reflection will convince any one how desirable it is to keep the nerves of the eye as nearly in their right position as possible ; for an undue strain is caused if they are strained, and the sight is injured if much work is done. The object-glass or objective (Fig. 3 A) is the most important part of the microscope ; conse- quently it is necessary to have good lenses to do satisfactory work. The objectives are known as low and high powers — but for bacteriological work, the microscope should be provided with the following objectives (Zeiss') : — D and E (dry lenses), J (water immersion), ^ (oil immersion). Zeiss' lenses give perfect defini- tions, and everything there is to be seen can be made out with the highest powers. Oil-immersion lenses are taking the place of water high powers, as they need no correction for the thickness of the cover-glass, and are therefore much easier to use ; 1 the only drawback is that the essential oil (e.g. cedar oil) used will dissolve Canada balsam, Dammar varnish, and many of the sealing fluids, and it is necessary to cover them with Hollis' glue, which is not acted on by cedar oil.' Of Zeiss' high powers the ^ oil-immersion lens is the best, and may be thoroughly recommended for bacteriological research. Oil-immersion lenses possess far greater brilliancy and definition than the water and dry lenses [such as Zeiss' K and L (water), and F (dry)]. In using THE BA GTE RIO L OGICA L LA BORA TOR Y 1 7 oil-immersion lenses, a drop of cedar oil is placed on the front glass, the lens in use is then lowered on to the slide until contact is made. The lens is then focussed by the fine adjustment (Fig. SF) until the object is seen sharply defined.1 It is desirable that the bacteriologist's microscope should be fitted with a revolving triple nose-piece (Fig. 3 A); by this means three objectives (of different magnifying power) can be brought succes- sively into position, without unscrewing. The eye-piece or ocular is also an essential part of the microscope (see Fig. 3c); and for bacterio- logical research, the whole (five in number) of Zeiss' huyghenian eye-pieces are recommended. The fol- lowing table shows the magnifications of Zeiss' objectives and eye-pieces with a tube of 155 milli- metres in length — i.e. the Continental microscope with a short tube : — 1 Zeiss no longer makes the ^ oil-immersion objective ; this lens has now been superseded by the introduction of a new series of objectives — the apochromatic lenses — made of the new glass. These lenses are said to excel the ordinary objectives, by giving almost perfect achromatism and sharpness of image over the whole visual field (see Abbe's paper in Sitzungxberichte d. med.-naturw. Gesettschaft zu Jena, 1886). 18 A MANUAL OF BACTERIOLOGY EYE-PIECES. i. II. III. IV. V. Oil-immersion Water-immersion -P. , . ,. objectives. objectives. Drv obJectlves- «i a 2' «« a aa A,AA B, BB C,CC D, DD E F G H J K L * A A 7 11 15 22 12 17 24 34 20 27 38 52 4-12 30 7-17 41 10-24 22 56 75 38 52 71 130 97 175 130 235 70 95 120 145 195 270 360 175 230 320 435 580 270 355 490 670 890 1350 405 540 745 1010 260 340 470 640 855 320 430 590 805 1075 430 570 570 760 785 1070 1430 1900 2570 1045 1425 1930 770 1030 1415 260 340 470 640 855 380 505 810 695 950 1265 605 1110 1515 2020 Zeiss' J objective is equal to an English TV inch, while his B, 0, D, DD, E, and ^ oil-immersion are equal to an English 1, J, J, J, J, and ¥V inch respec- THE BACTERIOLOGICAL LABORATORY 19 lively. The medium objectives are issued in two different forms, with a greater or less aperture according to the purpose for which they are required. Those with a large aperture (distinguished by double lettering), possess with equally perfect definition a considerably higher resolving power, and permit of greater magnification being obtained by the use of the stronger eye-pieces. Nevertheless, the work- ing distance in BB, CO, DD, although relatively large, is perceptibly less than in the corresponding series of smaller aperture, and the former are more sensitive to differences in thickness of the cover-glass and object than the latter. Therefore, B, C, and D are recommended as the more suitable for working glasses in histological and anatomical research, particularly when the next stronger dry lens is available for higher magnification. The magnifications of the English objectives and eye-pieces with a ten-inch tube (i.e. the English microscope with a long tube), are given in the following table : — 20 A MANUAL OF BACTERIOLOGY EYE-PIECES. OBJECTIVES. A B c D 4 inch. 10 14 28 40 3 20 27 40 52 2 30 40 60 75 1 60 80 120 150 \ 75 100 150 190 i 100 133 200 250 A 170 227 350 440 \ 254 333 500 625 270 360 540 675 i. 450 600 900 1125 ft 500 666 1000 1225 ^ 700 940 1350 1640 The above will serve as approximately correct tables for ordinary work, but if the exact magnifying power of any objective is required it must be specially tested. The proper illumination of microscopic objects is of the highest importance, and that suitable for one class may be altogether unfit for another. Daylight is the best light to use for bacteriological work ; but if one is working at night or in the winter, a paraffin lamp is required. It is essential that the flame should be steady and of moderate size. Parallel rays may be advantageously thrown on the mirror (Fig. SD) of the microscope by means of a bull's-eye condenser, placed so that the flame is nearly in the focus. For comparatively low powers, a fla.t or concave mirror may be used to reflect the light, but for higher powers it is essential that the light should be concentrated by means of an Abbe's THE BACTERIOLOGICAL LABORATORY •21 substage condenser (Fig. 3fi). This condenser, first described by Professor Abbe (of the University of Jena),1 is of very short focus, and collects the light reflected by the flat mirror into a cone of rays of very large aperture, and projects it on the object. Abbe's condenser is focussed on the object by coarse and fine adjustments. ' When the whole field is to be examined, the lamp is used with the whole breadth of the flame, but when a small portion is to be K FlG. 4. MlCROPHOTOGRAPHIC APPARATUS. (After Carl Zeiss.) specially examined with a high power, it is necessary to turn the lamp so that the edge of the flame is pre- sented, by which the light is very much intensified. The correct distance at which to place the lamp can only be found out by practice. A piece of blue glass should be interposed between the lamp and the con- denser : this can be done by having it fitted into the condenser or by having a separate stand ; different shades of blue will be found useful for various objects. 1 Archivfur Mikr. Anat. vol. ix. p. 496. 22 A MANUAL OF BACTERIOLOGY The blue colour is a great help to the eyes, and also throws up the stained specimen with more dis- tinctness.' Microplwtograpliic Apparatus. — The application of photography as a means of illustrating microscopic preparations has been, on the whole, successful. Koch, Crookshank, Van Ermengern, and others, have produced beautiful photographs of microbes and sections of diseased tissues. For this purpose many different kinds of apparatus have been devised ; but one of the best is represented in Fig. 4. It consists of two tables (A and S) for the microscope and camera respectively ; two diaphragm carriers (E and F) for use with sunlight ; an electric lamp (C) ; a holder for taking absorption cells (H); a water chamber for absorbing heat rays (T); a camera (K); a collective lens-system for projecting the image of the carbon points on the focussing screen (L) — this is required when the electric lamp is used ; a micro- scope (M) ; and focussing apparatus (a, b, b', h). This apparatus can be used with sunlight,1 lime- light, electric-light, and lamp-light. For micro- photographic purposes, microscopic preparations are best when stained yellow, black, or brown, and mounted in either Canada balsam (dissolved in xylol) or a saturated solution of potassium acetate. Several authors have recommended the use of the isochromatic dry-plates, and first-class photographs have been obtained by them.2 1 When sunlight is used, a heliostat is also necessary. 2 See Crookshank's Photography of Bacteria; and Van Enn- engem in Bulletin de la Soc. Beige de Microscopie, No. 10, 1884. THE BACTERIOLOGICAL LABORATORY 23 Another method for obtaining illustrations of microscopic preparations is by means of the camera lucida. Among the best of these instruments, suitable for bacteriological purposes, are those of Zeiss and Nachet. ' Combined with the use of a micromillimetre objective, the camera lucida affords also a simple method for the measurement of bacteria.' The third and last method for obtaining illustra- tions of microscopic preparations is drawing by hand. If a white piece of card-board or smooth drawing-paper is fixed at the same level as the stage of the microscope ; by keeping both eyes open — one for looking at the object through the microscope, and the other for looking at the piece of card-board — an image of the object is seen on the card, which can be readily traced with a pencil. For drawing bacteria, etc., no pencil is so well adapted as Windsor and Newton's HHHH; the blacklead being brought to its final point by gentle rubbing on the surface of the finest ground glass, or, better still, a very fine hone. For inking the pencil drawings, the finest etching pens should be used — perhaps the best are those made by Joseph Gillott ; and the same maker's No. 303 is also a very fine- pointed pen. In addition to the pencils and pens —Indian-ink, water-colours, and brushes are neces- sary. With practice and patience, very accurate drawings of microscopic preparations can be made by hand. Dissecting Instruments. — For the dissection of diseased organs, tissues, etc., certain instruments are 24 A MANUAL OF BACTERIOLOGY necessary. Figs. 5 and 6 represent various kinds of scalpels, microscopic needles, knives, scissors, and FIG. 5. DISSECTING KNIVES AND NEEDLES. A to F are used in microscopic dissections. G to J are used in ordinary dissections. forceps ; and Fig. 7 illustrates a very useful form of dissecting microscope. THE BA CTERIOLOGICAL LA BORA TOR Y 25 The mode of carrying out a dissection for bac- teriological purposes is as follows : Animals either artificially inoculated with pathogenic microbes or those naturally suffering from infectious diseases FIG. 6. DISSECTING SCISSORS AND FORCEPS. FIG. 7. DISSECTING MICROSCOPE. should be dissected as soon after death as possible. In dissecting, every precaution must be adopted to exclude putrefactive or other microbes. The dis- section should be performed in a perfectly still 26 A MANUAL OF BACTERIOLOGY room with closed doors ; and the instruments used in the dissection must be previously sterilised in the hot-air steriliser or the Bunsen flame. The animal under examination (e.g. a mouse, rabbit, guinea-pig, etc.) is pinned out on a slab of gutta- percha previously washed in a solution of mercuric chloride (corrosive sublimate). It is now bathed in a stream of the same germicidal agent ; and after having cut away the hair with sterilised scissors, the seat of inoculation, etc., should be examined first, and any pathological characteristics should be noted. If there is any exudation, it should be used for inoculating purposes and microscopical examination. To examine the internal organs, place the animal on its back and make an incision extending (if necessary) from the abdominal to the thoracic region. The organ under examination should be re- moved from the body-cavity, with sterilised scissors and forceps ; and after removal it should be washed with mercuric chloride. The organ is now incised, and the fluid, or a portion of the organ itself (i.e. from the cut) should be used for inoculating various cultivation media. If the blood of the animal is required, it is best obtained from a vein by making an incision with sterilised scissors, and then insert- ing a sterilised capillary pipette or a platinum needle. The blood so obtained should be examined microscopically, and various cultivation media inoculated with it. If the cultivations are con- taminated by the presence of other microbes, frac- tional plate-cultivation must be resorted to, in order to isolate the pathogenic microbe. THE BACTERIOLOGICAL LABORATORY 27 After dissection, the organs, etc., may be pre- served in absolute alcohol, i.e. if they are required for future examination ; and all useless matter should be destroyed, and, finally, the hands, instru- ments, and table disinfected. FIG. 8. SCHANZE'S MICROTOME. Such is the mode of carrying out a dissection on a dead animal; but to obtain microbes from the living animal or from man, these may be isolated from pus and other discharges, or from the blood. These fluids must be obtained with all the necessary 28 A MANUAL OF BACTERIOLOGY precautions to prevent external microbes gaining an entrance. Microtomes. — These instruments are used for cut- ting sections of organs, tissues, etc. ; and there are many forms in use. Fig. 8 represents Schanze's microtome, and it is a most useful instrument for cutting sections imbedded in celloidin. The Cambridge rocking microtome 1 is an instru- ment for producing ribbons of sections imbedded in paraffin. The razor is supported and clamped in front of a brass tube containing the imbedded object. This tube fits tightly on to the end of a cast-iron lever ; and is made to slide backwards or forwards so as to bring the imbedded object near to the razor. By an arrangement of pivots, milled screws, and a milled wheel, the lever is moved for- wards, and the object to be cut is therefore brought across the edge of the razor : when the lever is made to move backwards the section is cut. The values of the teeth on the milled wheel are as follows : 1 tooth of the milled wheel = ^57 of an inch = '000625 mm. 2 teeth ,, ,, = ^J^ ,, ='001250 mm. 4 j, ,, ,, = 10000 55 — '0025 mm. 16 ,, ,, 15= urfto 55 = '01 mm. On working this microtome the sections should adhere together so as to form a ribbon. The work- ing of this instrument requires very little skill on the part of the operator ; consequently it is to be recommended to those who require very thin sec- 1 Made by the Cambridge Scientific Instrument Company, St. Tibb's Row, Cambridge. THE BACTERIOLOGICAL LABORATORY 29 tions of diseased organs, etc. Dr. Sims Woodhead has somewhat modified the Cambridge rocking microtome by adding a solid end to the brass tube into which ' dies ' of various sizes, with roughened surfaces, can be screwed. ' This does away with the inconvenience of having to " melt in " the imbedded tissue into the tube. A dozen of the dies may be used, and to each of these a piece of tissue may be fused, and kept ready for cutting at any time. ' Besides the microtomes just mentioned there are those of Korting, Eeichert, and Jung, which are principally used in France and Germany. When tissues are to be examined in the fresh state, either Koy's or Williams' freezing microtome should be used for section-cutting. In the former instru- ment the tissue is frozen by means of an ether spray ; while in the latter the frozen tissue is pro- duced by a mixture of ice and salt. There is no doubt that Eoy's microtome is the better instrument of the two, as the freezing of the tissue only occupies from thirty to forty seconds ; and this microtome may also be used for cutting objects imbedded in paraffin and not requiring freezing — in other words, the instrument can be used both as a freezing and a non-freezing microtome. Tissues imbedded in paraffin or a mixture of white wax and olive oil may be cut by hand with a hollow-ground razor. The razor is dipped in dilute alcohol and then drawn diagonally across the mass (containing the specimen) with a steady sweep. Before cutting each section the razor A MANUAL OF BACTERIOLOGY should be dipped in dilute alcohol. 'Great care is required in cutting sections by hand, to hold the razor firmly yet lightly, so as to cut them thin and at the same time even, and this cannot be done without a great deal of practice/ The author has, in his possession, sections of the human brain vary- B FIG. 9. KOCH'S 'STEAM STERILISER. ing from the y oV o to the T^¥ of an incn in thick- ness which were hand-cut by Dr. E. Palmer, who was formerly the resident physician to the Lincoln County Asylum, Lincoln. To obtain such sections requires skill and practice, therefore it is better to use the microtome. THE BACTERIOLOGICAL LABORA TORY 31 Sterilisers. — In a study like bacteriology, all vessels, instruments, etc,, used in the cultivation of microbes, must, before use, be rendered perfectly sterile. It cannot be too firmly impressed upon the mind that the only way to obtain pure cultiva- tions of microbes, is the complete sterilisation of all vessels arid instruments used by the experi- menter. For the accomplishment of this object steam, hot-air, Btmsen or spirit flames, and germi- cides are used as sterilising agents. Fig. 9 represents Koch's steam steriliser ; and is used for sterilising test-tubes, flasks, and for cook- ing potatoes. It is a cylindrical vessel of stout tin plate, with a copper bottom, provided with a conical lid, brass tubulure for the insertion of a thermometer, a grating, water gauge, tap, and a receiver with perforated bottom for cooking potatoes (b). The cylinder (which is 20 in. high and 10 in. diameter) is divided into two compartments (a and c). The lower one contains boiling water, while the steam therefrom passes into the upper or sterilising compartment. The cylinder is heated from below by a Bunsen's or Fletcher's burner. Steaming is usually kept up for from fifteen to twenty minutes ; and this operation is repeated on three successive days each time for twenty minutes. By such steaming the various cultivation media, etc., are rendered sterile, i.e. free from microbes. A later form of this steriliser contains three compart- ments instead of two. Two of these are used as sterilising compartments, while the lowest one con- tains the boiling water, which is always kept at a 32 A MANUAL OF BACTERIOLOGY constant level. Both forms of Koch's ' steriliser are covered externally with felt. The so-called steam digesters or ' autoclaves ' are chiefly used in France. They are made of stout copper, and are used for sterilising sealed flasks (containing bouillon) under pressure. The temperature in these digesters often rises as high as 120°C. Besides the above - mentioned steam sterilisers, there are those of Herrmann and Hirschberg, Ost- walt, Muencke,1 and Woodhead.2 All these are use- ful instruments, and are to be re- commended for the bacteriological FIG. 10. HOT-AIR STERILISER. laboratory. Two forms of hot-air sterilisers are represented in Figs. 10 and 11 respectively, and are used for the sterilisation of flasks, test-tubes, cotton- wool, etc. The former consists of a double wall of sheet-iron, and the inner dimensions are 12 in. x 10 in. x 10 1 Dingler's Polytechnisches Journal, 1885, Bd. 257, p. 283. '2 Proceedings of Royal Physical Society of Edinburgh, vol. ix. p. 524. THE BACTERIOLOGICAL LABORATORY 33 n. It is heated by a gas-burner, and is made to hang against the wall of the laboratory. The steriliser represented in Fig. 1 1 l is either heated by paraffin-oil or by gas. It consists of a I I /O Q '&<^L> 4j f H/g? 5> 0 & Q O Q -^FFTT'^W Fio. 11. HOT-AIR STERILISER HEATED BY PARAFFIN OIL OR GAS. (Devised by Mrs. A. B. Griffiths.) A, Thermometers. B, Copper Shelf with Holes of different sizes. C, Mica Window. D, Iron support for Oven over Flame. E, Paraffin Oil Lamp. F, Screw to raise Wick. G, Wire Gauze. sheet-iron chamber, provided with shelf containing 1 First described by the author in the Proceedings of the Poyal Society of Edinburgh, vol. xiv. p. 105. C 34 A MANUAL OF BACTERIOLOGY a series of holes of different sizes; by this means the tubes or flasks are placed in a vertical posi- tion in the steriliser. It may be stated that all good hot-air sterilisers should allow the tubes to be placed in a vertical rather than a horizontal position. By this means the heated air rises in the inverted tubes, flasks, etc., and the current so formed (in each tube, etc.), destroys all the microbes and spores present therein. The hot-air sterilisers of Koch, Muencke, Pasteur, and Klein are all good sterilisers. Dr. Klein's con- sists of an iron chamber with double wall and double folding-doors. In the inner chamber are placed the test-tubes in a horizontal position, and the cotton-wool above them. After closing the doors the steriliser is heated by a Fletcher's gas-burner. ' Test-tubes (to be sterilised) should be exposed to the full heat of the chamber for several hours. After this they should be taken out of the steriliser while hot, plugged with sterilised cotton-wool, and then reheated for a few hours longer. Beakers and glass funnels may also be sterilised in the hot-air steriliser, or by being heated over a Bun sen flame. To prepare sterilised cotton-wool, place the wool in a loose condition, and heat it in the hot-air steri- liser to a temperature of about 150°C. for several hours on several successive days.'1 Over-heating the cotton-wool in the hot-air steriliser to the above temperature until singed has proved invariably and absolutely safe for all cultivations. 1 See Dr. A. B. Griffiths' book, Researches on Micro-Organisms, p. 14(Bailliere&Co.). THE BACTERIOLOGICAL LABORATORY 35 To use cotton-wool, flasks, and tubes disinfected by prolonged steeping in alcohol, carbolic acid solu- tion, and other chemicals, is not absolutely reliable ; and many failures have been the results of such methods of sterilisation. Therefore, 'it cannot be too thoroughly insisted on that the flasks and test- tubes, and especially the cotton- wool used as plugs for the vessels, should be thor- oughly sterilised by over-heating, for cultivations are as often con- taminated by this not being properly carried out as by the non-sterility of the nourishing fluids or the acci- dental entrance of organisms from the air.' For the sterilis- ation of scalpels, forceps,platiuum needles, etc., the Bunsen flame is the best way of cleansing them ; but, unfortunately, the naked flame is most destructive to the blades of scalpels; to obviate this, Israel's case was devised. It is a sheet- iron box (with lid), in which the scalpels, etc., are exposed to a temperature of 150°C. in the hot-air FIG. 12. SERUM STERILISER. 36 A MANUAL OF BACTERIOLOGY steriliser for an hour or so. By this device the blades are not injured. For the preparation of solidified sterile blood serum two pieces of apparatus are necessary ; these are represented in Figs. 12 and 13. Fig. 12 is the serum steriliser, and consists of a double-walled cylinder, 13 inches in height and 11 inches in dia- meter, made of stout tin, with a copper bottom. FIQ. 13. SERUM INSPISSATOK. This cylinder is provided with a double-walled lid, having a tubular prolongation of stout copper, tap, and gauge : the whole being surrounded with thick felt. The apparatus is divided internally into four compartments ; and into these are placed the test- tubes, or glass capsules, containing the blood serum. Between the two walls of the cylinder is a layer of water, which is heated from below ; while the water THE BACTERIOLOGICAL LABORATORY 37 in the lid (i.e. between the two walls) is heated by means of the prolongation (see Fig. 12). It will be seen that the whole apparatus is essentially a hot- water jacket. The temperature of the steriliser should be maintained for an hour at 60° C. on five or six successive days. By this means the fluid serum is completely sterilised, but it is not solidi- fied. To solidify the serum the piece of apparatus represented in Fig. 13 is required. It is a double- walled case, also made of stout tin (13J in. longx 13 J in. widex4j in. deep). It is provided with a copper bottom, glass cover, water-gauge, and ther- mometers ; there is also an arrangement by which this inspissator, as it is called, can be fixed at the angle required; this being necessary to give the serum a sloping surface. The tubes, etc., containing the sterile but fluid serum, are placed in the inspis- sator ; and this apparatus (like the serum steriliser) containing water (between the two walls) is heated from below. To coagulate the serum, and to solidify nutrient agar-agar, a temperature between 65° and 68° C. should be maintained ; but as soon as solidi- fication takes place the tubes should be removed from the inspissator. Solidified blood serum is used for the cultivation of Bacilhis tuberculosis, Bacillus mallei, and a few other microbes ; but we shall refer, in detail, to the various cultivation media and the methods of cultivation, in the next chapter. Incubators. — Many microbes are capable of being cultivated at the ordinary temperature of the laboratory; but certain microbes require a higher temperature for their proper development and mul- 38 A MANUAL OF BACTERIOLOGY tiplication. For the latter purpose various ovens or incubators have been devised. One of the most FIG. 14. BABES' INCUBATOK. convenient forms is the incubator of Dr. Babes (Fig. 1 4). It consists of a rectangular, double-walled THE BACTERIOLOGICAL LABORATORY 39 chamber, covered on five sides with felt, but in front the felt forms a loose flap, which can be raised. The interspace between the two walls is filled with water, which is heated from below. The incubator has two glass doors, a moveable shelf, a water-gauge, and a gas-regulator. Among other good incubators are those of Pas- teur,1 Eohrbeck,1 Klein,2 Gautier, Abel, D'Arsonval. and Hueppe. Whatever form of in- cubator is preferred, it is essential that it should be provided with a gas or heat regulator. The acting agent in most regulators of this de- scription is either a membrane, mercury, or electricity. Tieftrunk's, Giroud's, and Elster's are membrane gas -regulators ; Eeichert's, Page's, Schiitz's, Fraenkel's, and Meyer's are mercury heat - regulators ; and Schlosing's is a membrane heat-regu- lator. Fig. 15 represents Keichert's mercury heat - regulator. It is a Flo> 15> tube with two lateral arms (a and REICHERT-S ,x ,, .. f , \ , . REGULATOR. 0); the upper portion of which is extended into a funnel-like arrangement, bearing the arm b. Into this funnel-like opening fits a hollow T piece. ' One arm of the T piece is open, and connected with the gas supply; the vertical 1 For figures of these incubators see Dr. Griffiths' Researches on Micro-Organisms, pp. 19 and 20. 2 See Dr. Klein's Micro- Organisms and Disease (3d ed.), p. 15. 40 A MANUAL OF BACTERIOLOGY portion terminates in a small orifice, and is also provided with a minute lateral opening.' The tube and arm a contain mercury. Reichert's regulator is fixed into the roof of the incubator, so that its FIG. 16. KARL ABEL'S INCUBATOR. (With thermo-electric regulator.) lower portion projects either into the water chamber or into the interior of the incubator. 'When the incubator reaches the required temperature, the THE BA CTERIOLOGICA L LABOR A TOR Y 41 mercury is forced up by means of the screw in the lateral arm, until it closes the orifice, at the extre- mity of the vertical portion of the T piece. The gas which passes through the lateral orifice is suffi- cient to maintain the apparatus at the required temperature. If the temperature of the incubator falls the mercury contracts, and the gas passing through the terminal orifice of the T piece, increases the flame of the burner, and the temperature is restored.' Page's regulator resembles somewhat the regulator just described : both are simple and useful forms for the bacteriological laboratory. By such devices the various incubators, may be maintained at a temperature which is almost constant; the slight differences (say of one or two degrees Centi- grade) are due to the variations in the pressure of the gas supply ; but this inconstancy is remedied by first passing the gas through a pressure - regulator (such as Moitessier's). In addition to the above-mentioned regulators, there are two forms which are worked by the agency of the electric current. Babes'1 and Abel's2 are thermo-electric regulators ; the latter being repre- sented in Fig. 16. These are useful regulators ; but for general work those of Keichert and Page are specially recommended. Cultivation Tubes, etc. — Fig. 17 represents an im- portant series of glass tubes, flasks, etc., used in the cultivation of microbes in liquid media. These 1 Centralblatt fur Bakteriologie und Parasitenkunde, 1888. 2 Ibid,, 1889, p. 707. 42 A MANUAL OF BACTERIOLOGY are first carefully washed with soap and water, then with a boiling solution of potassium per- manganate, to which a few crystals of oxalic acid are added. They are then rinsed with distilled water, and are allowed to drain on a rack for some time, after which they are carefully plugged with FIG. 17. CULTIVATION TUBES, FLASKS, ETC. A, Gayou's Tube. B, Gayon and Dupetit's Tube. C, Chamber-land's Tube. D, Sternberg's Bulb. E, Aitken's Tubes. F, Pasteur's Flask with Cap. G, Pasteur's Bulb Pipette. H, Pasteur's Test-tube. I, Miquel's Double Tube. J, Lipez's Tube. K, Miquel's Bulb Tube. L, Pasteur's Pipette Flask. MO, Pasteur's Flasks. N, Lister's Flask. P. Chamber-land's Pipette. R, Duclaux's Tube. S, Miquel's Filter Flask. T, Pasteur's Double Tube. cotton-wool, care being taken that the wadding inside the neck is perfectly smooth and firm, the tuft outside being large enough to overlap well the lip of the test-tube (Woodhead). They are THE BACTERIOLOGICAL LABORATORY 43 then ready for the nutrient fluid and subsequent sterilisation. Fig. 17 F and H represent Pasteur's flask and tube, both of which are provided with caps. The narrow portion of each cap contains a plug of cotton-wool. De Freudenreich's flask is somewhat similar to that of Pasteur. These are used for the cultivation of microbes in bouillon. In Pasteur's pipette flask (Fig. 17 L), the tube above the bulb is contracted twice, and on either sides of these con- tractions there are plugs of cotton-wool. The portion below the bulb is bent twice and is drawn out to a capillary point. The flask is charged with bouillon and inoculated by aspiration ; and then the capillary point is sealed in the Bunsen flame. Miquel's1 bulb tubes (Fig. 17 K and i) are similar devices. The tubes and flasks T, M, K, P (Fig 17) are pro- vided with lateral arms drawn out to fine points, and with necks plugged with cotton-wool. They are filled by aspiration and are convenient for storing sterilised bouillon. ' The sealed end of an arm is nipped off with sterilised forceps, the sterile bouillon aspirated into each limb, and the arm again sealed in the flame; a series of such tubes and flasks can be arranged upon a rack on the working table.' Sternberg's bulbs (Fig. 17 D) are generally kept in stock in the bacteriological laboratory. They are readily prepared by blowing a bulb on a piece of glass-tubing, and then drawing the tube out to a fine point which is hermetically sealed. To fill a bulb, it is first slightly heated, then the sealed 1 Lea Organismes Vivants de I' Atmosphere, 1883. 44 A MANUAL OF BACTERIOLOGY point nipped off, and the open end dipped beneath the surface of the culture fluid. As the bulb cools the fluid is drawn into it. The neck of the bulb is again sealed, and the fluid contained therein is sterilised by repeatedly boiling the bulb in a water- bath. It is then placed in an incubator for three or four days. If the contents remain transparent and clear, there is no doubt that the fluid has been properly sterilised. Many of these bulbs, containing sterilised bouillon, should be kept in stock. It may be mentioned that Chamberland's tube (Fig. 17 c) is filled and sterilised in the same way as Sternberg's bulb. Sir Joseph Lister's flask (Fig. 17 N) is used for the storage of culture fluids. The fluid is introduced into the flask, the neck plugged with cotton-wool, and the fluid sterilised by repeated boiling. When a portion of the sterile fluid is required, all that is necessary is to pour it through the lateral arm of the flask : this is done by simply tilting the flask. When the flask regains its erect position a drop of the fluid remains behind in the fine opening of the arm ; and thereby prevents the regurgitation of unfiltered air. After the removal of a portion of the fluid, a cap of cotton- wool is tied over the lateral opening, and the residue in the flask is kept for future use. Aitken's tube (Fig. 17 E) is a modification of the ordinary test-tube. It has a lateral arm whose ex- tremity is hermetically sealed. The nutrient fluid is introduced through the open end of the tube, THE BACTERIOLOGICAL LABORATORY 45 which is then plugged with cotton-wool. The fluid is sterilised by heating in the usual way ; and is inoculated by nipping off the sealed end of the lateral arm, and introducing the inoculating needle through the orifice. The needle deposits the material on the opposite side of the tube : it is then withdrawn and the lateral orifice again sealed. The fluid is then tilted so as to wash down the inoculating matter. The inoculated tube is then placed in an incubator. The remaining tubes, flasks, and pipettes (see Fig. 17) are ati used in the cultivation of microbes. Some are used for storage purposes ; while others are used as culture tubes, flasks, etc. A very good stor- age flask has been recently described by Dr. Sims Wood- head.1 This flask (Fig. 18) was devised in order to do away FIG. is. .., ., , , ,11 WOODHEAD'S STORAGE FLASK. with the troublesome method of filling test-tubes, etc., with a pipette. A large flask (containing bouillon) is fitted with an india- rubber stopper with two holes. ' Through these pass two tubes, one with a thistle-head tube run- ning to near the surface of the fluid, i.e. about two-thirds of the distance down into the flask, the other passing just through the stopper. To the shorter tube is fitted a piece of india-rubber tubing 1 Proceedings of Royal Physical Society of Edinburgh, vol. ix. p. 537. 46 A MANUAL OF BACTERIOLOGY on which is a Mohr's clip, and to the other end of this tubing is fitted a piece of glass tubing with a constricted orifice. A plug of carefully sterilised cotton wadding is pushed into the thistle- head, the india-rubber stopper is pushed into the neck of the flask, and then a sheet of cotton wadding is placed over the whole of the tubes and the mouth of the flask, and is held in position by an india-rubber band. The flask is placed in a steam steriliser, where it may be left for a sufficient length of time to allow of it becoming perfectly sterilised. It is filled nearly a third full with bouillon or gelatine, after carefully removing the sheet of wadding and the stopper ; these are then replaced, and the whole is again sterilised as usual. When the gelatine or bouillon is to be drawn off into test-tubes, the flask is inverted and held in a retort stand, the sheet of wadding is carefully re- moved and folded, the glass nozzle is inserted into the mouth of the test-tube, the clip is opened and the gelatine or bouillon escapes ; all the air passing into the flask, being filtered through the wadding in the thistle-head tube, is thoroughly sterilised. If the whole of the gelatine or bouillon is not with- drawn, all that is necessary is to replace the sheet of wadding (care having been taken to preserve the inner surface, by folding it inwards). There is no necessity to sterilise after this has been once done, all that is necessary subsequently is to heat sufficiently to render the peptonised gelatine fluid ; but this is not required if the stock flask con- tains bouillon. This apparatus is specially useful if ' ^^ ITJiriVERSITYf THE BACTERIOLOGICAL LABORATORY 47 for milk, as the cream always rises to the surface and is so left to the last/ In all the vessels previously mentioned the nutrient fluid is sterilised by heat ; but in certain cases it is necessary to sterilise the fluid without the application of heat : this is performed by means of the apparatus devised by M. Chamberland, of the Pasteur Institute. The fluid is forced by a hand- pump through porous porcelain ; and by this means it is sterilised. In addition to the apparatus, etc., already men- tioned in this chapter, a well - fitted laboratory should contain: gas-burners with mica chimneys, water-baths, hot-water filters, platinum needles, wire cages for test-tubes, test-tube stands, glass damp chambers, graduated cylinders, glass dishes and capsules, thermometers, syringes, meat press, ' glass benches,' desiccators, anatomical jars, iron box for glass plates, mouse cages, beakers, glass rods, glass and india-rubber tubing, chemical balance and weights, as well as the various nutrient materials, stains, hardening, imbedding, and mounting mate- rials, and chemical reagents. The last are useful for the extraction and analysis of ptomaines and similar bodies. Although we have detailed the most important pieces of apparatus for the bacteriological labora- tory, there are others of importance, but as these are only used for special purposes they will be described later in the volume, i.e. in their proper places. 48 A MANUAL OF BACTERIOLOGY It only remains for us to say on this subject that all the apparatus, etc., used by the English, French, and German schools of bacteriologists may be ob- tained from Messrs. F. E. Becker & Co., 33 Hatton Wall, Hatton Garden, London. CHAPTER III THE METHODS OF CULTIVATING, STAINING, AND MOUNTING MICROBES, ETC. Cultivation Media. — There are two forms of media used in the cultivation of microbes — one fluid ajid the other solid. The fluid media were first used by the French school, while the latter (i.e. the solid media) were originated by Dr. E. Koch and his fol- lowers. Both fluid and solid media have their own special advantages, and both are now used in every bacteriological laboratory. Of the fluid media, the first to be described is bouillon (beef, pork, or chicken broth). This medium is prepared in the following manner : — one pound of lean beef (pork or chicken) is minced by passing it through an ordinary mincing or sausage machine. The minced beef is thoroughly mixed with lOOOcc. of distilled water, and the mixture allowed to stand for twenty-four hours. It is again thoroughly mixed, and then boiled for about an hour. As the fluid is always more or less acid, it is ^ necessary to render it neutral or slightly alkaline, this being done by the addition of a solution of pure sodium carbooate. The point at which the fluid D 50 A MANUAL OF BACTERIOLOGY becomes neutral or slightly alkaline is easily ascer- tained by the ordinary test-papers (litmus and tur- meric). It is essential to neutralise any acids, because they are well known to interfere with the growth of many microbes. The extract so obtained is strained through fine linen, and finally filtered through Swedish filter paper. If the filtrate is still acid, add a little more sodium carbonate solu- tion ; remove the fat by skimming; add distilled water to make up to the original bulk; and again filter (by means of a hot- water filter, Fig. 19) into a large storage flask or into sterilised test-tubes provided with sterilised plugs of cotton -wool. These vessels and their contents are then heated in the steam steriliser for half-an-hour on each of three successive days. Sometimes the beef ex- ria. iy. HOT-WATER *ILTEB. tract or bouillon is modi- fied by the addition of other materials. Dr. P. Miquel adds common salt in such proportions as to make a 0'5 per cent, solution. MM. Eoux and Nocard add glycerine to the bouillon before it is finally sterilised. This glycerine-bouillon is an excellent medium for the growth of Bacillus tuberculosis. THE METHODS OF CULTIVATING MICROBES 51 Liebig's extract (5 to 1000) and Cibil's extract of beef (20 to 1000) may also be used for the same purposes as bouillon; but these extracts require very careful sterilisation by Professor Tyndall's method of discontinuous heating. Liquid blood serum is used in drop-cultivations, etc. It is obtained by collecting the blood of a healthy sheep, calf, or horse, in sterilised flasks or glass cylinders with stoppers. The vessel or vessels containing the blood are placed in an ice-box or in ice-cold water for about twenty-four hours, when the separation of the clot will be completed. The fluid serum is then transferred, by sterilised pipettes (see Fig. 1 7 G), into sterilised test-tubes provided with cotton-wool plugs. The test-tubes and their contents are then heated in a serum steriliser for an hour or two at 60° C. on six successive days. Up to this point the serum forms a fluid medium ; but in the majority of cases blood serum is used as a solid medium. To solidify it, the serum (contained in test-tubes, watch-glasses, or capsules) is placed in an inspissator, kept at a temperature between 65° and 68° C., until solidification takes place. Milk is also used as a fluid medium. It is best sterilised at 120°C. in a steam digester or an auto- clave. By this means it is readily sterilised in about fifteen minutes. Milk can also be sterilised in the steam steriliser at 100°C., but it is necessary to heat it for an hour on the first day, and for thirty minutes on each of the following two days, that is (unless an autoclave is used), milk must be sterilised by discontinuous heating. 52 A MANUAL OF BACTERIOLOGY Various infusions of hay, wheat, cucumber, and turnip, and decoctions of malt, prunes, raisins, and horse-dung are used as cultivation media. They are sterilised by being heated in the steam steriliser for thirty minutes on three or four successive days. The mucors grow well in decoctions of malt and horse-dung ; various Aspergilli in a decoction of malt and prune-juice ; and an infusion of hay is a useful medium for the growth of Bacillus suUilis. Urine and other fluids of the body are used as cultivation media: these are sterilised after the manner described for bouillon. Besides the above-mentioned fluid media, there are two others which are useful for the growth of certain microbes and moulds. One of these is Pas- teur's fluid, which contains 10 parts of pure cane- sugar, 1 part of ammonium tartrate, the ash of 1 part of yeast, and 100 parts of distilled water. The other is known as the Cohn-Mayer fluid, which con- tains in 100 cc. of distilled water half a gramme each of magnesium sulphate and potassium phosphate, one gramme of ammonium tartrate, and 0*5 gramme of tricalcium phosphate. Pasteur's and Cohn- Mayer's fluids are sterilised by the method of dis- continuous heating • or if they are placed in sealed flasks and sterilised in an autoclave, the sterilisation is complete in about fifteen minutes. Both of these fluids are useful media for the cultivations of the various species of Torulce or yeasts. Test-tubes, flasks, etc., are filled with fluid media by means of sterilised pipettes ; or, better still, the fluid media can be run directly into the cultivation vessels by THE METHODS OF CULTIVATING MICROBES 53 using Woodhead's storage flasks, which have already been described. For the inoculation of various media, pieces of platinum wire, either mounted in sealed glass-tubing, or unmounted, are used. They are sterilised by being heated in the Bunsen flame. To inoculate any medium, the sterilised needle, or a capillary pipette (Klein), is first dipped into the A. :B. -JT FIG. 20. INJECTION SYRINGES. (A, Dr. Petri's. B, Dr. Klein's. C, Dr. Koch's.) inoculating substance, and then transferred to the medium. Where Aitken's tubes and similar devices are used the medium contained therein is inoculated by using unmounted sterilised needles. These are dipped into the inoculating substance and then dropped into the fluid medium. To inoculate 54 A MANUAL OF BACTERIOLOGY animals, platinum needles or injection syringes (Fig. 20) are used; but in every case these instruments must be thoroughly sterilised before use. As Petri's and Koch's syringes cannot be heated without de- struction, they are sterilised by being immersed in a solution of mercuric chloride or mercuric iodide ; and after this these syringes should be washed with sterilised hot water. In addition to the above, ' glass needles are espe- cially useful when anaerobic microbes are being dealt with, as the smooth surface of the glass does not allow of oxygen (air) being carried down with it along the track, which closes up as soon as the needle is withdrawn.' We now proceed to describe the solid media beginning with nutrient gelatine. This is made according to the process already described for the preparation of bouillon, except that after the filtra- tion of the neutral or slightly alkaline fluid, 100 grammes of the best gelatine,1 10 grammes of pep- tone (albumin), and 5 grammes of common salt are added. The gelatine is allowed to soften and dissolve gradually by gently heating the mixture in a water bath. The nutrient gelatine is then steril- ised as usual, and filtered into tubes or flasks where it solidifies. The tubes and flasks being filled with the nutrient gelatine must be sterilised in a steam steriliser for a quarter of an hour on three successive days. If these tubes show no signs of turbidity after about a week's incubation, they may be con- sidered sterile. 1 Coignet's gold label gelatine is the best for this purpose. THE METHODS OF CULTIVATING MICROBES 55 Solid egg albumin is sometimes used as a cultiva- tion medium. The white of an egg is poured on to a slab of glass (sterilised), where it is coagulated and sterilised by being heated in the steam steriliser, or in the hot-air steriliser if tho temperature be properly regulated. Solid egg albumin (Fig. 21) is FIG. 21. MICROCOCCUS CHLORINUS. Growing on sterilised white of egg, after a fourth attenuation. (The white of egg coagulated and sterilised upon a slab of blackened glass.) readily inoculated by means of a platinum needle containing the inoculating material. Micrococcus chlorinus grows very well on this medium. Dr. F. Hueppe's method of cultivating on egg albumin is different from the above. It is as follows: — The shell is first disinfected with a solu- 56 A MANUAL OF BACTERIOLOGY tion of mercuric chloride ; a hole is chipped at one end of the egg, and the membrane cut through with a pair of sterilised scissors. The exposed egg albumin is inoculated by means of a platinum or glass needle. The opening is covered with a piece of sterilised paper or cotton wool, which is then painted over and sealed with surgical collodion. The egg is then placed in an incubator. Cooked potatoes are also used for cul- tivation purposes. The potatoes (smooth -skinned) are scrubbed and the so-called eyes removed by a sharp knife. They are now soaked for twenty minutes in a solu- tion of mercuric chloride (1 in 1000); washed in water, and then cooked in a steam steriliser for thirty minutes. After cooling, the potatoes are cut by a knife previously sterilised in the naked flame, or in Israel's box placed in a hot-air steriliser. The potatoes are cut 1 through the middle, and the two halves of each potato are then placed in previously sterilised damp chambers (Fig. 22). 1 The hands during this operation should have been pre- viously dipped into a solution of mercuric chloride. FIG. 22. DAMP CHAMBER. (For plate-cultivation, etc.) THE METHODS OF CULTIVATING MICROBES 57 The potatoes are inoculated by means of a plati- num needle or scalpel containing the inoculating material which is streaked over the surfaces of the potatoes. Second, third, and fourth attenuations may be made from potato-cultivations. Sometimes these cultivations require placing in an incubator, while at other times the growth readily forms at the ordinary temperature of the laboratory. Potatoes form a good medium for the cultivation of numerous microbes, especially the putrefactive and chromo- genic forms. Another solid medium for the cultivation of microbes is agar-agar.1 This substance is an excel- lent substitute for nutrient gelatine ; for the latter melts at about 26° C., consequently it cannot be used for the cultivation of certain microbes requir- ing a much higher temperature for their proper growth and development. Agar-agar remains solid up to 50° C. Sterilised nutrient agar-agar is pre- pared by a similar method to the one already described for preparing nutrient gelatine, with the exception that 20 grammes of agar-agar are used instead of the 100 grammes of gelatine. Although nutrient agar-agar remains solid up to 50° C., it is surpassed in this property by blood serum. Blood serum solidifies at 70° C., and always remains solid. The method for preparing solid blood serum has already been described. Both nutrient agar-agar and solid blood serum are suitable media for the growth of certain microbes requiring a higher tem- perature than usual. 1 Consists of the dried fragments of certain Algae. 58 A MANUAL OF BACTERIOLOGY A good medium for the growth of chromogenic microbes is made of ground rice. The late Dr. Isidor Soyka's formula for the preparation of this medium is as follows : — 1 0 grammes of ground rice, 15 cc. of milk, and 5 cc. of neutral beef bouillon. These ingredients are made into a paste, which is transferred to covered glass dishes or small flasks. The dishes or flasks are then sterilised (in the steam steriliser) for half an hour on three successive days. Bread-paste is also used as a medium for the cultivation of microbes. It is prepared in the fol- lowing way : — The crumb of a loaf is broken into small pieces, dried in an oven, and rubbed through a fine sieve. The finely-divided bread is then placed in a sterilised flask, to the depth of half an inch, sterilised water being added until the bread is thoroughly moistened. After replacing the cotton- wool plug the flask (or flasks) is sterilised in the steam steriliser for the same length of time as rice- paste. The flask containing either bread- or rice- paste can be reversed, and is readily inoculated by means of a platinum needle. To inoculate solid culture media ' the test-tube or flask is held inverted in the left hand, and the plug of cotton wool is twisted once or twice in the mouth of the test-tube to break down any adhesions between it and the neck of the vessel. If the plug is at all dusty, it is well to singe the surface by passing it rapidly through a flame before removing it from its position. The plug is removed and held between two of the unoccupied fingers of the left hand, great care being taken that no part of the THE METHODS OF CULTIVATING MICROBES 59 plug that passes into the test-tube shall come in contact with any source of infection other than the air itself. At the same time this portion of the plug is directed downwards, in order to avoid any falling germs that may be present in the atmosphere. The platinum or glass needle, with its charge of seed material, is plunged straight into the gelatine mass, then carefully withdrawn and the plug replaced. Where the seed material is also in solid gelatine, the two tubes may be held inverted in the left hand, one between the thumb and finger, the other between the first and second, the plugs being held between the second and third and third and fourth fingers ' (Woodhead). The macroscopical appearances of the test-tube cultivations should always be noted, for many microbes give rise to characteristic growths. Some microbes wholly or partially liquefy the nutrient medium, while others have not this property ; but may give rise to pigments, etc., in the medium or media in which they are growing. Cultivation Methods. — If the original fluid under examination contains different microbes, and it is desired to separate them, so as to obtain pure culti- vations of one or all of the microbes present in the original fluid, one of three methods may be used for this purpose. The three methods are known as — plate-cultivations, fractional cultivations, and the dilution method. In order to utilise the method of plate-cultivation, about three tubes containing sterilised nutrient gelatine or agar-agar are placed in a water-bath 60 A MANUAL OF BACTERIOLOGY heated to 40° C. or 55° C. respectively, so as to melt the medium in each tube. The tubes are then care- fully inoculated with a mere trace of the original fluid. The cotton-wool plugs are replaced, and the tubes rolled about so as to distribute the microbes throughout the media. The contents of the tubes are quickly poured into the lower portion of the same number of Dr. Petri's double dishes (Fig. 23 B) or glass plates (Fig. 23 A). The dishes or plates (which should have been previously sterilised) are then placed in a damp chamber (see Fig. 22). The damp chamber, with its contents, are removed to an FIG. 23. APPARATUS FOR PLATE-CULTIVATION. A, Glass Bench with'Plates. B, Petri's Double Dish. incubator, and remain there for several days at about 23° C., or higher if agar-agar is used (i.e. according to the temperature required for the growth of the microbes). In a few days or so each species will have started a separate growth or colony in different parts of the solidified plate of nutrient gelatine, or agar-agar. The individual colonies are recognisable according to certain macroscopical appearances, such as colour, shape, liquefaction or non-liquefaction of the medium, and the size of the colonies. By plate- cultivation the different species of microbes (i.e. in THE METHODS OF CULTIVATING MICROBES 61 a microbian mixture) separate themselves from each other ; and from these colonies pure cultivations of each microbe may be obtained by carefully re- inoculating a number of tubes containing sterilised nutrient gelatine or agar-agar. Plates of gelatine or agar-agar (Fig. 24) may also be reinoculated in a FIG. 24. A PLATE-CULTIVATION OF SPIRILLUM TYBOOENUM. A, Colonies growing on nutrient gelatine (sterilised). B, The spirillum x 1265. similar manner. First, second, and third attenua- tions may be obtained by this mode of cultivation. Both the macroscopical and microscopical appear- ances should be noted. To examine the growth under low power one of the plates should be placed upon the stage of the microscope, and the appear- 62 A MANUAL OF BACTERIOLOGY ances carefully observed under Zeiss' B, C, and D objectives, or any similar low powers. After this a cover-glass preparation should be made by rubbing a needle, previously dipped into the growth on the plate, on a clean cover-glass. A drop of sterilised water is now added; the cover-glass is allowed to dry ; then passed three times through the Bunsen flame ; and finally stained with a drop of fuchsine or some other aniline colour. The cover-glass pre- paration should be temporarily or permanently mounted, according to the methods described later in this chapter. After mounting, the preparation should be examined under high powers, such as Zeiss' J and T^. It should be borne in mind that the eye has to be trained in order to see objects distinctly with such high powers ; but, it may be remarked, that ' in all extremely delicate work with high-power lenses, the first difficulty is the greatest. If once an object has been seen, however difficult, it is immensely easier to see it again. On the other hand, there is as great a diversity in different individuals in the sensitiveness of the retina, as there is in the sensitiveness of the olfactory, or auditory nerves. It is impossible to enable some persons to see objects beyond a certain limit of minuteness ; as it is to enable others to detect certain scents, or hear notes pitched higher or lower than a given point.' The fractional cultivation method consists in the attempt to isolate, by successive cultivations, the different organisms that have been growing pre- viously in the same culture. A number of tubes containing various cultivation media (sterilised) are THE METHODS OF CULTIVATING MICROBES 63 inoculated with a mere trace of the original microbian mixture, and are then placed in an incubator for a couple of days or so. It will then be noticed that the different species of microbes (sown in each tube) will not have increased equally in numbers in all the tubes (due, of course, to the nature of the medium, the temperature, and the period of incuba- tion). It is possible that only one species will have developed, so far, in each tube. With these tubes a similar number of tubes are re-inoculated, and so on. By this fractional method of cultivation pure growths are ultimately obtained. For further information concerning this method the reader is referred to Dr. Kleb's paper in the Archiv fur Exper. Pathologic, 1873. The dilution method consists in greatly diluting a drop of the original microbian mixture with some sterile saline solution (0*5 °/0). A series of tubes, containing different cultivation media (sterilised), are each inoculated, by means of a platinum needle or glass pipette, with a mere trace of the diluted mixture. After about thirty hours' incubation, growths (most likely of one species only), make their appearance in some of the tubes. The original microbian mixture or fluid maybe diluted a thousand- or even a million-fold, if the original fluid teems with different microbes. The dilution method has been largely used by Dr. P. Miquel in his examina- tions of the different waters in and around Paris. By the fractional, dilution, and plate methods, cultures containing many different species of microbes are capable of being separated one from 64 A MANUAL OF BACTERIOLOGY another. Sometimes a combination of the fractional and dilution methods is used for the same purpose. The methods of cultivating anaerobic microbes are somewhat different from the above ; as the air I must be excluded from the cultivation apparatus. In the cultivation of Bacillus cholerce Asiaticce, Koch made use of plate cultivations on which very thin sheets of glass or mica were placed before the gelatine was perfectly set. By this means the colonies of microbes grow out of contact with the o air. A second method for excluding air (i.e. oxygen) is to allow the microbes to grow under the receiver of an air-pump which has been exhausted of air. A third method is to allow the microbes to grow in an atmosphere of carbonic anhydride or hydrogen gas. Another method consists in inoculating a cultivation tube with the anaerobic microbe, and then covering the surface of the medium with a layer of sterilised oil. Dr. Eoux has also devised two methods for this object. One of these methods is to fill a sterilised pipette with sterilised nutrient gelatine. Both ends of the pipette are hermetically sealed. To inoculate the gelatine, one end of the pipette is nipped* off, the inoculating material introduced, by a fine glass needle, into the gelatine, and finally the open end of the pipette is again sealed. By this device the microbes grow anaerobically. The second method of Dr. Eoux is to boil a quantity of agar-agar in a test-tube ; and after quickly cooling, the medium is inoculated with the anaerobic microbe. A layer of THE METHODS OF CULTIVATING MICROBES 65 melted nutrient gelatine is now poured on the surface of the agar-agar, and when it is cooled a drop of a bouillon cultivation of Bacillus siibtilis is run on to the surface from a capillary pipette. The tube is then sealed, or the cotton-wool plug is rendered impervious by being luted with warm paraffin- wax. The object of growing Bacillus subtilis is that it uses up the oxygen at the surface ; con- sequently the microbe below receives none, or, in other words, it is able to grow anaerobically. To obtain inoculating material from Roux's tube, it is broken at the bottom and a sterilised needle inserted into the lower growth. FIG. 25. DROP-CULTURE CELL. (With arrangement for admitting Gases into the Cell) In place of the Bacillus subtilis, the layer of nutrient gelatine is covered with a solution contain- ing one part of pyrogallic acid to ten parts of a solution of potassium hydroxide (10 per cent.). The potash solution of pyrogallic acid may be replaced, with advantage, by a 3 per cent, solution of ferrous sulphate, or a 2 per cent, solution of cuprous chloride ; both of these compounds (the author has found) prevent the entrance of air. A drop culture forms a useful method for study- ing the growth and multiplication of microbes under low or high power objectives. For this purpose a glass cell is required. This is made by cementing 66 A MANUAL OF BACTERIOLOGY a sterilised glass ring ( j in. diam. x | in. high) to a microscopic slide, which has been thoroughly cleaned and sterilised (Fig. 25). The upper edge of the ring is moistened with olive oil or vaseline ; and the cell is covered over by means of a thin cover-glass, previously sterilised by passing it through a Bunsen flame. The surface of the sterilised cover-glass (A) contains a drop of bouillon or other medium, along with the microbes for examination. A drop or two of sterilised water should be deposited at the bottom of the cell ; i.e. upon the upper surface of the glass slide. This arrangement forms a miniature damp chamber, in which the growth of microbes may be watched even under the highest powers. After the examination of the cell and its contents, it may be placed in an incubator until it is required again for microscopical examination. To study the action of heat on drop cultures, the warm stages of Schafer, Eanvier, Israel, Schultze, Strieker, etc., are often used upon the fixed stage of the microscope.1 The action of various gases on drop-cultures may be watched by a modification of the glass cell as represented in Fig. 25. The gases enter through B. The author has used this device during his researches on the action of certain gases on Bacillus tuberculosis. The action of the voltaic current or discharges of faradaic electricity may be observed by simple modifications of the drop- culture cell. 1 An excellent piece of glass apparatus is used by the Rev. W. H. Dallinger, F.R.S., for ascertaining the thermal death point of microbes. (See Proc. Roy. Soc., 1878.) THE METHODS OF CULTIVATING MICROBES 67 Drop-cultures form ready means for studying the complete life-history of any microbe. The methods for examining fluids and fresh tissues are as follows: (1) blood, urine, saliva, pus, tears, culture-fluids, and other liquids containing microbes, are easily examined microscopically by placing a drop of the liquid on a glass slide and covering it with a thin cover-glass; (2) when microbes for examination are growing on plates of nutrient gelatine, a small portion of the culture should be taken up on a sterilised platinum or glass needle and placed in a drop of sterilised water on a glass slide. After thinning, the preparation is covered with a cover-glass and examined under low and high powers ; (3) for the microscopical examination of fresh tissues, they should be teased out with sterilised needles in dilute glycerine or salt solution (sterilised), then temporarily mounted, in either liquid, on a glass slide and covered with a thin cover-glass. If there is an excess of glycerine or salt solution round the edges of the cover-glass, it must be removed by placing small pieces of filter or blotting- paper in contact, which will soon absorb the super- fluous fluid, but the paper must not be left too long or it will drain the fluid from under the cover-glass. In the examination for micrococci and other small microbes, the tissues should be first treated with acetic acid, and then with a solution of potassium hydroxide (potash), the object being to dissolve and disintegrate fatty and albuminous globules which might be mistaken for microbes. Alcohol and ether are also useful agents for dissolving small globules of fat. 68 A MANUAL OF BACTERIOLOGY It has been recorded that a certain foreign medi- cal professor mistook minute globules of fat for so many micrococci ; and certainly the illustrations in his paper indicated that such was really the case. Therefore, let all bacteriologists, young and old, be very sceptical at times as to what they think they see with the highest powers of the microscope. Without wishing to detract an iota from the honesty of purpose and truth of our fellow-workers, we are sure that a good deal unintentionally has been said to have been seen with the microscope which has never been seen at all. We set to work longing to discover something newer than the last new thing. We hope to find it, we begin to think we have found it, and we may go so far as to make ourselves believe we really did see it once. The event must be recorded ; we proclaim it, and in so doing pro- pagate error. Therefore, let it be borne in mind that to use the highest powers with accuracy re- quires continual practice ; even when the retina of the eye is sensitive enough to appreciate light-waves proceeding from such organisms as the smallest micrococci. Staining Cover-glass Preparations and Tissues. — To prepare a cover-glass preparation for staining, a sterilised cover-glass is smeared with the microbian matter (solid or liquid), or with blood, pus, etc., by means of a sterilised needle or capillary pipette. The excess of material is squeezed out by means of an additional cover-glass placed over the original one. The two glasses are then separated, each bear- ing a small portion of the microbian matter. After THE METHODS OF STAINING MICROBES 69 drying for a few minutes, they are passed rapidly (three or four times) through a Bunsen flame. To stain the preparations they are allowed to float (with the prepared side downwards) on the surface of an aqueous solution of methyl violet, gentian violet, or magenta, for a short time. After this the cover-glasses are washed with water, then spirit, and finally with sterilised distilled water. They are then drained, dried, and mounted in Canada balsam or any suitable medium. The preparation must now be set aside to dry, and when thoroughly dry it is ' ringed ' or sealed with Hollis' glue. Before continuing the description of the various methods of staining, we describe the preparation of several staining fluids : — (1) Gentian violet stain is prepared by rubbing 2 grammes of gentian violet in a glass mortar with 10 cc. of alcohol (sp. gr., 0*83), in which has been dissolved 2 cc. of aniline oil. To this is added 90 cc. of distilled water ; (2) Koch's methyl violet stain contains the following ingre- dients:— Aniline water, 100 cc. ; an alcoholic solu- tion of methyl violet, 11 cc. ; and absolute alcohol, 10 cc. ; (3) the stain known as Bismarck brown is prepared by dissolving 2 grammes of Bismarck brown in 15 cc. of alcohol, and then adding 85 cc. of distilled water ; (4) haematoxylin solution con- tains 2 grammes of haematoxylin, 2 grammes of alum, and 100 cc. each of alcohol, glycerine, and distilled water; (5) the methylene blue stain is prepared by dissolving 2 grammes of methylene blue in the same quantities of alcohol and water as are required to prepare the Bismarck brown stain ; 70 A MANUAL OF BACTERIOLOGY (6) Eanvier's picro-carmine stain contains 1 gramme of carmine, 3 cc. of ammonia, 10 cc. of distilled water, and 200 cc. of a cold, saturated solution of picric acid ; (7) vesuvin stains are prepared by dis- solving 3, 4, or 5 grammes of vesuvin in 100 cc. of distilled water ; (8) Dr. Gibbes' solution for double staining contains 2 grammes of magenta and 1 gramme of methyl violet, which are triturated in a glass mortar with 15 cc. of alcohol (in which has been dissolved 3 cc. of aniline oil). To this mixture is added 15 cc. of distilled water; (9) Gram's iodine solution is prepared by dissolving 1 gramme of iodine and 2 grammes of potassium iodide in 300 grammes of distilled water; (10) LofHer's stain con- tains 30 cc. of a concentrated alcoholic solution of methylene blue, and 100 cc. of an aqueous solution of potassium hydroxide (1 in 10,000) ; (11) an eosin solution is prepared by dissolving 5 grammes of eosin in 100 cc. of distilled water. We now continue the description for staining microbes and tissues. To stain tissues containing microbes, place them in either an aqueous solution of methyl violet (2 -2 5 grammes in 100 cc. of water), or one of gentian violet (containing the same strength of solution), and allow them to remain in the solution for some hours. When deeply stained, wash in water to remove the excess of the stain, and then lay them out flat in methylated spirit, and let them remain until no more colour comes away. Transfer them to absolute alcohol, and then oil of cloves, and mount in Canada balsam (Gibbes). To double stain bacilli which produce spores, the THE METHODS OF STAINING MICROBES 71 cover-glass preparation should be floated for half an hour on the surface of a small quantity of hot magenta and aniline stain.1 The magenta is dis- charged from the bacilli by washing in water, in alcohol, or weak nitric acid, according to the species. The preparations are then treated (for three or four minutes) in a solution of methylene blue, and finally washed with water, drained, dried, and mounted in Canada balsam or other mounting media. By this method the spores are stained red, while the bacilli are blue. Koch's method for staining tubercle bacilli is as follows: — Cover-glass preparations of the sputum, etc., are placed in a solution containing 1 part of a concentrated solution of methylene blue, 2 parts of a potash solution (10 per cent.), and 200 parts of dis- tilled water. The preparations remain in the solution (heated to 40° C.) for twenty-four minutes. They are then washed in water, and placed in an aqueous solution of vesuvin for two or three minutes ; again washed, and subsequently treated with alcohol, oil of cloves, and finally mounted in Canada balsam. Koch's method stains the bacilli blue, and the nuclei, etc., brown. ' All the other forms of bacteria which Koch has as yet examined in this way are stained brown, with the exception of the bacilli found in leprosy, which also retain the methylene blue in preference to the vesuvin. These bacilli 1 This stain is prepared by mixing together 5 cc. of aniline oil and 100 cc. of distilled water. The mixture is filtered, and to the filtrate is added a concentrated alcoholic solution of fuchsine or magenta, until a precipitate begins to ba formed. 72 .A MANUAL OF BACTERIOLOGY may also be stained by other aniline dyes if the solution be made alkaline by the addition of caustic potash or soda.' To stain the flagella of certain microbes, Koch recommends that the cover-glass preparations should be floated on a concentrated aqueous solution of hsematoxylin. They are then transferred to Muller's fluid,1 or to a five per cent, solution of chromic acid. By using either of these reagents the flagella are stained a brownish-black colour. On the other hand, Dr. Dallinger 2 does not think that Koch's method of staining brings out the flagella well. Dallinger uses high powers and the microbes alive. Dr. Crookshank has, however, succeeded in photo- graphing the flagella by staining with a concentrated alcoholic solution of gentian violet. The prepara- tion is then rinsed in water, dried, and mounted in Canada balsam. Gram's method for staining microbes in tissues is as follows : — The sections containing the microbes are soaked in absolute alcohol for twelve minutes, and then placed in a gentian-violet-and-aniline solution 3 for about three minutes. The sections are then placed in a solution of iodine (in potassium iodide) for several minutes, or until they are of a brown colour. After this they are transferred to absolute 1 This fluid contains 2 grammes of potassium bichromate, 1 gramme of sodium sulphate, and 100 cc. of distilled water. 2 Journal of Royal Microscopical Society, 1878, p. 172. 3 This is similar to Koch's methyl-violet-and-aniline stain, except the methyl violet is replaced by gentian violet. THE METHODS OF STAINING MICROBES 73 alcohol until decolourised ; they are then placed in oil of cloves, and finally mounted in Canada balsam. As Gram's method only gives a faint colour to the tissues, they may be stained a deeper colour by immersing the sections (after decolourising with alcohol) in an aqueous solution of vesuvin, eosin, or Kanvier's picro-carminate of ammonia. They are finally washed in alcohol, and mounted as already described. One of the best methods for staining cover-glass preparations is the one devised by Ehrlich. The cover-glass preparations are made to float (with the prepared face downwards) in a solution of fuchsine made in the following manner : 5 cc. of aniline oil and 100 cc. of distilled water are mixed together and filtered. To the filtrate is added a concentrated alcoholic solution of fuchsine. The preparations re- main in this solution for fifteen minutes ; they are then washed in nitric acid (one part of nitric acid to two parts distilled water) and rinsed in distilled water. An after-stain of methylene blue or vesuvin gives the nuclei, etc., a blue or brown colour, while the tubercle-bacilli or other pathogenic microbes are stained red. The elegance of this method is that the tubercle-bacilli impregnated with fuchsine resist the action of nitric acid, whilst the saprophytic microbes (present in sputum and saliva), nuclei, etc., are immediately decolourised by the acid. Both Ehrlich's and Koch's methods are also applic- able for staining tubercular and other tissues. The Ehrlich- Weigert is another method for stain- ing microbes in situ. The tissues are placed in a 74 A MANUAL OF BACTERIOLOGY warm solution of aniline-methyl-violet,1 and then decolourised with nitric acid (one in two). The tissues may be stained brown by immersing them in an aqueous solution of Bismarck brown or vesuvin. In this case the microbes are blue and the tissues brown. Other aniline colours may be used, but the decolouriser is nitric acid. The stained sections are washed, cleared in oil of cloves, and then mounted in Canada balsam. In the Baumgarten method cover-glass prepara- tions of sputum are placed in a very dilute solution of potassium hydroxide (potash), and after being slightly pressed on the microscopic slides they are ready for examination. By this method the bacilli (tubercle) are seen in the unstained condition. This is a quick method of examining phthisical sputum, as it does not take more than ten minutes. Gibbes' rapid double-staining method is applicable for staining sections as well as cover-glass prepara- tions. No decolourising agent is used, while the double-staining process is performed in one opera- tion. The preparations are allowed to remain in a warm aniline-magenta-methyl- violet solution for five minutes, or in the case of sections for several hours. They are washed in methylated spirit until no more colour comes away. The preparations are now dehydrated in absolute alcohol, dried and mounted in Canada balsam dissolved in xylol. By this 1 The above solution is prepared by mixing together 100 cc. of a saturated aqueous solution of aniline and 11 cc. of a satu- rated alcoholic solution of methyl violet. The filtered mixture is the Ehrlich-Weigert stain. THE METHODS OF STAINING MICROBES 75 method the tubercle-bacilli and certain other patho- genic microbes are stained red, while the putrefac- tive bacteria and micrococci are blue. This method is a rapid one, and is, consequently, recommended for the busy medical man. The Ziehl-Neelsen method of staining the tubercle- bacilli is a modification of the Ehrlich-Weigert method already described. The cover-glass prepara- tions or sections are stained in the following dye : 1 gramme of fuchsiue is dissolved in 10 cc. of absolute alcohol, and to this is added 100 cc. of an aqueous solution of carbolic acid (5 per cent.). The mixture is then heated. In the hot dye sections are stained in six or seven minutes ; and cover-glass preparations are stained in about three minutes. The preparations or sections are now placed for a second or so in 90 per cent, alcohol, then in dilute sulphuric acid (25 per cent.), when the pink colour is replaced by a yellowish brown. The preparations, etc., are then transferred to a solution of lithium carbonate. They are afterwards stained in an aqueous solution of methylene blue, cleared in oil of cloves, and mounted in Canada balsam. This method (also known as the carbol-fuchsine method) gives excellent results. To ascertain the presence of tubercle-bacilli in tuberculous milk, the best plan is to pass the milk through one of the ordinary centrifugal machines used in the dairy ; and then to take the sediment (after the separation of the cream and skim milk) for examination. In lieu of a centrifugal machine, the milk should be allowed to stand for about 76 A MANUAL OF BACTERIOLOGY twenty-four hours in a chemical separator (Fig. 26) surrounded with ice. The sediment (containing the bacilli) is drawn off from the separator by means of a tap (see Fig. 26) ; and a few drops of the sediment are dried on a cover-glass, and examined in the ordinary way. Dr. W. Ktihne's methylene blue method is one of the best means of staining for general purposes. It ^ is prepared by dissolving 1'5 grammes of methylene blue in 10 cc. of absolute alcohol; and 100 cc. of an aqueous solution of carbolic acid (5 per cent.) are added. Preparations are stained in this dye from five minutes to two hours ; and sections remain in it for twenty-four hours. They are washed in water, followed by acidulated water,1 and are then transferred to a solution of lithium carbonate (5 per cent.). They are again washed in water, dehydrated in abso- lute alcohol, placed in aniline oil, and transferred to terebene for two or three minutes. After this treatment the preparations are washed in xylol, and finally mounted in Canada balsam. This stain is useful for the bacilli of leprosy, glanders, tuber- culosis, arid almost any microbe. Cover-glass preparations of anthracic blood, etc., are floated on a hot alcoholic solution of fuchsine FIG. 26. CHEMICAL SEPARATORS. 1 Two or three drops of hydrochloric acid to 100 cc. of distilled water. THE METHODS OF STAINING MICROBES 77 for thirty minutes. They are then decolourised in weak hydrochloric acid, and after - stained with methylene blue. By this means the spores are stained red and the bacilli blue. Anthrax- bacilli and spores may also be stained with an aqueous solution of gentian violet, fuchsine, or any of the aniline dyes ; if the cover-glass pre- paration is first passed ten or eleven times through the Bunsen flame. Sections of anthracic tissues are well stained by Gram's method, and after - stained with picro- carminate of ammonia, or eosin. The bacillus of glanders is stained by the method of Schlitz. The sections are placed in an alcoholic potash solution of methylene blue 1 for twenty-four hours. They are then washed in acidulated water,2 transferred for five minutes to 50 per cent, alcohol, ten minutes to absolute alcohol, clarified in oil of cloves, and finally mounted in Canada balsam. As already stated, the bacillus of glanders (Bacillus mallei) may be stained by Klihne's methylene blue method. There are three principal methods for staining the bacillus of syphilis. (1) Lustgarten's method con- sists in placing the sections of syphilitic tissues, etc., for about twenty-four hours in a solution containing 100 cc. of aniline-water (5 per cent.) and 11 cc. of a saturated alcoholic solution of gentian violet. They are now heated for two hours at 60° C. After this 1 This stain contains equal parts of a concentrated alcoholic methylene blue solution and a solution of potash (1 in 10,000). - Water containing 5 per cent, of acetic acid. 78 A MANUAL OF BACTERIOLOGY treatment, the sections are placed for three or four minutes in absolute alcohol, transferred to a solution of potassium permanganate (1*5 per cent.) for ten minutes, and decolourised by immersion in con- centrated sulphurous acid. The sections are then dehydrated in absolute alcohol, clarified in oil of cloves and mounted in Canada balsam. (2) The next method is that of Doutrelepont and Schiitz. The sections of syphilitic tissues containing the bacilli are immersed in an aqueous solution of gentian violet (1 per cent.), and are after-stained with an aqueous solution of safranin (1 per cent.). (3) The last method is that of De Giacomi, in which the preparations are immersed in a hot solution of fuchsine containing a drop or two of ferric chloride. They are then decolourised in a concentrated solu- tion of ferric chloride, and after - stained with Bismarck brown or vesuvin. In both the Doutrele- pont-Schiitz and De Giacomi methods, the prepara- tions (after staining) are dehydrated, clarified, and mounted in the usual way. Sections of tissues containing the Bacillus leprce are stained by immersion in a solution of fuchsine in aniline-water. They are then decolourised in hydrochloric acid (33 per cent.), and after-stained with methylene blue. Another method is, first to tie a piece of thread around the base of one of the leprosy nodules, so as to cut off the blood supply ; then with a fine-pointed scalpel (see Fig. 5) a small puncture is made, when a clear fluid exudes. From this fluid, cover-glass preparations are made. Cover- glass preparations and sections of leprosy tissues THE METHODS OF STAINING MICROBES . 79 may be stained by the methods of Ehrlich, Ziehl- Neelsen, and Gram. The method (devised by Dr. Loftier) for staining the Bacillus diphtherice consists in placing the sections in Loffler's alkaline methylene blue (already described) for about five minutes. The excess of stain is removed by very dilute acetic acid (0'5 per cent). They are then dehydrated in alcohol, clari- fied in cedar oil, and mounted in Canada balsam. Sections may also be stained by Gram's method; and Dr. Klein has produced beautiful stained sec- tions of diphtheritic membranes * by staining them with rubin 2 and methyl blue. By this method the bacilli are stained blue, while the nuclei and necrotic substances of the membranes are stained red. To stain the Bacillus typhosus (the microbe of typhoid fever), there are several methods in use. For tissue-staining, the method of Gram may be used. Some bacteriologists recommend steeping the sections for twenty-four hours in methylene blue; but this stain possesses the disadvantage of quickly fading. The colour, however, may be fixed by placing the sections either in a solution of picro- carminate of ammonia, or of iodine dissolved in potassium iodide, or in ammonium picrate. Dr. Kiihne's method consists in allowing the sections to remain for some time in a concentrated aqueous solution of oxalic acid, washing them in water, and afterwards staining with methyl blue dissolved in a 1 See Report of Medical Officer of the Local Government Board, 1889-90, p. 143. 2 Rubin is rosaniline nitrate. 80 A MANUAL OF BACTERIOLOGY solution of ammonium carbonate (1 per cent.). To demonstrate the spores of this bacillus, cover-glass preparations and tissue-sections must be placed in a hot solution of fuchsine. They are then decolourised with nitric acid (see Ehrlich's method), after-stained with methylene blue, and mounted as usual, after dehydration and clarification in the media already described. The most important methods for staining the Micrococcus pneumonice are as follows: — (1) By the method of Gram. (2) Cover-glass preparations of pneumonic sputum and exudations are treated with acetic acid, stained with gentian violet, and temporarily mounted in distilled water, or water and glycerine, i.e. for immediate examination; or they may be dried and permanently mounted in Canada balsam. Cover-glass preparations of gonorrhceal pus, blood, or of artificial cultivations of the Micrococcus gonorrhcece are readily stained with an aqueous solution of fuchsine. This method may be also used for demonstrating the presence of the same micrococcus in the tears of new-born infants suffer- ing from purulent ophthalmia of gonorrhoeal origin. The cholera bacillus (Bacillus cholerce Asiaticce) is stained by the following methods : (1.) The dis- charges, etc.. containing the microbe are spread and dried on a cover-glass. They are then stained with an aqueous solution of fuchsine, washed with water, dried, and mounted in Canada balsam. (2.) The hardened sections x of the intestines are placed for 1 Hardened in absolute alcohol. THE METHODS OF STAINING MICROBES 81 twenty-four hours in a strong aqueous solution of inethylene blue ; and finally treated in the usual way. (3.) 'The best method yet described of de- monstrating the cholera bacillus in the discharges of the intestines is that recommended by Cornil and Babes, who spread out one of the small white mucous fragments on a microscopic slide, and then allow it to dry partially ; a small quantity of an exceedingly weak solution of methyl violet in dis- tilled water is then flowed over it, and it is flat- tened 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. Cholera bacilli so prepared and examined with an oil-immersion lens (Zeiss' ^o homog., Oc. 3 or 4) 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 speci- men were dried, stained, and mounted in the ordinary fashion/ For staining cover-glass preparations of the blood of patients suffering from relapsing fever (i.e. con- taining the Spirillum Obermeieri), fuchsine, gentian violet, and Bismarck brown have been used with considerable success. Sections of the brain, liver, lungs, kidneys, etc., of monkeys or human beings dead of the disease, are best stained with Bismarck brown, vesuvin, or chrysoidine. Cover-glass preparations of the blood, exuda- tions of the throat, etc., from cases of scarlatina F 82 A MANUAL OF BACTERIOLOGY (i.e. containing the Micrococcus scarlatince) are stained with a saturated solution of methyl violet. The micrococci, adhering to the scales of the desqua- mating epidermis in such cases, are also stained with the same dye. As the scarlatina micrococcus has been found in diseased cow's milk,1 such milk should be treated by the method described for the examination of tuberculous milk (see p. 75). Bacillus lutyricus is best stained with a solution of iodine in potassium iodide. Actinomyces is usually stained by Plant's method. Sections of nodules, tumours, etc. (from cases of Actinomycosis) are immersed for ten or twelve minutes in a stain containing two grammes of magenta, 3 cc. of aniline oil, 20 cc. of alcohol (sp. gr. 0*83), and 20 cc. of distilled water. The stain (with the sections) is warmed to 45° C. The sections are rinsed in water, and after- stained in a strong alcoholic solution of picric acid for about eight minutes. They are then immersed in water for five minutes, in alcohol (50 per cent.) for fifteen minutes ; and finally passed through absolute alcohol and oil of cloves, and mounted in Canada balsam. The tissues containing this fungus may be examined in the fresh state. A little of the tissue, etc., is transferred to a microscopic slide, teased out with needles, and then temporarily mounted in a drop of glycerine and water. We have given most of the principal methods for the examination of microbes. It may be re- 1 See Dr. Klein's Reports to the Local Government Board, 1885-8. THE METHODS OF MOUNTING MICROBES 83 marked that nearly all microbes can be stained with the various aniline dyes ; although their capacity for absorbing these dyes differs consider- ably. This capacity or affinity for aniline dyes is of great use to the bacteriologist to ascertain the presence of microbes, and to differentiate in many instances morphological details which in the un- stained condition are not discernible. Hardening, Imbedding, Cutting, and Mounting Preparations. — Many medical men and students on reading the different staining, hardening, imbedding, cutting, and mounting processes ' which any tissue has to undergo before it can be examined with the microscope, will be inclined to think it very tedious work. It is, however, a mere matter of routine, and when once this routine is established, the whole thing is comparatively simple. It takes very little time to change the hardening fluid, and if the student gets into the habit of looking over the bottles on the shelf every morning where he keeps tissues in the process of hardening, a glance at the labels will show those requiring a change. When the sections are mounted and examined under the microscope, he will find himself amply repaid for all his trouble if he has faithfully carried out the different processes in every detail. It is always better to have one or two shelves devoted to those preparations which require changing; and those which require fresh fluid often, as for instance those hardening in chromic acid should be kept by themselves. Each bottle should be labelled, and the tissue, date, and hardening fluid clearly 84 A MANUAL OF BACTERIOLOGY written on the label. Every morning this shelf should be examined, and the hardening solution changed in those requiring it, the date being each time written on the label, so that it may be seen at a glance how long the tissue has been in the fluid, and whether the hardening agent ought to be renewed. Muller's fluid and bichromate of potash preparations may be placed by themselves, and need only be looked at occasionally/ The best hardening agents are absolute alcohol, methylated spirit, Muller's fluid, chromic acid solu- tion, potassium bichromate solution, and osmic acid. (1.) Pieces of an organ, etc., should be cut from J in. to 1 in. cubes, and placed in one of the hardening solutions. If absolute alcohol or methy- lated spirit is used the tissues should remain in the spirit from two to three days. Many delicate tissues, however, cannot be placed in strong spirit without shrinking ; to obviate this such tissues are first placed in dilute spirit (one part of water to two parts of spirit). In this mixture the tissues remain about twenty-four hours and are then trans- ferred to the strong spirit for one or two days. After this they are ready for imbedding and cutting. (2.) Muller's fluid is an excellent hardening agent. To prepare it, dissolve two parts of potassium bichromate, one part of sodium sulphate, and 100 parts of distilled water. The preparations to be hardened should remain in the fluid from two to three weeks. When the fluid becomes cloudy it re- quires changing ; but it retains its hardening pro- perties for a long time. The preparations, after THE METHODS OF MOUNTING MICROBES 85 being hardened in Miiller's fluid, should be washed in water, and then placed in dilute spirit (one of water to two of spirit) for about twenty-four hours. Sometimes the treatment with dilute spirit is dispensed with, especially if the sections are to be cut immediately. (3.) Chromic acid solution is really a mixture of chromic acid and spirit. It is prepared by dissolving one gramme of chromic acid in 600 cc. of distilled water. Two parts of this solution is then mixed with one part of methylated spirit. The material to be hardened is placed in this fluid for twenty-four hours ; the fluid is then changed, and again every third day; the material being hardened in from eight to twelve days. The material should not be allowed to become brittle, which it does if it remains too long in this fluid. After hardening the material is washed in water, and the sections cut immediately (i.e. after imbed- ding), or it is placed in dilute spirit for twenty-four hours, and then transferred to strong methylated spirit. In this fluid the material may remain for an indefinite time ; that is, if it is not required for immediate use. (4.) A two per cent, solution of potassium bichromate is sometimes used, especially where tissues require slow hardening. ' This solu- tion takes from three to seven weeks to harden, according to the size of the specimen, and the fre- quency with which the solution is changed.' (5.) A 0'5 per cent, solution of osrnic acid is used for hardening certain preparations — such as the in- ternal ear. This solution must be protected from light ; for this purpose the bottle in which it is 86 A MANUAL OF BACTERIOLOGY kept should be painted externally with black oil paint. To decalcify small bones or teeth, they are placed in Ebrier's or Kleinberg's solution. Ebner's solution contains five grammes of sodium chloride (salt), 5 cc. of hydrochloric acid, 20 cc. of distilled water and 100 cc. of alcohol. Kleinberg's solution is made as follows: 100 cc. of a saturated aqueous solution of picric acid are added to 2 cc. of strong sulphuric acid. The mixture is filtered and 300 cc. of distilled water are added. In either solution the materials (to be decalcified) remain until sufficiently softened ; they are then allowed to soak in water, and finally passed through weak spirit to absolute alcohol. For cutting sections either by hand or by the microtome, it is necessary (as a rule) to imbed the material in one of the imbedding mixtures. If the material to be cut has been preserved in alcohol, it is better first soaked in water for about ten hours to remove the spirit, and then placed in mucilage T for about five hours. For cutting with the non-freezing microtomes, the material is imbedded in celloidin or paraffin, mounted on cork.2 To imbed in celloidin the hardened material is first placed in a mixture of alcohol and ether for thirty or forty minutes ; then transferred to a solution of celloidin (dissolved in equal parts of alcohol and ether) from two to twenty hours.3 A cork placed in the clamp of the micro- 1 Mucilage is prepared by making a solution of gum Acacia. 2 If the material is firm enough it is sometimes mounted on cork without being imbedded. 3 The length of time depends on the nature of the material. It is longer for spongy structures like the lungs. THE METHODS OF MOUNTING MICROBES 87 tome is smeared on its upper surface with a solution of celloidin, which is left to harden. When the material is ready, it is mounted upon a prepared cork (i.e. it is placed on the smeared surface) ; and a little celloidin solution is poured over the material so as to cover it. The mounted material is now placed in 70 per cent, alcohol in order to harden the celloidin (which has a pasty consistence). In a few hours or so the imbedded material will be ready for cutting with one of the microtomes already described. Schanze's microtome is a useful instru- ment for cutting sections of materials imbedded in celloidin. In cutting a tissue imbedded in celloidin or mounted directly on cork, the razor and tissue should be kept wet with alcohol, and the sections carefully transferred to alcohol. The sections (if from celloidin material) are placed in oil of cloves in order to dissolve out the infiltrated celloidin. They are then ready for staining, etc. For fixing pieces vifirm materials directly on corks either glycerine-gelatine l or gelatine is used. These substances are liquefied by the application of heat. Paraffin wax for use as an imbedding material is first dissolved in chloroform, and then used in a similar manner to the solution of celloidin already described. The imbedded material must be cut perfectly dry, and the sections removed to xylol. 1 Glycerine-gelatine is prepared as follows : to 10 parts of gelatine add sufficient water to allow the gelatine to swell up ; pour off the water, and melt the gelatine. To the melted gela- tine add 10 parts of glycerine, and finally a few drops of some germicidal agent, preferably carbolic acid. The latter is added in order to preserve the glycerine-gelatine. 88 A MANUAL OF BACTERIOLOGY The xylol dissolves out the infiltrated paraffin, and the sections are then placed in alcohol to extract the xylol. After this treatment they are ready for the staining process. Instead of celloidin and paraffin, wax-and-oil mixture l and vaseline-and-paraffin mixture are used for imbedding purposes. Before alcohol-hardened tissues are cut with the freezing microtomes they must be soaked in water for ten minutes, this process to be followed by five hours' soakage in mucilage. After this they are frozen and cut with the microtome, whose razor must be perfectly sharp and free from notches. Tissues hardened in Muller's fluid (if they have not been subsequently placed in alcohol) are at once dried with blotting-paper, then frozen, and finally cut. Fresh tissues are covered with mucilage, frozen, and cut. The razor should be moistened with a solution of gum, and the sections transferred with a camel-hair brush to warm distilled water for fifteen minutes — the object being to dissolve out the mucilage. They are then ready for staining, etc., with the exception of sections of fresh tissues, which should be placed, before staining, in a 0'6 per cent, saline solution, so as to prevent too much shrinking of the sections. In cutting sections with the microtome ' very little force is required in pushing the razor or knife through the material, and if it is sharp a very slight turn of the screw each time will enable one to cut a 1 Equal parts (by weight) of white wax and olive oil are melted together. THE METHODS OF MOUNTING MICROBES 89 section, which ought to be so thin as to be almost invisible.' After staining, etc., the sections are mounted in various media on glass slides (3 in. X 1 in.), and covered with thin cover-glasses.1 ' When high- power lenses are to be used it facilitates the work very much to know the exact thickness of the cover- glass under which the specimen is mounted, and with very high powers, or those with wide angles of aperture, the cover-glass must be at least 0'004 in. to enable the lens to work through it.' All Zeiss' objectives in fixed mounts are corrected for a cover- glass of medium thick- ness (between 0'15 and 0'2 mm., or 0'006 and 0-008 in.). In the higher series from CC upwards the thickness of the cover-glass con- sistent With the mOSt Flo> .27, zEIS8- COVER-GLASS TESTER. perfect correction is indicated on the side of the mount by small figures (mm.). As a rule, it is sufficient for ordinary work to use cover-glasses of an estimated medium thick- ness. Oil-immersion objectives are within wide limits independent of the thickness of the cover-glass. But considerable variations in the thickness of the cover-glass may be compensated for — by slightly lengthening the body-tube for thinner cover-glasses ; and by slightly shortening the body-tube of the 1 The round ones are better than those that are square. 90 A MANUAL OF BACTERIOLOGY microscope for thicker cover-glasses. Zeiss makes a good tester (Fig. 27) suitable for the exact measurement of the thickness of cover-glasses. The measurement is effected by a clip projecting from a box ; the reading is given by an indicator moving over a divided circle on the lid of the box. The divisions show hundredths of a millimetre, and the instrument is capable of measuring up to five milli- metres. Before use the glass slides and cover-glasses should be perfectly clean. Many methods for permanently mounting tissues and cover-glass preparations have already been described. For fresh tissues glycerine is often used, while for hardened tissues the following mounting media have each their special advantages : — (a) Canada balsam dissolved in xylol. (&) Canada balsam dissolved in benzol. (e) Canada balsam dissolved in chloroform and turpentine. (d) Dammar varnish. After the tissues have been stained, they pass through the following processes : — Washing off the excess of stain, dehydrating, clearing or extracting the infiltrated material used in the imbedding pro- cess, etc. ; mounting, cementing, or sealing ; and finally, labelling the slides. The following list gives the various agents for the above-mentioned pro- cesses : — (Water. (1) Washing agents, . . . J Dilute spirit. I Absolute alcohol. (2) Dehydrating agent, . . Absolute alcohol. THE METHODS OF MOUNTING MICROBES 91 (3) Clearing agents, . Oil of cloves. Oil of cedar. Xylol. Aniline oil. Terebene. ( Canada balsam. (4) Mounting agents, . . . \ Dammar varnish. I Glycerine. f Hollis' glue. «3, Cementing agent,, iBlack asphalte varnish. To mount afresh specimen, the section should be placed with the utmost care in the centre of a glass slide. The section should not be folded in any part, therefore it must be carefully spread out with needles. This must be performed without stretch- ing the specimen. After this has been done, wipe off all moisture with a clean cloth. Now take up ' a cover-glass and place a drop of glycerine in the centre, invert and place it horizontally on the pre- paration, leaving the weight of the cover-glass to spread out the glycerine.' If there is an excess of glycerine round the edges of the cover- glass, it must be carefully absorbed by filter or blotting- paper, but on no account should the cover-glass be removed. To seal, ring, or cement the preparation, paint round the edge of the cover-glass and a little way on the slide, a ring of Hollis' glue or Dammar varnish. Hollis' glue1 is better than Dammar varnish, for it is not acted upon by the cedar oil used with oil-immersion lenses. The sealing of 1 Gold size is sometimes used for sealing glycerine prepara- tions. 92 A MANUAL OF BACTERIOLOGY microscopic preparations with Hollis' glue or any other cementing agent is performed with a camel- hair brush and a turn-table (Fig. 28). The slide is fixed with the clips of the turn-table, the table re- volved, and the brush containing the cement is held in a vertical position, so as to touch the edge of the cover-glass. By this means a ring of the cement is deposited, which dries in a day or two. The pre- paration is permanently sealed, and should now be labelled and placed in the cabinet. The various preparations of Canada balsam and Dammar varnish are prepared as follows : — (a) To prepare xylol balsam it is necessary to dissolve Canada balsam in xylol until it has the consistency of treacle ; (&) benzol balsam is prepared by first drying the Canada balsam until it is brittle. It is then dissolved in benzol until it has the same consist- ency as the xylol balsam. If these mounting fluids get thick on keeping, they are thinned by the addition of xylol and benzol respectively; (c) chloroform- turpentine balsam is prepared by dissolving 3 ozs. of Canada balsam in 1 oz. of chloroform and 1 oz. of turpentine. If this medium gets thick, it is thinned by the addition of chloroform ; (d) Dammar varnish is prepared by first dissolving 1J oz. of powdered gum Dammar in 1 J oz. of turpentine, and filtering. At this point \ oz. of gum mastic is dis- solved in 2 ozs. of chloroform, and the solution filtered. The two solutions are finally mixed to- gether, and again filtered. THE METHODS OF MOUNTING MICROBES 93 These fluids are used for mounting hardened •tissues, and they should be preserved in stoppered or well-corked bottles ; while for daily use a small drop-bottle of each fluid should be placed on a table set apart for mounting purposes. It may be mentioned that xylol balsam is the best mounting fluid for stained microbes ; chloroform-turpentine balsam acts well with hardened sections ; and benzol balsam is the most useful solution for general micro- scopic purposes. These mounting fluids are all used in the same manner, therefore a description of mounting in xylol balsam will also apply to the other fluids. The sections having been stained and washed, they are placed for twelve minutes in absolute alcohol contained in a watch-glass : the alcohol dehydrates them. They are now drained, and then placed in oil of cloves to clarify them. While in this medium they should be carefully straightened out with needles.1 Having now placed a drop of xylol balsam in the centre of the slide, it is spread out with a needle ; then a section is carefully lifted 2 out of the oil of cloves, drained, and placed in the xylol balsam. A small drop of xylol balsam is placed on the under surface of a clean cover-glass, which is lowered on to the section. With practice and perseverance, slight pressure with the forefinger is all that is required to produce a slide devoid of 1 Ordinary steel needles mounted in wooden handles. 2 A lifter is made by beating out one end of a copper wire, and then turning up the broad portion. Lifters made of German silver may be purchased at Messrs. F. E. Becker & Co., of Hatton Garden, London. 94 A MANUAL OF BACTERIOLOGY air-bubbles. To remove these bubbles small air- pumps have been devised, but they are not to be recommended ; ' the only thing to be done when an air-bubble lodges in a cavity of the section, and refuses to move in any way by gentle pressure, is to lift the cover-glass, and transfer the section to oil of cloves, and then remount it.' As the mount- ing of sections may be performed in the summer, the xylol balsam is much thinner than usual (due to the heat), and therefore takes a much longer time to set. In such cases a mounting clip (Fig. 29) is useful to keep the cover-glass from moving, i.e. until the balsam sets. After this the slide should be sealed with Hollis' glue, or some other ce- menting agent, as already described.1 FIG. 29. MOUNTING CLIP. . Methods of Introduc- ing Microbes into Living Animals. — In such experiments guinea-pigs, rabbits, mice, fowls, etc., are used. Pure cultivations of microbes and infectious matter are introduced into the animal body by the following methods : — (a) Inhalation. (b) Swallowing. (e) Direct inoculation. (d) Special operations. (a) An animal is made to inhale the infectious matter, etc., disseminated by means of a spray ; (b) 1 For further information see Martin's Manual of Microscopic Mounting. THE METHODS OF MOUNTING MICROBES 95 the infectious matter is mixed with the animal's food; (c) the infectious matter is introduced into the animal body by cutaneous or subcutaneous in- oculation or injection; (d) by the fourth method mentioned above (i.e. special operations), the infec- tious matter may be injected into the duodenum, or introduced into 'the peritoneal cavity by the per- formance of abdominal section/ These and other operations are used as means of introducing micro- bian matter into the living animal. But it cannot be too firmly impressed upon the mind that all operations should be performed with antiseptic pre- cautions ; and the instruments, as well as the hands of the operator, should be thoroughly disinfected. Before closing the present chapter we give a few remarks on what is known as the unit of micro- scopical measurement. It has been the general prac- tice among bacteriologists to give the dimensions of microbes in terms of a thousandth part of a milli- metre, which is called a micro-millimetre, and is known by the symbol fi.1 This unit is of great importance, for ' it is always easier to conceive the size of any object, and especially to realise the com- parative sizes of two objects, when their dimensions are given in terms of a unit smaller than either; for instance, it is difficult exactly to comprehend the length represented by -^ of an inch, and few people can readily compare such dimensions as TV and 75^ of an inch. This difficulty vanishes when the dimensions are expressed as multiples of a small, properly chosen unit, and not as fractions of a large 1 1 /u=0'001 mm.=Tjr^nr in., or 0*0000393 in. 96 A MANUAL OF BACTERIOLOGY one. For this purpose a fraction of an inch might be adopted instead of a fraction of a millimetre (mm.) ; but, at any rate, in measuring the spores of fungi, TWOTT °f an incn is too large a unit, and TWO-OIF of an inch would be inconveniently small. It happens that, if we take TFOTJ- of a millimetre as our unit, we can express the size of the spores of all fungi, etc., in the fewest possible figures. For in- stance, many micrococci measure about 1 //,, the spores of Penicillium about 3 //,, the spores of many Myxomycetes about 1 0 //,, and so on. If we compare these figures with the following: O'OOl mm., 0'003 mm., O'Ol mm.; or, still more, with these: 0*00004 in., 0*00012 in., 0'0004 in. — we see the great saving effected in the trouble of writing down the dimen- sions, quite apart from the greater readiness with which they can be compared with one another. But perhaps the difficulty with some is that of realising and actually applying this unit; we will therefore give an easy method by which the size of the micromillimetre may be obtained. Place the micro- scope in such a position that the image projected upon a piece of white paper is magnified 254 times : this can easily be done by a quarter-inch objective, with the use of the draw-tube, or by placing the paper at a greater distance than ten inches from the eye-piece. Let this position be marked, so that the microscope can be placed in it again at any time. Now copy on the paper, from a scale, an inch divided into ten parts, and with a line pen subdivide each tenth into five equal parts. Then the value of each of these subdivisions will be 2 /A, and of the THE METHODS OF MOUNTING MICROBES 97 whole tenth of an inch, 10 /-t. If this scale be care- fully copied on a piece of thin cardboard or other suitable substance, the dimensions of any microbe, etc., drawn by the camera lucida or otherwise on the paper in that position of the instrument, can be easily read off in /*s. With the aid of a deeper eye- piece or higher objective we can magnify the image 508 times, and then each small division of the scale will represent 1 CHAPTEE IV THE ORIGIN, CLASSIFICATION, AND IDENTIFICATION OF MICROBES SCIENTISTS and non-scientists are agreed that there was a lifeless period in the history of the earth — therefore that life had a beginning. But when, where, and how did life begin? 'As to the time, there is no evidence whatever. Life is enormously older than any record of it. Even the higher forms were developed long before the periods in which we first find their remains. As to the place, probably in the polar regions, as Buffon suggested in his Epoques de la Nature. The earth being a cooling globe, those regions would be the earliest to reach a temperature under which life is possible.' During the past twenty years or so, Buffon's theory has been supported by Comte de Saporta * and others ; and it is highly probable that in the earliest zoic epochs (especially the north polar) regions of the earth were of a hot and humid nature. Moisture and heat are essential to life ; therefore life had its beginnings in water.2 It is probable that lowly plants (possibly microbes) were the first organised 1 UAncienne, Vtg&ation Polaire. 2 See Professor Moseley in Nature, September 3, 1885. THE ORIGIN OF MICROBES 99 beings which made their appearance on the earth, for it is well known that all microbes require mois- ture, while many live m water or similar media. From these and other facts it is probable that the Schizomycetes were the forms of life which originated in the polar regions of the earth — the other parts of the earth, at that remote time, being too hot for life to exist. But if life originated in the particular part of the earth indicated, this does not explain the origin of life. How did life begin ? This question has occupied the thoughts of men in all ages, but if we regard living and non-living matter as composed of elements which are common to both kinds of matter, wherein lies the difference which gives as one result non-living matter, and as another result living matter? The difference must lie in the mixing of these elements. If the first form of living matter were a microbe it originated either by a creative act or by spontaneous generation. Both the theory of creation and that of spontaneous genera- tion account for the origin of life : in fact, the be- ginning of life can only be explained theoretically, for there is no practical or direct proof of how life originated. On this point Professor Huxley 1 says : ' If it were given me to look beyond the abyss of geologically recorded time to the still more remote period when the earth was passing through physical and chemical conditions, which it can no more see again than a man can recall his infancy, I should expect to be a witness of the evolution of living protoplasm from not living matter. I should expect i Critiques and Addresses, p. 238. 100 A MANUAL OF BACTERIOLOGY to see it appear under forms of great simplicity, en- dowed, like existing fungi, with the power of deter- mining the formation of new protoplasm from such matters as ammonium carbonates, oxalates, and tartrates, alkaline and earthy phosphates, and water without the aid of light. That is the expectation to which analogical reasoning leads me ; but I beg you to recollect that I have no right to call my opinion anything but an act of philosophical faith.' Besides the two great theories which account for the origin of life from mineral matter,1 there are others, which we now describe. It has already been stated that putrefaction is the result of life, not of death — the result of microbian activities 2 — but formerly many naturalists believed that by putrefaction the organic elements which had com- posed the body of the dead animal formed them- selves by free creative power into independent beings, which differed entirely from those from which their material was produced, yet are in every case animated, and have the power of propagation ; thus the albumin and fat globules take the form of microbes, perhaps also of yeasts and moulds, or even of those little infusorial animals, whose presence never fails in corruption. This mode of origin has been called equivocal generation or generatio cequi- voca.3 Other naturalists dispute the possibility of 1 Those of creation and spontaneous generation. 2 The microbes being introduced from the air, water, etc. 3 The equivocal origin of microbes must be distinguished from the spontaneous generation, which we have already alluded to ; for in the latter case there existed no organisms on the earth. THE ORIGIN OF MICROBES 101 living beings, however small and simple, ever origi- nating in any other way than from germinal matter which sprang from the same form of life ; and they insist that the belief in the equivocal origin of microbes is that last remnant of an old superstition, which the light of science has not entirely banished. In ancient times it was thought that serpents and frogs originated from slime, that caterpillars were generated from decayed leaves, vermin from filth, and worms from spoiled meat. Now-a-days every child knows that all these things are fables ; every housewife knows by experience that no maggots originate in meat if the blow-fly is prevented by a wire-screen from entering and depositing its eggs. They have learned, through careful covering, to keep away the minute mould- spores, which settle with other dust from the air, and which colonise on their preserved fruits ; they know that trichina and tape- worm only originate from raw or half-cooked pork, in which these animals were already present in the embryonic stage. Even the farmer no longer believes that the grain rust (Puccinia graminis) originates from chilling, but that it springs from spores which are scattered by the barberry bushes (Berberis vul- garis), or other fallen stalks, and that the blight may be prevented in corn crops, if the seed (before sow- ing) is steeped in a solution of iron sulphate or copper sulphate, in order to kill the spores which cling to it.1 Concerning microbes and their related ' fermenta- tions/ the above-mentioned observations lead without 1 See Dr. Griffiths' book, The Diseases of Crops, pp. 128-132 (Bell & Sons). 102 A MANUAL OF BACTERIOLOGY doubt to the conclusion that they do not originate through equivocal generation ; for when nitrogenous material from the animal or vegetal world is heated in flasks, even at as low a temperature as 70° C., all the microbes are killed, and if the entrance of new germs from outside is in every way prevented, and it were possible to keep the flasks for ever, no microbes would ever originate of themselves. On the contrary, the entrance of a single germ, in each flask, is sufficient to cause multiplication, and with it putrefaction. If microbes originate from putrid matter through equivocal generation, putrefaction must appear before the microbes ; but experience shows the contrary, that putrefaction is a conse- quence of the development and growth of microbes. "Within the last few years a theory has been advanced to account for the origin of microbes, which has caused some sensation, viz., that under certain conditions the ordinary mould-fungus will give rise to moving germs of extraordinary minuteness ; such germs are capable of developing into microbes, into yeasts, and finally again into the mould-fungus. When microbes are found in the blood or organs in certain diseases, the authors of this theory of pleo- morphism are satisfied that the spores of the com- mon mould germinate in the human body; that these spores first swarm as microbes, but under suitable culture may be nourished into different species of moulds. However, unprejudiced research has not given the slightest proof that microbes stand in any connection with the dev^pment of yeasts, moulds, or other fungi. They always originate, THE ORIGIN OF MICROBES 103 as far as we know at present, from spores, etc., of the same kind (Cohn). Concerning the doctrine of pleomorphism, it may be stated that Lankester,1 Van Tieghem, Zopf, Cienkowski, Billroth, Neelsen, Hauser, and others have noticed that certain microbes pass through various phases during their life-histories. And Sattler, Gravitz, and Blichner, believe that they have transformed certain non-pathogenic microbes into pathogenic forms by simply cultivating the former in different media or under different physical condi- tions. For instance: Sattler2 states that he has transformed the non-pathogenic Bacillus subtilis into a pathogenic form, capable of producing infectious ophthalmia, by cultivating the microbes (at 35° C.) in an infusion of jequirity seeds. Gravitz believed that he had transformed the non-pathogenic moulds — Aspergillus glaucus, Penicillium glaucum — into pathogenic forms by cultivating them in alkaline media at about 40° C. Biichner states that he has transformed Bacillus subtilis into Bacillus anthracis and vice versd : ' that by successive cultivation of Bacillus anthracis under constant variation of the nutritive material, he saw it assume the morphological and physiological characters of Bacillus sultilis! Klein, Koch, Cohn and others do not accept the theory of pleomorphism, or the transformation of microbes ; and Klein 3 has proved most conclusively 1 Quarterly Journal of Microscopical Science, 1873, p. 408. - Wiener Medic. Wochenschrift, 1883. 3 Micro-Organisms and Diseate, pp. 207-231 (3d ed.). 104 A MANUAL OF BACTERIOLOGY that no pathogenic microbe is ever transformed into a non-pathogenic form, or vice versd. In fact, he says that 'those organisms which are connected with morbid processes possess this pathogenic power ab initio ; and are not due to any peculiar condition of growth.' If a harmless microbe could be proved capable of transformation into a harmful form, ' the whole doctrine of the infectious diseases is involved in such a case ; for if in one case it can be unmistakably proved that a harmless microbe can be transformed into a pathogenic organism, i.e. into a specific virus of an infectious disease, and if this again can, under altered conditions, resume its harmless property, then we should at once be relieved of searching for the initial cause in the outbreak of an epidemic. But in that case we should be forced to contemplate, as floating in the air, in the water, in the soil, everywhere, millions of microbes which, owing to some peculiar unknown condition, are capable at once to start any kind of infectious disorder, say anthrax (Buchner), in- fectious ophthalmia (Sattler), and probably a host of other infectious diseases, and thus to form the starting-point of epidemics. And the only redeem- ing feature, if redeeming it can be called, in this calamity, would be the thought that the par- ticular microbe would by-and-by, owing to some accidental new conditions, again become harmless ' (Klein). The transformation of microbes into different forms is entirely opposed to the Darwinian law. To one who has fully comprehended the meaning THE ORIGIN OF MICROBES 105 and the operation of this law, it will be at once apparent that there must be error somewhere in the matter. ' If the law of actual variation/ says Dr. Dallinger, ' with all that is involved in the survival of the fittest, could be so readily brought into complete operation, and yield so pronounced a result, where would be the stability of the organic world ? Nothing would be at one stay. There could be no perman- ence in anything living. The philosophy of modern biology is that the most complex forms of living creatures have derived their splendid complexity and adaptations from the slow and majestically progressive variation and survival from the simpler and the simplest forms. If, then, the simplest forms of the present and the past were not governed by accurate and unchanging laws of life, how did the rigid certainties that manifestly and admittedly govern the more complex and the most complex come into play ? If our modern philosophy of biology be, as we know it is, true, then it must be very strong evidence indeed that would lead us to conclude that the laws seen to be universal break down and cease accurately to operate, where the objects become microscopic, and our knowledge of them is by no means full, exhaustive, and clear. Moreover, looked at in the abstract, it is a little difficult to conceive why there should be more uncertainty about the life-processes of a group of lowly living things, than there should be about the behaviour, in reaction, of a given group of molecules. The triumph of modern knowledge is a knowledge — which nothing can shake — that Nature's processes 106 A MANUAL OF BACTERIOLOGY are immutable. The stability of her processes, the precision of her action, and the universality of her laws, are the basis of all science, to which biology forms no exception. Once establish, by clear and unmistakable demonstration, the life-history of an organism, and truly some change must have come over Nature as a whole, if that life-history be not the same to-morrow as to-day ; and the same to one observer, under the same conditions, as to another. 'But the fact that there is no evidence of any direct relation evolutionary between two such forms as Bacillus subtilis and Bacillus anthracis, the fact that there is no ready way either naturally or artificially of their being changed into each other, must not blind us to the fact that such an evolutional relation in the past is eminently probable, nay almost certain. It may, in all probability must, have taken an indefinite time in the past to effect ; but being once effected, the specificity is continued as in every other form by inheritance.' There are certain conditions under which a microbe may appear to have altered its properties. For instance, Chauveau1 has shown that Bacillus anthracis loses its virulence when submitted to the action of compressed oxygen ; but it does not lose its vaccinal property after this treatment. This new character is said to be maintained by suitable cultivation. Although Bacillus anthracis may lose its virulence under such abnormal conditions as already alluded to, it does not become a non-patho- genic microbe, for it still preserves one of the most 1 Comptes Rend/us de P Academic de* Sciences, tome 109. THE ORIGIN OF MICROBES 107 essential attributes that indicate the infectious nature of the pathogenic microbe, viz., its vaccinal property. Besides, Chauveau has further shown that the non-virulent Bacillus anthracis may be revivified by degrees when grown in suitable media. These researches do not point to any transformation of Bacillus anthracis into a non-pathogenic species, but simply show that oxygen under pressure is capable of modifying the microbe's pathogenic power. In fact, microbes have the power of adapt- ing themselves to considerable variation of external conditions; but this does not involve permanent change in the organism. Microbes belong to the vegetal kingdom ; in other words, they are fungi. As they multiply by re- peated subdivision, and also frequently reproduce themselves by spores, which are formed endogen- ously, they are grouped together in a class called the Schizomycetes, splitting fungi, or Spaltpilze, as the German naturalists term them. The forms of microbian cells vary considerably — they are round, ovate, elliptical, cylindrical, etc. Microbes live isolated, singly, or in larger or smaller colonies, or, in many cases, united in pairs, or many together in threads or groups. Nearly all microbes possess two different modes of life : one of motion and another of rest. In certain conditions they are extremely motile, and when they swarm in a drop of water or other fluid they move among each other in all directions; sometimes rotating round their longitudinal axis, while in other cases the move- ment is an oscillating one, or the threads alternately 108 A MANUAL OF BACTERIOLOGY bend and straighten themselves, etc. At other times the motile microbes become motionless. In this state many of them aggregate together and excrete a gelatinous material which entirely envelopes them. This colony is termed a zoogloea, in fact it is the resting stage of the particular microbes. In the zooglcean stage, microbes often produce spores. Microbes multiply by fission (i.e. division) and spore-formation. The warmer the air, etc., the faster proceeds the division, and the stronger the multiplication ; in a lower temperature it becomes slower, and ceases entirely at the freezing-point of water. Their fecundity is enormous, and would, in a very short time, choke up the earth ; but this rapid rate of increase is kept in check by the limited supply of food, climatic conditions, and the struggle for existence. As an example of the enormous fecundity of mic- robes we describe the rate of reproduction of a common form, viz., Bacillus sultilis. This bacillus attains a certain length and then divides across into two. 'Each half grows to the size of the parent, and then similarly divides, and so on as long as food and other conditions of their life are present. Bacillus sultilis has been observed to divide in this way every half-hour, a rate which gives in twenty- four hours more than three hundred billion of in- dividual microbes as the offspring of one parent. They are extremely minute, varying from 20^0^th of an inch to the TzyWth of an inch in length.' As already stated, microbes propagate by fission and by spore-formation. The following table gives THE REPRODUCTION OF MICROBES 109 those that are produced only by fission, and those that multiply by fission and spores : — Mode of Propagation. General Remarks. Micrococci . Fission Spherical and oval in form. Bacteria . Fission . ~ .'' Rod- shaped microbes, generally smaller than bacilli, and devoid of spore-formation. Bacilli . . Fission and spores Rod-shaped microbes, many are provided with flagella. Vibriones . Fission and spores Curved or more or less wavy rods provided with flagella. Spirilla . . Fission and spores Spiral-shaped microbes. Spirochaetae Fission and spores Filamentous and wavy microbes. Concerning the reproduction of the micrococci, if the division takes place in one direction only, the resulting form (if the two cells remain together) is a diplococcus, dumb-bell, or colon (:). The diplo- coccus may again divide, without separation, form- ing a streptococcus or chain, which may become curved or even twisted in appearance. Sometimes the division of these microbes is in two directions, resulting in four cocci (::), which is termed a meris- mopedia, or in three directions forming a sarcina- coccus or sarcina.1 All microbes require for their nutrition and growth 1 A division into a large and an indefinite number of cells is termed an ascococcus. 110 A MANUAL OF BACTERIOLOGY oxygen, carbon, nitrogen, certain salts, and water. Although some microbes are anaerobic, they require oxygen, which is obtained from the carbohydrates and albuminoids of the medium in which they live, or from the free oxygen which may be dissolved in that medium. Before considering the various classifications of microbes, we mention the fact that microbes in general are sometimes called Bacteria, but as there is a genus of that name, it is better that the word should be applied only when one is alluding to microbes of that genus. The study of microbes (which includes all forms of Schizomycetes) has been consequently termed Bacteriology ; but it is an un- fortunate name, which, at the present time, cannot well be replaced by another. Microbes may be simply divided into aerobic1' and anaerobic 2 forms. Bacillus spinosus and Bacillus cedematis maligni are examples of the former ; while Micrococcus candicans and Bacillus sultilis are ex- amples of the latter kind. Microbes may be also divided into pathogenic (disease-producing), septic (putrefactive), zymogenic (fermentive), and chromogenic (pigment-forming) forms. The Schizomycetes, which Sachs includes in his group the Thallophytes, have been classified by Cohn3 into five genera : — (1) Spherobacteria or micrococci. 1 Those requiring free access of oxygen (air). 2 Those which do not require free oxygen. 3 Beitrdge zur Biologie der Pjlanzen, 1872 et seq. THE CLASSIFICATION OF MICROBES 111 (2) Microbacteria or bacteria. (3) Desmobacteria or bacilli and vibriones. (4) Spirobacteria or spirilla. (5) Spirochsetae. This classification is founded upon the idea that all the various morphologically or physiologically distinct forms belong to different species. Koch's researches with plate-cultivations have given great support to the classification of Cohn, which, in our opinion, is the best, that is, from the bacteriologist's point of view. In such a classification a micro- coccus produces nothing but a micrococcus, a bacil- lus nothing but a bacillus, and so on. Zopf (who is the great apostle of the doctrine of pleomorphism) divides microbes into four groups :* — (1) Coccacese. (2) Bacteriaceae. (3) Leptothricheae. (4) Cladothrichese. The first group contains streptococcus, merismopedia, sarcina, micrococcus, and ascococcus forms ; in fact this group only contains cocci. The second group contains the following genera : — Bacterium, Spiril- lum, Vibrio, Leuconostoc, Bacillus, and Clostridium. Most of these forms, according to Zopf, pass through a coccus stage. The third group contains four genera : — Crenothrix, Beggiatoa, Phragmidiothrix, and Leptothrix. This group (like the second) is believed to possess coccus, rod, and thread forms. The fourth and last group only contains the genus 1 Die Spallpilze, 1885. 112 A MANUAL OF BACTERIOLOGY Cladothrix, which shows coccus, rod, thread, and spirillar forms. Baumgarten divides microbes into two groups, each containing three genera : — Monomorphic Group. Pleomorphic Group. Coccus. Bacillus. Spirillum. Spirulina. Leptothrix. Cladothrix. The genus Bacterium is entirely dispensed with in this classification ; and Fliigge, who modified Cohn's classification, has submerged the genus Bacterium into the genus Bacillus, as both these forms were rod-shaped ; but it should be borne in mind that the bacteria do not produce spores, whereas in the bacilli spore-formation is of common occur- rence. Hueppe's classification is based on the mode of reproduction, or, rather, fructification ; and the late Dr. De Bary divided them into two groups : Mic- robes which produce endospores, and microbes which produce arthrospores. But as we know so little about spore-formation in the Schizomycetes, Hueppe's and De Bary's classifications are of very little practical value at the present time. 'The determination of species rests upon the accumulated evidence afforded by a thorough know- ledge of their life-history.' The form of the mic- robe, the physiological, pathological, and other THE IDENTIFICATION OF MICROBES 113 changes it effects, and the microscopical and macro- scopical appearances under cultivation, must be collectively taken into account. This determina- tion or identification of species will be considered in the next chapter. CHAPTEE V THE BIOLOGY OF MICROBES, ETC. IN this chapter we describe nearly all the more im- portant microbes ; but the microbes present in such diseases as tuberculosis, cholera, diphtheria, scarla- tina, etc., will be described in Chapter vi. MICROCOCCI. Micrococcus prodigiosus. — This microbe, which measures from 0*5 to 1 //, in diameter, gives rise to a blood-red pigment when grown on boiled potatoes, white of egg, starch-paste, bread, agar-agar, and other media. Fig. 30 represents the macroscopic and microscopic appearances of -this microbe. It grows well on agar-agar, which it liquefies. The pigment, which M. prodigiosus gives rise to, is in- soluble in water, but soluble in alcohol; and in many of its reactions it resembles certain aniline colours.1 This pigment is only produced under cer- tain conditions, viz., at a temperature of from 20° to 22° C., and after the gelatine or agar-agar has lique- 1 Erdmann in Journal fur Praktische Chemie, 1866 ; and Schroter in Beitrdge zur Biologie der Pfldnzen, vol. i. p. 109. 114 THE BIOLOGY OF MICROBES, ETC. 115 fied. As the temperature rises to blood-heat M. prodigiosus loses its power of forming the red pig- ment ; but forms casein, lactic acid, and probably other substances. ' When growing and kept in the depth of a solid nourishing material, i.e. removed A, Macroscopic appearance of Mic- rococcus prodigiosus on sterilised potato after four days' cul- tivation. From a fifth fractional cul- tivation. C, Microscopic appearance of M. prodigiosus. X 1265.? B, A growth of Micrococcus prodigiosus in nutrient agar- agar. Fio. 30. MICROCOCCUS PRODIGIOSUS. from the free surface, colonies of this microbe grow as colourless micrococci.' They are always present in the atmosphere, and give rise to the phenomena known as ' bleeding bread,' ' blood-rain/ ' bloody sweat,' etc. 116 A MANUAL OF BACTERIOLOGY A desiccation of four months at 32° C. (dry heat) l does not destroy the vitality of M. prodigiosus; but when exposed to the action of ozone the microbe is killed.2 From these facts one can readily under- stand how it is that M. prodigiosus (as well as other aerial microbes) is always present in the air of towns, villages, etc. ; but is never in the air at sea, for the ozone present in sea-air destroys the microbes. Micrococcus luteus. — This is another chromogenic aerial microbe. It is found as single cells, dumb- bells, or in packets. The cells are 1 '2 p in diameter ; and they grow rapidly on nutrient gelatine plates (plate-cultivations) giving rise to a yellow pigment. The colonies, so produced, are round and slightly granular in appearance. M. luteus grows in nutrient agar-agar, bouillon ; on steamed potatoes ; and as drop -cultures. The pigment produced by this microbe is insoluble in water, and is unchanged by sulphuric acid and alkalies. It is also destroyed by the action of ozone. Micrococcus chlorinus. — This microbe produces a yellowish green pigment when grown on sterilised white of egg (see Fig. 21) and fluid media. The cells are about 1 ft in diameter. The pigment is soluble in water, and is decolourised by acids. Micrococcus aurantiacus. — The cells of this aerial microbe are 1*5 //, in diameter ; and they occur singly, in pairs, or in zooglea. On plate-cultivations they form orange-coloured drops and spots, which 1 Griffiths in Proceedings of Royal Society of Edinburgh, vol. xvii. p. 262. 2 See Griffiths' Researches on Micro-Organisms, p. 184. THE BIOLOGY OF MICROBES, ETC. 117 ultimately coalesce into equal-sized patches. On fluid media they form an orange-coloured pellicle. M. aurantiacus also grows on steamed potatoes and white of egg. The pigment is soluble in water. Micrococcus fulvus. — Cells 1/5 fi in diameter; they form rusty-red drops and gelatinous masses on horse-dung. Micrococcus violaceus. — The cells are 1*4 JJL in diameter, and occur as bright violet-coloured gela- tinous drops or patches on the surface of steamed potatoes exposed to the air. Micrococcus cyaneus. — The cells are elliptical and grow on potatoes and fluid media, giving rise to a blue pigment when in contact with air. The pig- ment is soluble in water, and the solution is at first green, but afterwards becomes an intense blue. Acids convert this pigment into a red colouring matter, while alkalies turn it green. There are no characteristic absorption bands shown when a solution of the blue pigment is examined by the spectroscope. Micrococcus rosaceus. — The cells are from 1 to 1*5 fjL in diameter, and give rise to a rose-coloured growth on the surface of nutrient gelatine and agar-agar. Micrococcus cinnabareus. — This microbe grows very slowly on the surface of gelatine. At the end of eight days the colonies appear as small drops of a red colour ; but ultimately the colour becomes red-brown. This microbe occurs in twos, threes, and fours ; and very rarely as an isolated coccus. The pigment is soluble in water. Micrococcus hcematodes. — This microbe is some- times found in human sweat. It grows on steamed- 118 A MANUAL OF BACTERIOLOGY egg albumin in a damp chamber placed in the incubator (see Figs. 22 and 14). The pigment pro- duced is a red colour. Micrococcus flavus tardigmdus. — Colonies of this microbe form raised drops of a chrome-yellow colour. In test-tube cultivations, they form small yellow beads along the track of the needle. This microbe does not liquefy the gelatine. Micrococcus flavus liquefaciens. — The microbe grows in colonies of a yellow colour, and the cells form diplococci and zooglea. They liquefy the gelatine. Micrococcus versicolor. — Small cocci forming iri- descent colonies. The colonies are flat, not raised \ and in test-tubes the yellowish colonies have the appearance of small beads, i.e. along the needle track. These cocci are found in pairs or in masses. Micrococcus flavus desidens. — This microbe occurs in the dust of the atmosphere. The cells are 0*8 p in diameter, and occur singly, as diplococci, and in short chains. They form yellow colonies, which ultimately sink down in the gelatine. The yellow pigment is only formed at the surface of the gela- tine, for in the track of the needle the colonies are white. Micrococcus citreus conglomerate. — The cells are 1'5 fju in diameter, and occur in the atmosphere and in blennorrhoeic pus. On gelatine plates they form citron yellow colonies. Micrococcus cereus flavus. — The cells are 1'5 //, in diameter, and occur singly, in lemon-yellow groups, or in short chains. They are found in pus. THE BIOLOGY OF MICROBES, ETC. 119 Micrococcus subflavus. — The cells are 0'8 //, in diameter, and occur singly, in pairs, in tetrads, and zooglcea groups. On gelatine they form white dots, which ultimately become yellow and confluent. This microbe was originally found in vaginal secretions and lochial discharges. Micrococcus radiatus. — The cells are 0'8 p in diameter, and occur singly and in short chains. They form ' whitish colonies with a yellowish-green sheen.' The colonies liquefy the gelatine and sink down in it ; there developing, in the course of a day or two, a circlet of rays. Micrococcus pyogenes citreus. — The cells occur singly, in chains, and masses. They grow on nutrient agar-agar and gelatine, giving rise to a lemon-yellow pigment. They are obtained from pus. Micrococcus pyogenes. — The cells are 1 p in diame- ter, and occur in chains or diplococci. They form small colonies which grow slowly ; on plate-cultiva- tions they are first white, then pale yellow, and finally become brown. They (the colonies) have no tendency to run together in either plate, stroke, or puncture cultivations, except on agar-agar or blood serum where the mass is thicker in the centre. They do not grow on potatoes ; and do not liquefy any medium. They occur in the pus of acute abscesses. Micrococcus pyogenes aureus. — This coccus occurs in osteomyelitis. It grows on boiled potatoes, nutrient gelatine, agar-agar, and blood serum, giving rise to orange cultures. This microbe liquefies gelatine, and the colonies remain limited to the 120 A MANUAL OF BACTERIOLOGY centre of the liquefying area. M. pyogenes aureus is 0*8 to 0'9 fj, in diameter, and occurs as diplococci, tetrads, short chains, and in irregular masses. It is fatal in large doses to guinea-pigs, mice, and rabbits if injected into the veins or into the peritoneal cavity. According to Becker, ' when a small quantity of a cultivation was introduced into the jugular vein after previous fracture or contusion of the bones of the leg, the animal died in about ten days, and abscesses were found in and around the bones, and in some cases in the lungs and kidneys/ This microbe peptonises albumin. Micrococcus urece. — The cells are round or oval, and measure 1/25 to 2 //, in diameter. They occur isolated or concatenate or forming a zooglcea on the surface of the fluid. M. urece secretes a ferment which causes the ammoniacal fermentation of urea : CH4N20 + 2H20 = (NH4)2 C03. The ferment has been isolated (in aqueous solu- tion), and it is proved that it has the power of converting urea into ammonium carbonate.1 Besides this well-known microbe, there are certain bacteria, and possibly bacilli, which produce a similar re- action.2 Micrococcus pyogenes albus. — The cells are 0*8 to 0*9 //, in diameter, and occur as diplococci, tetrads, short chains, or irregular masses. They grow rapidly on gelatine plates, producing colonies which 1 Dr. Musculus in Comptes Rendus, vol. Ixviii. ; and Dr. Sheridan Lea in Journal of Physiology, 1883 and 1885. 2 See Dr. MiqueFs paper in the Annuaire de I' Observatoire de Montsouris, 1889. THE BIOLOGY OF MICROBES, ETC. 121 are white. In test-tube cultivations, a white mass is formed along the needle track. About the third day of growth liquefaction sets in, and ultimately a white deposit settles at the bottom of the liquefied gelatine. This microbe is associated with suppura- tion. It is found in pus, necrotic tissues, etc. From what has been already stated in this chapter it will be seen that many micrococci are associated with wounds, abscesses, etc. Concerning the action of these microbes, Dr. W. Watson Cheyne l says : — (1.) There are various kinds of micrococci found in wounds treated aseptically, differing markedly from each other in their effects on animals. They agree in growing best at the temperature of the body, and in causing acidity and sweaty smell in the fluids in which they grow. The experiments (Cheyne's) show that cultivations may be carried on in fluid media with accuracy. (2.) The micrococci examined grew best in media exposed to oxygen gas ; and they grew only with difficulty in the absence of oxygen. Dr. A. Ogston 2 stated that these micrococci were anaerobic; but there is no doubt that this statement is erroneous. (3.) Their effect on animals was not altered by growth with or without oxygen. (4.) The effects of these micrococci on rabbits and man were not similar, some of the most virulent forms for rabbits causing no deleterious effect in wounds in man. (5.) The kidney is apparently an important 1 British Medical Journal, 1884. 2 Ibid., 1881. 122 A MANUAL OF BACTERIOLOGY excreting organ for microbes (Fig. 31); and microbes incapable of growing in the blood, may cause serious effects by growing in the excretory canals. This may explain some cause of pyelitis. (6.) Micrococci are always present in acute FIG. 31. SECTION OF KIDNEY CONTAINING MICROCOCCI (after Watson Cheyne). To the left is a mass of micrococci ; to the right an inflammatory ring, and intermediately the necrotic area, infiltrated with micrococci. What are evidently remains of two kidney-tubules are seen full of micrococci and leucocytes. X 375. abscesses, and are probably the cause of them. In some cases, the micrococci are the primary cause of the inflammation and suppuration, as in pyeernic THE BIOLOGY OF MICROBES, ETC. 123 abscesses; generally, however, they begin to act after inflammation has been previously induced. This inflammation may be caused by an injury, by the absorption of chemically irritating substances from wounds, by colds, etc. (7.) There are several different kinds of micrococci associated with suppuration. (8.) Micrococci cause suppuration by the produc- tion of a chemically irritating substance (probably a ptomaine), which, if applied to the tissues in a concentrated form, causes necrosis of the tissue, Fio. 32. MICROBES IN PURPURA. (Watson Uheyne.) A, Micrococci. B, Bacilli. X 2500. but, if more dilute, causes inflammation and sup- puration. Micrococcus in purpura hcemorrhagica. — Watson Cheyne1 has observed cocci (measuring 1'15 /* in diameter) in certain cases of purpura hsemorrhagica. This microbe forms colonies in the blood ; and the haemorrhages are due to the plugging of the small vessels by masses of these microbes. The microbes occur in chains (Fig. 32), and stain well with 1 Transactions of the Pathdogkal Society of London, 1884. 124 A MANUAL OF BACTERIOLOGY methylene blue. In another case of the same disease, Watson Cheyne found that certain bacilli plugged the vessels and gave rise to haemorrhages. Concerning this disease, he remarks that ' we may have to do with an infective disease of which the essence is the entrance of certain specific organisms into the blood, and their growth in it. It may, however, be that in these two cases, and in others, the primary affection is something quite distinct from microbes, resulting, however, in such an altered constitution of the fluids of the body, that of the innumerable organisms present in the mouth and intestinal tract, certain of them may be able to penetrate into and live in the blood, form emboli, and thus lead to the haemorrhages which are so marked a feature of these diseases.' Micrococcus variolce et vaccinice. — Micrococci (0'5 p in diameter) have been found in the lymphatics of the skin (in small-pox,1 cow-pox, and sheep-pox2) in the vicinity of the pocks. The microbes were found by Cohn3 in the lymph of vaccina and variola. No doubt they are the active agent in small-pox and cow-pox, for if the lymph is filtered through a Chamberland filter, the filtrate loses its infectious properties. The author * has shown that a solution of salicylic acid acts upon vaccine lymph, and deprives it of the power of inoculation. 1 Weigert in Med. Centralblatt, 1871. 2 Klein in Philosophical Transactions of Royal Society, 1874. 3 Virchow's Archiv, vol. Iv. 4 Griffiths in Proc. Roy. Soc. Edinburgh, vol. xiv. p. 97. i° t <>>& V »-•'• ••H: V 12 \ :; v. ^. - I1 ^ '** ' 7* u V..- . 8 : / 19 Fio. 33. VARIOUS MICROBES. 126 A MANUAL OF BACTERIOLOGY According to Quist,1 artificial cultivations of M. vaccinice have been used, with success, for vaccina- tion purposes. M. vaccinice (Fig. 33, 1) occurs singly, in pairs, chains, and colonies. Micrococcus endocarditicus. — This microbe has been found by numerous observers in masses and chains in the granulations, blood-vessels, the valves and muscles of the heart in endocarditis ulcerosa ; and there is little doubt that the disease is due to this microbe. M. endocarditicus measures from 0'5 to 1 fjb in diameter, and occurs singly and in chains. This microbe is capable of assuming the zoogloean state, and no doubt when in this state it gives rise to embolism. The same microbe has been found in the spleen, kidneys, and urine. Micrococcus in Measles. — Dr. Keating2 of Phila- delphia, and subsequently Cornil and Babes,3 have observed the presence of micrococci (singly and as diplococci) in the capillary vessels of the skin, in the catarrhal exudations, and in the blood of persons suffering from measles. The same microbe has also been found in the urine during the course of the disease. This microbe has not yet been cultivated. Dr. Salisbury, in 1862, stated that measles was due to a certain fungus derived from musty straw. Since that date, the pathogenic nature of Salisbury's 1 St. Peter sburgh Med. Wochenschrift, 1883. 2 Philadelphia Medical Times, 1882. 3 Les Bacteries, 1885. THE BIOLOGY OF MICROBES, ETC. 127 straw-fungus has been generally discredited, until the year 1889, when Mr. C. Candler1 argued in favour of Salisbury's theory — that fungus-dust from mouldy straw produces a disease resembling measles ; and that this fungus-dust when introduced into the human body, develops into microbes (!). In the great epidemic of measles in Victoria during the years 1874-75, Candler states that he could not discover any instance of measles in a dwelling from which damp straw (in the form of bedding) had been excluded, but in every house where measles occurred, the presence of damp straw in the bed- rooms was easily made out. There is nothing impossible in the supposition that damp straw favours the growth of microbes ; and it might con- ceivably be proved by sufficient evidence that this is a favouring or even a necessary condition for the growth of the specific virus of measles. But the evidence which Candler adduces is quite inadequate to prove that the cause of measles is a fungus, since it might just as well be Keating's micrococcus or any other microbe. Micrococcus gonorrhcece. — Drs, Neisser,2 Bokai, and Finkelstein 3 have described micrococci in the urethral discharge and the pus of gonorrhoea. These microbes (Fig. 33, 16) measure 0'83 ^ in diameter, and occur singly, as diplococci, sarcinse, and in zooglcean groups. They frequently adhere to the epithelial cells and pus- corpuscles. Dr. 1 The Prevention of Measles, 1889. 2 Centralblatt fiir d. Med. Wissensch., 1879. 3 Prager Med. Chir. Presse, 1880. 128 A MANUAL OF BACTERIOLOGY Bockhart 1 has artificially cultivated these microbes ; and has reproduced the disease by inoculation, thus proving their pathogenic character. A similar micro- coccus is often found in the purulent ophthalmia of new-born infants ; and it is possible that such ophthalmia is, in the majority of cases, of gonorrhceal origin. * Aufrecht2 reports the case of an infant twelve days old who died with suppuration of the umbilical vein and liver. The liver cells and the interlobular tissue were crowded with micrococci. These micro- cocci corresponded in size to Micrococcus gonorrhcece, and he thinks it probable that they were derived from the vagina of the mother ; during birth they might have got into the umbilical vein, there caused inflammation, and thence passed into the liver ' (Klein). Microccus tetragonus. — This microbe is found in the sputum of patients suffering from phthisis. It is only saprophytic in man, but pathogenic in animals. Mice inoculated with a pure cultivation of this microbe die in a few days, the microbe after- wards being found in the various organs of the body. Micrococcus tetragonus (Fig. 33, 14) measures 1 //, in diameter, and occurs as tetrads surrounded by a hyaline membrane. This microbe forms small white points on nutrient gelatine in about twenty- four hours, which ultimately run together. Micrococcus intracellularis meningitidis. — This microbe has been observed in the pus found at 1 Sitzungsberichte der Pkys. Med. Gesell. Wiirzburg, 1882. 2 CentralUattfur d. Med. Wissensch., 1883. THE BIOLOGY OF MICROBES, ETC. 129 the base of brain after death in cases of acute cerebral meningitis. It occurs singly, as diplococci, chains, and zoogloea ; and it grows on a mixture of agar-agar and gelatine at the temperature of the body. This microbe grows better at the surface than in the deeper layers of the medium, and gives rise to finely granular and yellowish-brown colonies. The microbe, when cultivated artificially, only remains virulent for six days ; and it is said that it ' affects mice, guinea-pigs, rabbits, and dogs.' Like M. gonorrhcece, this microbe 'is almost invariably found within the cells contained in the exudation/ Micrococcus lomlycis. — The cells are oval, and measure 0'5 p in diameter. They occur singly, as diplococci and chains, and produce the ' flacherie ' or ' schlafsucht ' — one of the silkworm diseases. Another disease of the same larva is known as ' pebrine ' ' maladie des corpuscules,' and is caused by a microbe called Micrococcus ovatus, which measures about 1*5 //, in diameter. M. ovatus is pre- sent in large numbers in the blood and organs of affected silkworms. Micrococcus of cattle -plague. — Micrococci have been found in the blood and lymphatic glands of cattle dead of this disease. They occur singly, as chains and zoogloea, and grow rapidly in bouillon and other media at 37° C. Semmer and Archangelski * have shown that calves inoculated from a pure cul- tivation of this microbe died in seven days with all the typical symptoms of cattle-plague or rinderpest. By successive cultivations, or by exposing culti- 1 Centralblatt fiir d. Mod. Wissensch., 1883. I 130 A MANUAL OF BACTERIOLOGY vations for an hour to a temperature of 46° C., the virulence of this microbe is greatly reduced ; and in this attenuated or weakened form it has been used for the protective inoculation of sheep and cattle. Micrococcus of foot-and-mouth disease. — According to Dr. Klein, the microbe of this disease occurs singly, as diplococci, and in curved chains. ' It grows well in milk, in alkaline peptone broth, in nutrient gelatine, and in agar-agar mixture. Grow- ing on solid material, its growth, besides being extremely slow, is very characteristic; it forms a film composed of minute granules or droplets, closely placed side by side, but not confluent. It does not liquefy nutrient gelatine, and in liquids does not form a pellicle, but nevertheless when grown on solids, its growth remains limited to the surface. It does not curdle milk, although it turns the reaction of this latter slightly but distinctly acid.' The microbe has been observed in the vesicles of sheep suffering from the disease. Micrococcus septicus. — The cells are 0*5 //, in dia- meter, and occur singly, as diplococci and chains. Colonies are produced very slowly on nutrient gelatine; they are seen as minute dots on the fourth and fifth days in plate and tube cultivations. They are fatal to mice, rabbits, etc. ; the vessels in the various organs become plugged with these microbes, this ultimately forming purulent or necrotic foci. M. septicus is present in soil. Micrococcus in gangrene. — Small oval micrococci have been found in gangrene of the lungs. They live in colonies, form zoogloaa, and grow on nutrient THE BIO LOO Y OF MICROBES, ETC. 131 gelatine, giving rise to the characteristic but offen- sive gangrenous odour. The same microbes have been observed in various gangrenous tissues, and also in the blood of patients suffering from ' Clou de Biskra ' or ' Bouton d'Alep,' which excite gangrene when injected into rabbits. Micrococcus perniciosus. — According to Wolff1 this microbe is the cause of a disease of the grey parrot. The cells measure 0*8 p in diameter, and occur singly, in chains and zooglcea. They produce nodules in the liver, lungs, spleen, and kidneys; but inflammation around the nodules is entirely absent. The microbes also occur in the blood. The disease is said to be fatal to 80 per cent, of the grey parrots imported into Europe. Micrococcus insectorum. — This microbe occurs in the digestive organs of the chinck-bug (Blissus leucopterus) ; and is probably the cause of an infec- tious disease of this insect. The cells are obtusely oval (0'7 to 1 fj, long x 0'55 /JL broad), and occur singly, in pairs, chains^ or zoogloea. They may be cultivated in bouillon. Micrococcus of Tissue Necrosis in Mice. — Dr. E. Koch observed that a certain micrococcus, isolated from putrid fluids, when injected into the ear of a mouse, gave rise to tissue necrosis and death in about three days. The microbe was not found in the blood and internal organs. The cells measure 0'5 fi in diameter, and occur in chains and zoogloea. Micrococcus in whooping-cough. — Whooping-cough is undoubtedly an infectious disease, and, according 1 Virchow'a Archiv, 1883. 132 A MANUAL OF BACTERIOLOGY to Dr. Burger,1 oval micrococci are often present in the pearly phlegm ejected by patients suffering from this disease. They have not yet been culti- vated. Micrococcus in pernicious anaemia. — According to Frankenhauser2 the blood of pregnant women suffer- ing from pernicious anaemia contains a large number of micrococci which appear to be of a pathogenic character. These micrococci are comparatively of large size, ' about one-tenth of the broad diameter of a red blood-corpuscle.' These microbes have not been cultivated. Micrococcus of Nitric Fermentation. — Mr. E. War- ington, F.K.S.,3 has recently isolated from soil a micrococcus which converts nitrites into nitrates. But this micrococcus, as well as Frankland's nitrous bacillus, will be described later, i.e. under the head- ing of ' the microbes of the soil.' Micrococci in Pyaemia and Septicaemia. — A con- siderable number of micrococci (from O'l to I'O //, in diameter) have been found in various organs, blood, etc., in pyaemia and septicaemia in the lower animals and in man.4 Micrococci have also been described in haemo- philia neonatorium, in ozsena, in acute yellow atrophy of the liver, in closed abscesses, and in many other diseased conditions. 1 Berlin Klin. Woch., 1883. See also the Appendix. 2 Centralblatt fur d. Med. Wissensch, 1883. 3 Journal of Chemical Society, 1891, pp. 484-529. 4 See Dr. Watson Cheyne's papers in the British Medical Journal, Sept. 20, 27, Oct. 4, 1884, and July 31, 1886 ; also Dr. Crookshank's Manual of Bacteriology (2d ed.) THE BIOLOGY OF MICROBES, ETC. 133 BACTERIA. Bacterium termo. — The cells are oblong and measure 1'5 n in length and about 0'5 //, in breadth (Fig. 33, 15). Each cell is surrounded with a thick membrane of cellulose (C6 H10 05) and a flagellum at each end. Dr. W. H. Dallinger, F.K.S., has measured the diameter of the flagellum of this microbe, andjie finds that it is the aoAooth of an inch, or expressed in decimals 0*00000488526 inch.1 B. termo is one of the commonest forms in water and putrefying fluids, but it always disappears when putrefaction terminates, in fact it has been called the microbe of putrefaction. It has remarkable powers of vitality; it is most active between 32° and 36° C. ; at a temperature below 5° C. and above 46° C. it does not produce putrefaction in putrescible fluids ; however, above 50° C. it is killed, but even at so low a temperature as — 1 1 0° C. this microbe is not destroyed.2 It grows well on bouillon, agar-agar, etc., and after several days' incubation a pellicle is formed on the fluid medium. When grown on solid agar-agar it imperfectly liquefies the medium, and gaseous products are generated. On sterilised potatoes B. termo produces a slimy grey colony. It occurs singly, in pairs, and zoogloea, and it is readily obtained by placing a piece of meat in water kept in a warm place for a few hours. It may be remarked that this microbe has been considered to be only a phase-form of a protean species. 1 Journal of Royal Microscopical Society, 1878, p. 174. 2 Giglioli's Fermentive Microbi, p. 50t , „ -^ v. I w « IVZXwITj o»- 134 A MANUAL OF BACTERIOLOGY Bacterium lineola. — This microbe resembles B. termo in form and movement, only it is much longer and thicker than that microbe. Each cell measures from 3 to 5 /A in length and 1*5 //, in breadth, and is provided with two flagella, one at each end of the cell. This microbe occurs singly, in pairs, in zoo- gloea, but never in chains or ros- aries. It is found in well-water and stagnant water, where no distinct putrefaction is go- ing on ; it forms pellicles on steril- ised potatoes and various infusions. <^> — " Bacterium allii. — During the year 1887 the author1 discovered this microbe in the greenish slime of diseased or putre- fying onions and allied plants. It measures from 5 to 7 //, in length and 2 /JL in breadth, and occurs singly, in pairs, and forms zooglcea. It has been named Bacterium allii because it was originally found on Allium cepa (the onion). Bacterium allii grows tolerably well on nutrient agar-agar, and produces a bright green pellicle on 1 Proceedings of Royal Society of Edinburgh, vol. xv. p. 40. FIG. 34. BACTERIUM ALLII. A, Growing on agar-agar. B, The microbe isolated. THE BIOLOGY OF MICROBES, ETC. 135 the surface of the nourishing medium (Fig. 34). The green pigment is soluble in alcohol, and an alcoholic solution gives an absorption spectrum, consisting of a band extending from the extreme violet to the blue part (nearly to the Fraunhofer line F) of the spectrum. There is also an absorption band in the green, and one in the yellow, part of the spectrum. The end of the band in the yellow is exactly in the same position as the D line in the solar spectrum. Bacterium allii forms an alkaloid or ptomaine from albuminoid molecules. This ptomaine has the same chemical composition as hydrocoridine (C10 H17 N).1 Besides the pigment and ptomaine, small quantities of sulphuretted hydrogen gas are liberated from the medium on which the microbe lives. The sulphu- retted hydrogen was proved by the black stain (PbS) produced upon paper impregnated with a solution of lead acetate, and also by the yellow stain (CdS) produced on cadmium paper (CdCl2). B. allii is best stained with gentian violet. The vitality of this microbe is remarkable,2 for it still retains its vitality when exposed, in a dry state, to a tempera- ture of 32° C. for six months. A pure culture of this microbe exposed to —15° C. for three days proved that it was not killed ; but it was killed after fourteen days' exposure at the same tempera- ture. An E.M.F. of 3 -3 volts killed B. allii iii ten minutes. 1 See Dr. Griffiths' papers in Comptes Rendus de I'Acade'mie des Sciences, tome 110, p. 416 ; and Gentralblatt fur Bakte.riologie und Parasitenkunde, Bd. vii. (1890), p. 808. - Proceedings of Royal Society of Edinburgh, vol. xvii. pp. 262-264. 136 A MANUAL OF BACTERIOLOGY The microbe in question is quite distinct from the lacillus (giving a green fluorescence) which Heraeus obtained from soil.1 The bacillus of Heraeus converts urea into ammonia, while Bac- terium allii has no such action, for it decomposes albuminoids (vegetal and animal) with the forma- tion of a ptomaine among other products. Bacterium aceti. — This is the microbe which causes the acetic fermentation according to the well- known reaction : — C2H5OH + 02 = H20 + CH3COOH. It is about 1'5 //, in length, and occurs singly, in long chains, and forms a pellicle on the surface of the nutritive fluid. Although Pasteur maintained that B. aceti was the cause of the acetic fermenta- tion, and Cohn2 observed the microbe largely in sour beers, yet not until the commencement of 1886 could any one say with certainty that this microbe was the real cause of the acetic fermentation. In that year Mr. A. J. Brown 3 prepared pure cultiva- tions of the microbe in question, and found that it does convert alcohol into acetic acid or vinegar. The author4 entirely indorses the correctness of Brown's researches. After obtaining pure cultiva- tions of B. aceti by the fractional and dilution methods, it was found that these cultivations, when used to inoculate sterilised ethyl alcohol (6 per cent.) gave acetic acid in abundance. 1 Zeitschrift fur Hygiene, 1886. 2 Biol. d. Pflanzen, Bd. ii. p. 173. 3 Journal of Chemical Society, 1886, p. 172. 4 Proceedings of Royal Society of Edinburgh, vol. xv. p. 46. THE BIOLOGY OF MICROBES, ETC. 137 Bacterium lactis. — The cells measure from T5 to 3 IJL in length, and are constricted in the centre like the figure 8. They occur singly, in long chains, zoogloea, and like B. aceti, they are motile. Bac- terium lactis is the cause of the lactic fermentation or the souring of milk. The sugar of milk or lactose is converted into lactic acid by the growth of this microbe (Lister *). Unlike B. aceti, this microbe is anaerobic. B. lactis, along with other microbes, plays an important part in the preparation of sauer- kraut ; and Dr. Baginski has recently shown that it produces a powerful reducing action in pure cultiva- tions, where the nutrient fluid was coloured with methylene blue. Bacterium decalvaiis. — This microbe was discovered by Dr. G. Thin 2 in the roots of the hair in cases of Alopecia areata ; and he supposes that it penetrates downwards between the root-sheath and the hair, then passes through the cuticle of the hair, and ultimately ascends within its substance, causing it soon to fall off. It measures 1*6 /*, in length, and occurs usually in pairs. Bacterium cholercegallinarum. — This is the microbe of fowl cholera, and it is found in large numbers in the blood and organs of fowls dead of this disease. It measures 1*2 to 1-5/4 in length, and the ends are always stained more deeply than the middle part. B. cholerce gallinarum is easily cultivated in chicken broth (neutral) at 25° — 35° C., and when fowls are inoculated with a drop of this culture they always i Transactions of the Pathological Society, 1878. Proceedings of Royal Society, vol. xxxiii. p. 247. 138 A MANUAL OF BACTERIOLOGY die with the characteristic symptoms of the disease. If a culture of the microbe is kept for two or three months its virulence is lessened ; and an attenuated virus has been successfully used by Pasteur in the protective inoculation of fowls against this disease. This microbe is pathogenic in rabbits as well as fowls, but guinea-pigs have an immunity; it is aerobic, and is cultivated in contact with sterilised air or in aerated fluids. In fact, ' its toxic effect has been supposed to be due to the abstraction of oxygen from the blood, producing asphyxia.' B. cholerce gallinarum grows on gelatine as small, round, white colonies with lemon-yellow centre. It grows on potatoes at 37°C., producing yellow-grey drops. M. Duclaux1 has shown that this microbe produces a ptomaine; but when the ptomaine is separated, by filtration through porous porcelain from its microbe it does not produce fowl cholera ; for it causes a passing sleep, which does not gener- ally end fatally. From this fact the conclusion may be drawn that in fowl cholera the microbe is essentially the active agent in producing the disease. Bacterium pseudo-pneumonicum. — This microbe forms greyish-white layers in test-tube cultivations ; while on gelatine plates the colonies appear as white dots. It grows on sterilised potatoes at 37° C., giving rise to a white, viscid layer; it measures 1*16 fj, in length, and 0*8 p in breadth, and requires air for its growth. It is only slightly pathogenic. B. pseudo-pneumonicum occurs in pus taken from abscesses. 1 Ferments et Maladies. THE BIOLOGY OF MICROBES, ETC. 139 Bacterium xantMnum. — This bacterium is the cause of 'yellow milk/ which at first is acid, but soon becomes alkaline. The pigment produced by this bacterium is soluble in water, and insoluble in alcohol and ether. B. xanthinum measures from 07 to I'O ft in length, and forms colonies on pota- toes. Bacterium septicus agrigenum. — This microbe occurs in soils, and measures from 2 to 3 /z, in length. On plate cultivations it produces colonies of a greyish- yellow colour, with a yellowish-brown centre. This microbe is fatal to mice, rabbits, and guinea-pigs. It multiplies rapidly in the blood, and it adheres to the red blood corpuscles. Bacteriumcoli commune. — This bacterium measures 1-7 fi in length and 0'4 p in breadth, and it occurs in the faeces of infants fed on human milk. It grows on nutrient gelatine, forming granular colonies of a yellowish colour. It is fatal to rabbits and guinea-pigs, causing violent diarrhoea and fever. Bacterium fcetidum. — This microbe was discovered by Dr. Thin 1 in the alkaline serous exudation from the soles of the feet of a person who suffered from profuse sweating of the feet. It produces a foetid odour, which is also observable in artificial cultures of this microbe. B. fcetidum occurs singly, in pairs, and leptothrix threads. This microbe appears to be identical with Rosenbach's Bacillus saprogenes ; and it is readily cultivated in agar-agar and blood- serum. Bacterium Neapolitanum. — This microbe occurs as 1 Proceedings of Royal Society, vol. xxx. p. 473. 140 A MANUAL OF BACTERIOLOGY short-rods with rounded ends, measuring about 1 //, in length. On nutrient gelatine it forms circular colonies, which, however, become irregular, granular, refractive, and of a yellow colour. It was isolated from certain cases of cholera at Naples ; but it has nothing to do with the disease, for it is only sapro- phytic in man. Bacterium septicum sputigenum. — This microbe is identical with Sternberg's Micrococcus Pasteurianus and Frankel's Bacillus septicus sputigenus. It is present in human saliva, and occurs as short rods, frequently joined together in chains of five, six, or seven links. It is usually obtained from the rusty sputum of pneumonic patients and from severe cases of empysemia. It gro\^s well in bouillon and on agar-agar at 34° C., but slowly on gelatine plates. The colonies are granular and white. This microbe is pathogenic in rabbits, mice, and guinea-pigs, but fowls and dogs have an immunity. Dr. Watson Cheyne l says that it ' apparently loses its virulence when cultivated outside the body. The blood of rabbits which have died of this microbe is very virulent, a small quantity being sufficient to set up the disease in a fresh animal, but when grown in meat-infusion, agar-agar, etc., it rapidly (in three or four days, unless re-inoculated into fresh material) loses its virulence, and the dose necessary to cause death increases as the cultivation becomes older. When it does not cause death it may produce a slight local effect, and such animals are apparently protected from a subsequent inoculation with viru- 1 .British Medical Journal, July 31, 1886. THE BIOLOGY OF MICROBES, ETC. 141 lent material. The animals often do not die for three or four days after the injection, and generally exhibit nervous symptoms, sometimes ending in paraplegia.' Bacterium indicum. — Is an aerial microbe rod- shaped with rounded ends. On nutrient agar-agar it produces a scarlet-coloured growth, but after some days the growth loses its bright colour, and becomes purplish, like an old cultivation of Micrococcus pro- digiosus. On gelatine this microbe liquefies the medium, and colours it scarlet. It also grows well on the surface of potatoes. Bacterium merismopedioides. — Each rod measures from 1 to 1 '5 //, in thickness. It was first observed by Dr. Zopf in the river Panke, Berlin, and is said to divide into long and short rods, and finally into cocci. This microbe also exists in zooglean form.1 Bacterium Zopfii. — This bacterium, which was dis- covered by Kurth, measures from 2 to 5 p in length, and from 0*7 to 1 p in breadth. It is motile, and occurs in long threads. It grows on gelatine-plates, developing into thread-like growths in about thirty- six hours. This microbe was first isolated from the intestine of fowls. Bacterium oxytocum perniciosum. — First isolated from sour milk. Each rod has rounded ends, and forms yellowish colonies on gelatine plates. Needle cultures have the characteristic nail appearance. In milk this microbe produces curdling and an acid reaction. It measures 1 /JL in length ; and in large doses it is pathogenic in rabbits. 1 Zopf, Die Spaltpilze (1885) ; and Die Pilze (1890). 142 A MANUAL OF BACTERIOLOGY Bacterium phosphorescens. — The cells of this mi- crobe are almost circular, being from 1*3 to 1*9 /JL long, and lll to 1*7 p broad; each cell is motile, and surrounded by a gelatinous membrane. It is readily cultivated on fish broth containing a small quantity of peptone ; it grows slowly at the ordinary temperature in peptonised gelatine, or in peptonised gelatine containing 2 per cent, of glucose, but only at the surface, and the property of emitting light depends on the presence of oxygen.1 It also grows well in 2, 3, and 4 per cent, solutions of sea-salt, containing 0'25 per cent, of peptones. On shaking, the phosphorescence becomes intensified, but on cooling to 0° C. its intensity is somewhat dimin- ished. The phosphorescence disappears when the solution is heated to 35° C. for a few minutes, but re-appears on cooling; it is, however, completely destroyed by heating at 35° C. for fifteen minutes. After two or three weeks the culture solutions become yellowish, and gradually lose their phos- phorescence; after several weeks phosphorescence ceases entirely, but the microbes do not die. The phosphorescence is most probably caused by ferment action in the presence of oxygen. This microbe forms colonies on the surface of agar-agar, gelatine, and potatoes, and also grows in urine and milk. Bacterium P/£%m'.— This microbe is the most phosphorescent of all the light-emitting bacteria. It is distinguished from the preceding form by not emitting light with peptone and maltose, but it 1 See Hjelt's General Organic Chemistry (English translation), p. 94. THE BIOLOGY OF MICROBES, ETC. 143 emits light with peptone and glucose. It measures from 1'5 to T9 //, in length, and from 1 -3 to 17 //, in breadth ; these rods have rounded ends, and appear to divide exceedingly rapidly. The bacterium is motionless, and occurs singly, and sometimes in short chains. ' On plates prepared with peptone gelatine, to which a small quantity of glucose, and from 2 to 3 per cent, of common salt have been added, the microbe develops luxuriantly, giving rise to small, white, mother-of-pearl-like colonies, about the size of a pin's head, with no surrounding zone of liquefied gelatine.' This microbe is readily obtained by placing fresh cod or herring (with moist surfaces) between a couple of plates, and kept at about 20° C. for twenty-four or thirty-six hours. At the end of this time small phosphorescent points or dots are seen to glow on the surfaces of the fishes. These dots are colonies of the microbe in question. Bacterium Fischeri. — Unlike the preceding phos- phorescent microbes, B. Fischeri liquefies peptonised gelatine ; and by the addition of a small quantity of sugar the intensity of the phosphorescence is in- creased. The microbe is motile, and occurs singly and in short chains. It grows on agar-agar at a low temperature (from 0° C. to 15° C.). Bacterium Balticum. — Like the preceding microbe, B. Balticum was found in the waters of the Baltic, and also liquefies peptonised gelatine. The four forms of phosphorescent bacteria can- not develop their lighkemitting functions to their highest point without the presence of some substance from which carbon may be easily obtained, such as 144 A MANUAL OF BACTERIOLOGY glycerol, glucose, asparagine, sugar, etc., as well as peptone. For this reason they have been termed pep- tone-carbon-bacteria. Beyerinck,1 who has recently studied these microbes, states that they are best cultivated in fish broth with sea-water, to which are added 1 per cent, of glycerol, 8 per cent, of gelatine, and i per cent, of asparagine. Photo- Bacterium Indicum. — This microbe occurs in the West Indian Sea. It liquefies gelatine very rapidly ; and the greatest intensity of light is given off when the culture is kept between the tempera- tures of 30° and 35° C. Bacterium luminosum. — This microbe, which is most active at about 15° C., is found in the North Sea. Both the preceding and the present microbes give off light in peptonised gelatine without requiring the presence of sugar or any other carbohydrate, conse- quently they have been termed peptone-bacteria. In all these bacteria (phosphorescent) the develop- ment of luminosity is constantly accompanied by the transition of peptones into organised, living matter, under the influence of free oxygen, with or without the concurrence of another compound con- taining carbon. Certain other bacteria, although they do not emit light, are influenced by it, among these are the two following microbes : — Bacterium chlorinum. — The cells are from 2 to 3 fi in length, and are motile. This microbe accumu- lates in the light, but only when oxygen is absent. 1 Transactions of Royal Academy of Sciences of Amsterdam, 1890. THE BIOLOGY OF MICROBES, ETC. 145 Bacterium photometricum. — According to Dr. T. W. Engelmann,1 this microbe is influenced by light ; in fact, its movements are stated to depend on light. It produces a red pigment, but the amount of pigment formed varies with the action of light. Different coloured lights affect this bacterium differently, the most powerful being the ultra-red, the yellow, and part of the green. Bacterium crassum sputigenum. — This microbe was originally isolated from sputum ; it also occurs in the 'fur' scraped from the tongue. It measures 1 /* in length and 0*8 /A in breadth. Colonies on gelatine plates appear as grey, viscid drops, and in needle cultures develop a nail-shaped growth. This mic- robe is fatal to mice, rabbits, and dogs. Bacterium pneumonicum agile. — This is the mic- robe of vagus pneumonia of rabbits. The cells are short thick rods, which occur singly or in chains of three or four. This bacterium forms dark granular colonies on gelatine, which subsequently liquefies. It also grows on blood serum, bouillon, and potatoes. The growth on potatoes spreads very rapidly over the whole surface as a red layer. Pure cultures of this microbe are fatal to rabbits. Bacterium violaceum. — This microbe was dis- covered by Bergonzini,2 and it measures from 2 to 3 p in length, and from 0'6 to 1 p, in breadth. It occurs on egg-albumin, forming a violet pigment. This pigment is insoluble in water, and soluble "in 1 PJluger's Archiv, vol. xxx. p. 95; Revue Internal. Science, tome ix. (1882), p. 469. 2 Ann. Soc. Nat. Moden., vol. xiv. K 146 A MANUAL OF BACTERIOLOGY alcohol and ether. It is said that ether dissolves out a red-violet pigment, and alcohol a deep blue one. Bacterium brunneum. — ' Motile rods, producing a brown colour. They were observed on a rotting infusion of maize.' Proteus vulgaris. — This bacterium is found in abscesses, putrefying organic matter, meconium- fseces, and in water. The rods are from 1'25 to 3*75 /j, in length, and about 0'6 //, in breadth. The threads or chains are usually twisted and convo- luted, and, according to Hauser,1 involution forms are found — spherical bodies about 1*6 //, in diameter. This microbe grows rapidly in nutrient gelatine, causing liquefaction of the gelatine. In test-tube cultivations, the fluid gelatine, which is at first turbid, becomes subsequently more or less clear in the middle, with a' deposit of flocculi at the bottom, and a slight turbidity at the top. Growing on gelatine plates, this microbe rapidly forms greyish masses, which consist of motile and swarming colo- nies. After forty-eight hours' growth a foul odour and an alkaline reaction are developed. This microbe is pathogenic, and produces abscesses or death according to ,the dose or quantity of the microbian culture injected into the animal ; with rabbits and mice inoculation does not cause any effect, but the injection of quantities varying from ^ to T3o cc. causes death. On this point Watson Cheyne 2 states ' that TV cc. injected into the mus- cular tissue was a fatal dose, indeed ¥V cc. almost 1 Ueber Faulniss-Bacterien, 1885. 2 British Medical Journal, July 31, 1886. THE BIOLOGY OF MICROBES, ETC. 147 invariably killed, though some animals survived it : ^ cc. always caused an extensive abscess, of which the animals usually died in six to eight weeks. Doses of less than -g-J-^ cc. did not produce any effect. We have thus three results, according to the dose employed. A small dose (below -g-J^ cc.) pro- duced no effect ; from -^ to -^ cc. caused abscesses, while above ^V cc- caused death in from twenty- four to thirty-six hours. Further, the size of the abscess depended apparently also on the dose, -^ cc. causing only a slight trace of pus, which dis- appears, while TV causes a large and spreading abscess, ultimately resulting in the death of the animal; and the intermediate doses produce inter- mediate effects. On several occasions I have diluted the cultivation considerably, and made plate-cultiva- tions from this diluted material, in order to ascertain the number of bacteria present by counting the number of colonies which developed. The result is that on an average 1 cc. of gelatine cultivations contained 4,500,000,000 bacteria. Thus, doses up to 9,000,000 produced no effect; from 9,000,000 up to 112,500,000 caused abscesses, and above 225,000,000 caused death. It is difficult to under- stand the influence of dose in producing these effects, but the following seems to be a fair supposi- tion. Eabbits are not very susceptible to the action of this bacterium ; in other words, in the struggle for existence between the bacteria and the cells which follows the introduction of this bacterium, the victory will, in most cases, remain with the cells, and the bacteria will disappear. If, however, 148 A MANUAL OF BACTERIOLOGY along with the bacteria, a large quantity of their products (ptomaines) are introduced, these products interfere with the action of the cells, and enable the bacteria to get a foothold. If a large number of bacteria are introduced at one place they grow for a time till attacked by the cells, and each produces a small quantity of poisonous material. Where the number of bacteria is very large this material destroys the tissues in the neighbourhood, and enables the bacteria to spread over a large area before the layer of cells formed around them is able to form a barrier against their progress. The extent to which they spread — in other words, the size of the abscess which results — must, therefore, depend firstly on the number of bacteria and the quantity of products introduced in the first instance ; and, secondly, on the vitality of the animal. It may be that a very large amount of organisms is introduced in the first instance, producing such an amount of poison as to kill the animal in a few hours.' The investigations of Watson Cheyne, as well as those of Hauser, undoubtedly prove that a ptomaine of poisonous properties is formed by these bacteria (Proteus vulgaris), but there can be no doubt, also, that these bacteria themselves are truly pathogenic for rabbits under proper conditions. Watson Cheyne has also shown that when Proteus vulgaris is grown in bouillon it acts with less virulence than when it is grown in nutrient gelatine. Proteus mirabilis. — This bacterium is something like the preceding microbe, although somewhat shorter. It liquefies gelatine much slower than THE BIO LOOT OF MICROBES, ETC. 149 Proteins vulgaris, and forms granular colonies of a brownish colour. It also forms zoogloea. Protem Zenkeri. — This motile bacterium is 1'65 //, in length and 0*4 /j, in breadth. In plate-cultiva- tions it gives rise to greyish colonies, but no zooglcea are formed. There is only a very slight liquefaction of the gelatine, and no odour is given off from cultures on gelatine or blood serum ; but there is a strong smell given off when the microbe is cultivated in bouillon. BACILLI. Bacillus beribericm. — This microbe was discovered by De Lacerda1 in the blood of patients suffering from the disease known as beri-beri, kakke, etc. It occurs singly in long chains and produces spores. When cultivated in bouillon and then injected into rabbits this microbe is said to produce all the symptoms of beri-beri. The disease is characterised by anaemia, anasarca, degeneration of the muscular tissues, numbness, pain and paralysis of the extremi- ties ; and one of its chief habitats is in Japan. It is prevalent in the Malay Archipelago, the Molucca Islands, New Guinea, Burmah, Siam, Ceylon, and India (south and east) ; and it is endemic as well as epidemic in other parts of the world. According to Prof. B. H. Chamberlain,2 'kakke [i.e. beri-beri] is the national scourge of Japan, and attacks with special frequency and virulence 1 Lancet, February 9, 1884, p. 268. 2 Things Japanese, 1890, p. 188 (Kegan Paul, Trench, & Co.). 150 A MANUAL OF BACTERIOLOGY young and otherwise healthy men — women much less often.' De Lacerda believes that the bacillus is derived from rice which has undergone a peculiar alteration. The epidemic spread of this disease is probably influenced by climate, and seems to coincide with conditions of high atmospheric moisture and extreme thermometric variations.1 Bacillus alvei. — This microbe produces the disease known as ' foul- brood ' of bees, and it has been thor- oughly investigated by Cheshire and Cheyne.2 It mea- sures 4 fjL in length and 0 '5/4 in breadth, and the oval spores which it produces measure 2'1 //, in length and 17 JJL in FIG. 85. BACILLUS ALVEI. breadth. JB. alvei . blood and juices of the larvae, drones, workers, and queens, and is 1 For further information see Dr. Felkin's paper in Proceed- ings of Royal Society of Edinburgh, vol. xvi. p. 291 ; Dr. E. Baelz's paper in Mittheil. Deuts. Gesellschaft fur Natur- und Volkerkunde Ostasiens, Bd. iii. p. 301 ; Dr. Anderson's paper in Transactions of Asiatic Society of Japan, vol. vi. ; Dr. Wernich's Geographisch-medicinische Studien ; Dr. Scheube's Die Japan- ische Kak-ke; and the Japanese reports by Drs. Takaki and Miura. 2 Journal of Royal Microscopical Society, 1885, p. 582. THE BIOLOGY OF MICROBES, ETC. 151 also present in the ova. Numbers of this mic- robe are seen moving backwards and forwards in the blood, etc., of larva? attacked with the disease. Leptothrix forms of the microbe are common when the dis- ease is in rapid progress ; these sometimes measure 250 p in length (Fig. 35). In the juices of the larval bee during life these bacilli do not pro- duce spores, although after death spores abound. In test-tube cultivations the bacilli grow both on the surf ace of the gelatine1 and along the needle- track. At the surface the bacilli form a delicate ramifying growth, and along the track whitish irregular - shaped masses appear, which slowly in- crease in size and run together. In a few days processes are seen to shoot out from these masses, which may extend through the gelatine for long distances from the track, being thickened at various parts, and clubbed at the ends. If only a very few bacilli are introduced with the needle, a beautiful and characteristic growth is obtained, for by this i The best growth in gelatine is obtained at about 20° C. FIG. 36. BACILLUS ALVEI. (Cheshire and Cheyne,) 152 A MANUAL OF BACTERIOLOGY means groups of bacilli become planted at a con- siderable distance from each other (Fig. 36). This appearance is quite characteristic of B. alvei, and is not seen in the cultivation of any other microbe. 'The bacilli of anthrax and of mouse septicaemia also spread out from the needle track, but the appearance of their cultivation is quite different. In anthrax delicate threads, not clubbed, shoot out from the track, soon anastomosing with other threads and forming a delicate network throughout the gelatine. In mouse septicaemia the appearance is that of a delicate cloudiness spreading through the gelatine. These ' foul-brood ' bacilli, growing in this material, render it liquid after a time, the liquefaction beginning at the surface and only spreading slowly downwards, but ultimately the whole tube becomes liquid. The liquid becomes yellowish in colour after a time, and gives off an odour of stale, but not ammoniacal, urine. This colour and odour are distinctive of the diseased larvae.' In plate- cultivations, the bacilli grow out in series of rods in single file, or in rows of several side by side. The processes which are formed have a tendency to form curves and circles. Later on, the gelatine in the vicinity of the bacilli becoming liquid, forms a series of channels in which the bacilli move backwards and forwards. They grow most rapidly on the surface of nutrient agar-agar, forming a whitish layer, but the ramify- ing processes seen on the surface of gelatine do not occur, or only very imperfectly, in agar-agar. THE BIOLOGY OF MICROBES, ETC. 153 Here the bacilli arrange themselves apparently side by side, and producing spores in this position, we have as a result, after a few days' cultivation, long rows of spores lying side by side, with here and there an adult bacillus. In uiilk they grow well at the body temperature, and in a few days cause coagulation of the milk ; and on potatoes they form a dryish yellow layer. These bacilli also grow in blood serum and in bouillon. FIG. 37. BACILLUS ALVEI. (Cheshire and Cheyne.) A, Passage of spore into bacillus condition. B, Passage of bacillus into spore condition. B. alvei does not grow below 16° C. ; but it grows most rapidly in cultivating media kept at the body temperature. Cheshire and Cheyne sprayed a cultivation of the bacillus in milk over a honey- comb containing a healthy brood of larval bees, and succeeded in reproducing the disease known as ' foul-brood.' They also succeeded in infecting adult 154 A MANUAL OF BACTERIOLOGY bees by feeding them with material containing these bacilli. This microbe is best stained with methyl violet ; but the spores resemble the spores of other microbes in not taking on the stain. Fig. 37 represents the passage of a spore into the bacillus condition, and vice versd. Bacillus of Grouse Disease. — Dr. Klein 1 has re- cently proved the microbian nature of grouse disease. The disease, which is infectious, is caused by a bacillus measuring T6 x 0'6 //,. It grows well on agar-agar at 36° to 37° C.2 ; also on nutrient gelatine and in alkaline bouillon. Klein proved the patho- genic nature of the microbe by a series of inocula- tion experiments. The bacillus is readily stained by Weigert's method. Bacillus suUilis. — The hay-fever microbe was originally isolated from an infusion of hay. It measures 6 x 2 //,, and has slightly rounded ends. This bacillus occurs singly, in short chains, in lep- tothrix filaments, and in zooglcea. It forms oval spores (I'2x0'6 //) ; but spore-formation occurs only when there is an ample supply of air ; never- theless it is independent of any deficiency of nourishing material (Klein). The bacilli when single possess one or two flagella (Fig. 33, 10). ' The bacilli form a dense resistant pellicle on the surface of the nourishing medium, and in this copious spore-formation takes place. If shaken 1 Gentralblatt fur Bakteriologie und Parastienkunde, Bd. vi. pp. 36 and 593 ; Bd. vii. p. 82 ; and Bd. ix. p. 47. 2 That is, in from two to four days. THE BIOLOGY OF MICROBES, ETC. 155 when growing in a fluid the pellicle falls to the bottom, and soon a new pellicle is formed.' This microbe may be readily obtained by exposing a previously sterilised infusion of hay to the atmo- sphere for a short time : the spores being always present in the air. On plate-cultivations, white rounded colonies formed, which frequently give rise to radiating processes. On potatoes and agar-agar B. subtilis forms a moist, cream-coloured layer, which ultimately becomes granular and dry. It grows on blood serum and nutrient gelatine, both of which it liquefies. B. subtilis is a motile microbe, and is best cultivated at a temperature of about 30° C. This microbe can withstand a temperature of — 1 8° C. ; x and its spores have been proved to have a remarkable power of resisting the influence of high degrees of heat. For instance, a short ex- posure to 100° C. does not destroy the vitality of the spores. However, an E.M.F. of 2'72 volts destroys both the spores and bacilli.^ The action of ozone 3 on both the spores and bacilli is that they are com- pletely destroyed ; this fact explains the absence of this and other microbes in the air at sea — the latter containing an appreciable amount of ozone. Bacillus ethaceticus. — This small bacillus (1*5 to 5'1 x 0*8 to 1*0 /A) was discovered by Dr. P. F. Franklaud, F.E.S.,4 and has the power of decompos- 1 Griffiths in Proceedings of Royal Society of Edinburgh, vol. xvii. p. 263. a Griffiths, ibid., vol. xv. p. 45. 3 Griffiths' Researches on Micro -Organisms, p. 184. 4 Proceedings of Royal Society of London, vol. xlvi. p. 345. 156 A MANUAL OF BACTERIOLOGY ing solutions of mannite, glucose, sucrose, lactose, starch, glycerol, and calcium glycerate. It has no fermentive action on dulcite, the isomer of mannite, which thus furnishes a very striking instance of the selective power of microbes between the most closely allied isomeric bodies. The products of the fer- mentation of the above-mentioned compounds are essentially alcohol and acetic acid, with a small and variable proportion of formic acid, together with a trace of succinic acid. Frankland l represents the decomposition of glyceric acid (calcium glycerate) by this microbe as follows : 5C3H604 = C2H5OH + 4CH3COOH + H20 + 3H2 + 5C02. The alcohol and acetic acid are produced approxi- mately in the proportion of one molecule of alcohol to four molecules of acetic acid. Bacillus lutyricus. — This is the microbe of the butyric fermentation ; and it is found in the cells of laticiferous plants, in milk, and in decaying-plant infusions, etc. B. butyricus is morphologically like B. subtilis, but distinguished by the fact that at certain times it contains starch in its cells. It measures from 3 to 10 //, in length and 1 p in breadth : it frequently forms chains, and gives rise to well-developed spores. When spore-formation is about to take place the protoplasm of the cell becomes granular, and at certain points gives rise to oval spores. This microbe grows on gelatine-plates, in the deeper layers of the medium, as yellow or brown colonies of a granular appearance ; and ulti- i Journal of Chemical Society, 1891, p. 81. THE BIOLOGY OF MICROBES, ETC. 157 mately the gelatine is liquefied. On agar-agar it forms a viscid yellow layer ; while in test-tube cultivations it liquefies the gelatine which becomes cloudy. B. butyricus grows best between 35° and 40° C. It is the cause of the rancidity of butter and the ripening of cheese. It decomposes cellulose, and hence it is of great ' importance in the digestive process of herbivorous animals, in whose stomachs and intestines it is very common/ Bacillus ulna. — This species is closely allied to B. subtilis. It measures 10 X 2 /JL; and occurs singly, in chains, but it does not form leptothrix. It gives rise to spores which measure 2 '8 p x 1 p» This microbe is found in rotting eggs. On the surface of bouillon it forms thick colonies which ulti- mately unite, giving rise to a pellicle. It is readily cultivated on sterilised egg-albumin. Bacillus of Symptomatic Anthrax. — This microbe is the cause of the infectious disease known as quarter-evil, rauschbrand, charbon symptomatique, etc. The disease affects cattle, giving rise to the formation of an irregular tumour in the subcutaneous and intermuscular tissues. There is high fever, and death generally occurs in about forty-eight hours. This motile microbe (3 to 5 p x 0'5 to 0*6 //,) is found in the serous fluids, bile, tumours in this disease. It has been cultivated in fowl broth to which small quantities of glycerol and ferrous sul- phate have been added. It also grows on blood serum, nutrient gelatine, and vegetable albumin. As the microbe is anaerobic, it must be cultivated in an atmosphere devoid of free oxygen. It is best 158 A MANUAL OF BACTERIOLOGY cultivated at the temperature of the body. Spore- formation takes place at the ends of the cells. MM. Arloing, Cornevin, and Thomas l have shown that the virus is capable of giving immunity to animals inoculated with it. The following are the chief facts observed by them : (a.) Injection of a very small quantity of the virus into the loose con- nective tissue of any part of the body produces a temporary illness, and protects the animals, (b.) Injection of a moderate quantity into the scanty connective tissue of the tail produces a slight affec- tion, and confers immunity. Very large doses, however, may cause death. A moderate quantity injected into the cellular tissue in other parts of the body causes death, (c.) Injection into the veins does not kill, but confers immunity, and the same result follows injection into the respiratory tract. (d.) Cultivation does not deprive the microbe of its virulence, but heating the spores to 85° C. for six hours destroys their virulence. On page 117 of their book (loc. cit.), MM. Arloing, Cornevin, and Thomas state that the following sub- stances destroy or do not destroy the virulence of this microbe : — Do not destroy the virulence. Destroy the virulence. Alcohol (90 %). Glycerol. Sulphate of quinine (10 %)• Hydrogen peroxide. Sodium hyposulphite. Ammonia. Tannic acid (20 %). Salicylic acid (O'l %). Carbolic acid (2 %). Boric acid (20 %). Sodium salicylate (20 %). Potassium permanganate (5 °/0). Mercuric chloride (01 %). Silver nitrate (O'l %). Du Charbon Bacttrien (1883). THE BIOLOGY OF MICROBES, ETC. 159 Bacillus ianthinus. — This motile microbe was first found in water, and differs from B. violaceus (also found in water) by not liquefying gelatine. It occurs singly, and in threads. On nutrient gelatine, agar-agar, and potatoes it produces white spots, which rapidly become violet. The pigment, which is soluble in alcohol, is only developed in the pre- sence of air. Bacillus violaceus. — This bacillus is also found in water. It grows as small round colonies on gelatine plates. These are first white, but rapidly assume a violet colour. It also grows on agar-agar, blood serum, and potatoes ; giving rise, on each of these media, to the same pigment. The microbe is a motile rod about four times as long as broad, with rounded ends, and often contains spores. Bacillus cyanogenus. — This microbe measures 2'5 to 3'5 p x 0'4 IJL ; and occurs in chains and zooglcea. Spore-formation is also present. In test-tube culti- vations, it gives rise to a white head, while the surrounding gelatine becomes blue or dark brown. In alkaline milk, it gives rise to a slate-coloured pigment; while in acid or sour milk, a beautiful blue pigment is developed (in fact, it is called ' the microbe of blue milk'). On agar-agar, it forms a brown pigment. It also grows on potatoes, boiled rice, starch-paste, etc. ; and the colouring matter which is formed varies with the nourishing medium. These pigments are freely developed at from 15° to 18° C., but at 37° C. no colour is formed at all. Bacillus erythrospwus. — This bacillus was found 160 A MANUAL OF BACTERIOLOGY in putrefying albuminous fluids, potable water, etc. It occurs singly and as leptothrix. On gelatine- plates, white colonies are formed. The outer zones of these colonies are of a yellowish-green colour. On potatoes, this microbe forms brown patches which do not spread. It produces dirty-red spores. Bacillus cedematis maligni. — This microbe, ob- tained from soil, is a pathogenic microbe. Mice, rats, cats, etc., inoculated with a pure cultivation of this bacillus, die in a few hours. It measures from 3 to 5 fji x 1 //,, and has rounded ends. It occurs singly, in chains, and leptothrix (straight or curved) ; spores are formed ; and the microbe is anaerobic. It grows well on the surface of a neutral solution of Liebig's extract of meat at 36° to 38° C., or in nutrient agar-agar ; but air must be excluded from the cultivation tubes or flasks.1 For some recent work concerning the microbe of malignant cedema, see Dr. Klein's paper in Centralblatt fur Balderiologie iind ParasitenJcunde, Band x. (1891), p. 186. Bacillus of rhinoscleroma. — A microbe found in the tissues of patients suffering from rhinoscleroma — a disease which gives rises to tumours on the lips, and nasal and pharyngo-laryngeal regions. The bacillus measures from 1*5 to 3 //, x 0*5 to 0'8 //, ; it has rounded ends, produces spores, and sur- rounds itself with an elongated capsule (Fig. 33, 11). This microbe is readily stained with a solution of methyl violet. Bacillus of Indigo Fermentation. — This microbe is morphologically similar to the bacillus of rhino- 1 See Dr. Griffiths' Researches on Micro- Organisms, p. 235. THE BIOLOGY OF MICROBES, ETC. 161 scleroma ; and it has been proved by Alvarez 1 to be the cause of the indigo fermentation and the pro- duction of indigo-blue. Indigo-blue or indigotin is the product of several plants belonging to the Indigofera and other genera. It does not exist in these plants ready - formed, but is produced by the decomposition of a glucoside (C26H31N017) called indican. By the action of Alvarez's bacillus, indican yields indigo-blue (C8H5NO) and indiglucin (C6H1006):- ,, + 2 H20 = C8H6NO + 3 C6H1006. The bacillus of indigo fermentation has been shown to possess pathogenic properties, and oc- casions in animals a transient local inflammation, or death, with visceral congestion and fibrinous exudations. Bacillus pyocyaneus. — This microbe is a very minute, short, thin rod ; and it is said to produce spores. It occurs in chains of twos or threes, or collected into irregular masses ; and it has been isolated from pus of those cases in which the wounds exhibit a greenish-blue colour. According to Dr. Gessard,2 B. pyocyaneus produces a greenish pigment of a definite composition, which has been called ' pyocyanin.' Pyocyanin can be extracted from pus by means of chloroform. Dr. J. Kunz 3 has grown this microbe on nutrient gelatine kept for three or four days at the ordinary temperature, and then for 1 Comptes Rendus de I' Academic des Sciences, tome 105. 2 De la Pyocyanine et de son Microbe, 1882. 3 Monatsheftefur Chemie, Bd. ix. p. 361. T 162 A MANUAL <_.'/• Lt seven days at 35° C. It liquefies the gelatine, which shows a green fluorescence, and has the specific smell of blue pus. Kunz extracted from the lique- fied gelatine pyocyanin and pyoxanthose, but the liquid still showed a green fluorescence due to a distinct colouring matter, which is only soluble in water and alcohol, and is not destroyed by boiling. Concentrated solutions of this colouring matter transmit red and green light only, but dilute solu- tions have no absorptive power. According to Kunz, pyocyanin contains nitrogen and sulphur. The green pigment which is formed when this bacillus is grown on nutrient gelatine is most pro- bably produced by the oxidizing action of the air on a chromogen which is formed by the bacillus, as the pigment is not contained in the bacillary cells. In gelatine solutions, the green colour disappears gradually at the ordinary temperature in ten or fifteen weeks, giving place to a dark reddish-brown colour, and the reaction becomes strongly alkaline. B. pyocyaneus grows in milk, and produces a yellowish-green solution, which becomes intensely green when ammonia is added. The chemistry of the microbian pigments is a subject which has been very little investigated ; but these pigments are undoubtedly products formed from the decomposition of albuminoids by the agency of microbes. Bacillus septiccemice (rabbit). — This microbe, which is pointed at both ends, measures T4 x 0*7 p. It occurs singly and in chains; and it grows in bouillon, nutrient gelatine, and blood serum. On gelatine- THE BIOLOGY OF MICROBES, ETC. 163 plates, it produces 'dot-like colonies, and in test- tubes little spherical masses in the needle track, and a layer on the free surface/ This bacillus was isolated by Koch from putrid meat infusion and river-water. It is innocuous to guinea-pigs and white rats ; but rabbits, mice, and birds are very susceptible to the attacks of this microbe. Bacillus septiccemice (mouse). — This non-motile bacillus was isolated from garden soil and putrefy- ing fluids. It measures 1 x O'l /*, and occurs singly, in pairs, and chains of four or more. It grows on gelatine-plates, in the deeper layers of the medium, as delicate white clouds. In test-tube cultivations, it produces delicate branching growths along the track of the needle. On agar-agar, lemon- yellow colonies are formed. This bacillus kills house-mice in forty to sixty hours; but field-mice have an immunity. Bacillus septiccemice (man). — In human septicaemia, Klein x found in the blood-vessels of the lymphatic glands certain bacilli which form continuous masses in the capillaries and small veins. These bacilli measure 1 to 2*5 //, x 0*3 to 0*5 JJL, and occur singly or in short chains. Bacillus diphtlierice vitulorum. — This microbe measures 2 -5 to 3' 6 x 0*5 /&, and was described by Loffler as occurring in the diphtheria of calves. Mice inoculated from a calf died with all the characteristic symptoms of the disease. The microbe has not been artificially cultivated. Bacillus diphtheria columbarum. — This bacillus 1 Micro-Organisms and Disease, p. 120 (3d ed.). 164 A MANUAL OF BACTERIOLOGY was isolated from the false membrane of the diphtheria of pigeons. It is a short rod with rounded ends, and occurs in irregular masses. On the surface of gelatine, it forms light yellow films, while in the deeper layers of that medium white nodules are formed. This bacillus destroys pigeons, sparrows, mice, and rabbits ; but fowls, guinea-pigs, rats, and dogs have an immunity. Bacillus of Diphtheria of Rabbits. — This microbe measures 3 to 4 yu, X 1 /-t ; it has rounded ends, and occurs singly, in pairs, or in long chains. On gelatine-plates it forms grey colonies, which become brown. It was isolated during growth in 'the diphtheritic processes of the intestine ; ' and causes (in rabbits) an inflammatory exudation in the alimentary canal. Bacillus cavicida. — This microbe was discovered, by Dr. Brieger, in faeces and putrefying fluids. The rods are very small, and they form colonies com- posed of white concentric rings on gelatine plates. On potatoes they give rise to dirty yellow masses. They are fatal to guinea-pigs, but not to mice and rabbits. Bacillus pyogenes fatidus. — A microbe with rounded ends, and measuring 1*45 X 0'58 //,, was isolated from putrid pus. It occurs in pairs or chains, and is motile and produces spores. On the surface of gelatine and agar-agar it forms greyish films, and on potatoes a shining brown growth is developed. From all these media a strong putrid odour emanates, but no smell is given off when the microbe is cultivated in milk. It is fatal to mice and guinea-pigs. THE BIOLOGY OF MICROBES, ETC. 165 Bacillus of Swine Erysipelas. — This bacillus has been obtained from the blood of pigs which have died of the disease. It measures 1*1 //, x 0*2 p. In test-tube cultivations it produces a cloudy growth in the track of the needle. It is fatal to mice, pigeons, and rabbits, as well as pigs. Bacillus of Ulcerative Stomatitis in the Calf. — Drs. A. Lingard and E. Batt1 discovered certain bacilli in ulcerations on the tongue and mucous membrane of the mouth of calves (Fig. 33, 13). They measure 4 to 8 p, x 1 /i, and occur singly and as leptothrix forms, the filaments of which are either straight or more or less curved. They con- tain spores ; and when injected into a mouse or rabbit they produce a fatal result. These bacilli are best stained by immersion in a mixture of methylene blue and magenta. Bacillus of Swine Plague. — This microbe2 measures 2 to 3 //, in length, and produces spores. It was observed in the organs of pigs which had died of swine fever, or swine plague. It is readily culti- vated in broth and hydrocele fluid at temperatures ranging between 30° and 42° C. A drop of either of these cultures inoculated into pigs, rabbits, and mice produce the disease, with multiplication of the bacilli ; and ' the animals die with a character- istic swelling to the spleen, coagulative necrosis of tracts of the liver tissue, and inflammation of the lungs.' Pigs inoculated with artificial cultures of the microbe are protected against a fatal attack. 1 Lancet, 1883. 2 See Klein's Micro -Organisms and Disease, pp. 131-136. 166 A MANUAL OF BACTERIOLOGY Bacillus putrificus coli. — It was first isolated from faeces, and measures about 3 //, in length. On gela- tine it has an opalescent appearance, but finally becomes a yellow colour. It is motile microbe, which occurs in long or short threads. Spore- formation has been observed. Bacillus epidermidis. — It was discovered in the fragments of epidermis taken from between the toes- This microbe measures from 2 '8 to 3 yu, in length, and 0*3 //, in breadth; it forms spores from 1*2 to 1'5 p in length, and 0'3 to 0'4 p in breadth. It grows only sparsely on nutrient gelatine and agar- agar. On potatoes it forms a characteristic super- ficial skin. Bacillus of Nitrous Fermentation. — Dr. P. F. Frankland1 has recently isolated from soil a bacillus which converts ammonia into nitrites. This mic- robe will be described under the heading of ' the microbes of the soil/ Bacillus megaterium. — This microbe was dis- covered by the late Dr. De Bary on boiled cabbage. The rods are motile, and measure 10 p, x 2 -5 p. They occur singly and in chains, and grow on gela- tine and agar-agar, forming a whitish layer. On potatoes at 20° C. yellowish-white dots are formed. B. megaterium is an aerobic microbe, and produces spores. Leptothrix luccalis. — This microbe occurs in the slime of the teeth, on the epithelium of the mouth, etc. j in other words, it is one of the microbes of the mouth. It occurs as isolated bacilli or threads, 1 Philosophical Transactions of the Royal Society, 1890, p. 107. THE BIOLOGY OF MICROBES, ETC. 167 generally arranged in bundles (Fig. 38), which may be interwoven with one another. Each thread is divided into short rods, from 1 to 1'2 //, broad, and from 2 to 10 //, long. This microbe is believed to be connected with dental caries. Leptothrix innominata. — This microbe is usually found on the soft white matter which is deposited on the teeth. The threads are from 0'5 to 0*8 p in breadth. Leptothrix parasitica. — The threads are slender, not articulated, loosely felted, and for the most part curled. They measure from 100 to HO /* in length, and about 1 ^ in breadth, and occur both in still and running water. This bacillus (Fig. 33, 22) is best cultivated on in- fusions of rotting algae and animal substances. This microbe is believed by Plo. 38. LEPTOTHBIX BucCAUS. Zopf and others to give rise to micrococci, bacteria, etc. ; in other words, it is a pleornorphic form, but Zopf s observations were not made after exact methods. Beggiatoa roseo-persicina. — This is the ' peach- coloured bacterium ' of Ray Lankester,1 and is really a sulpho-chromogenic bacillus. It occurs 'on the surface of marshes, or on water in which algae are rotting, and sometimes these bacilli are in such 1 Quarterly Journal of Microscopical Science, vol. xiii. p. 408. 168 A MANUAL OF BACTERIOLOGY quantity that the whole marshes and ponds may be coloured blood-red by them/ B. roseo-persicina con- tains dark-coloured sulphur granules, the dark colour being due to the pigment — bacterio-purpurin — formed by the microbe. This pigment is in- soluble in water, alcohol, etc., and when examined spectroscopically it shows a strong absorption band in the yellow, a weaker band in the green and blue, and a darkening in the more refrangible half of the spectrum. Beggiatoa alba. — This bacillus occurs as threads without distinct articulations. The threads are longer and thicker than leptothrix, and they are found in marshes and sulphur springs. The cells (about 3*5 Abroad) of B.alba contain sulphur granules (Fig. 33, 20), and, according to Cohn and Cramer,1 these granules consist of crystalline sulphur, which is highly refractive. When these crystalline granules are disintegrated and examined microscopically, they are seen to be composed of a number of rhombic (octahedral) crystals. A variety (B. alba marina) of this microbe forms a delicate white gela- tinous membrane on decaying animals and algae in a marine aquarium. Beggiatoa nivea. — The threads of this bacillus are very slender, indistinctly jointed, and form undu- lated woolly tufts of milky-white colour. B. nivea occurs in sulphur springs. Beggiatoa miralilis. — The microbe occurs in sea- water, forming a white gelatinous scum on decom- posing algae, etc. The threads are very thick, 1 Beitrage zur Biologie der Pfltinzen, vol. i. THE BIOLOGY OF MICROBES, ETC. 169 motile, bent and curled in various ways, and they have rounded ends. They are distinctly articulated (16 //, broad), and contain sulphur granules. Besides the four last-mentioned microbes there are B. leptomitiformisy B. arachnoidea, and B. pel- lucida, each of which contains sulphur granules. These microbes play an important part in the elimi- nation of sulphur and the disengagement of sul- phuretted hydrogen. The sulphogenic or sulphur- forming microbes are found in certain waters, and many of the natural sulphurous waters are due to the action of these microbes on alkaline sulphates and organic matter present in such water. The decomposition of calcium sulphate by sulphogenic microbes may be represented by the following equations : — (a) 3 CaS04 + H20 = S2 + H2S + 3 CaO + 5 02. (ft) 2 CaS04 = S2 -|- 2 CaO + 3 02. Sulphogenic microbes are also capable of decom- posing animal and vegetal albumin with the libera- tion of sulphur. Bacillus septicus. — This microbe occurs in soil, putrid blood, and other fluids. Its breadth varies from 4 to 10 /i, and its length depends on the number of elements contained in a row : the shortest are about 4 /z. It is a non-motile bacillus, capable of forming leptothrix and spores. Bacillus of conjunctivitis. — This bacillus was obtained from the conjunctival sac in cases of con- junctivitis. It grows on agar-agar plates as pearly dots, and in bouillon. The latter medium is the 170 A MANUAL OF BACTERIOLOGY best for the cultivation of this microbe. It measures from 1 to 2 p in length, and 0*25 //, in breadth. Bacillus figurans. — This microbe was first de- scribed by Crookshank,1 and occurs in soil and in the atmosphere. It has rounded ends, and forms spores. On the oblique surface of agar-agar it forms a feather-like growth. On gelatine plates B. figurans causes ' a cloudy growth, spreading from various points/ When ' cultivated in nutrient gela- tine this bacillus forms on the surface visible wind- ings, from which fine filaments grow down into the gelatine. They spread out also in almost parallel lines transversely from the needle track.' Bacillus Hansenii. — The rods measure 2 '8 to 6 p x 0'6 to 8 //,, and are best cultivated on steamed potatoes, where they form a deep yellow layer, which has the odour of amylic alcohol. Ultimately the yellow layer dries, and changes to a brown colour, at the same time forming spores (1*7 JJLX 1-1 /*,). This bacillus occurs on bouillon, wine, and malt infusions, which have been kept at about 32° C. VIBRIONES. These microbes are rod-shaped, but not straight ; they are more or less wavy, and they are motile. Vibrio serpens. — This vibrio measures from 1 1 to 25 fju long, and from 0'8 to M //, in breadth. It occurs in various infusions. Vibrio rugula. — The rods measure from 6 to 16 yu, in length, and about 0*5 to 2*5 //, in breadth. They are curved or S-shaped, and bear a flagellum at each 1 Lancet, 1885. THE BIOLOGY OF MICROBES, ETC. 171 end. They swarm when causing decomposition in vegetable infusions. According to Prazmowski, Vibrio rugula develops a spore at one end of the cell. SPIRILLA. Spirillum tyrogenum. — This spirillum measures about 0*8 to 1*5 p, in length. On gelatine plates (see Fig. 24) it forms colonies of a greenish-brown colour. In test-tubes the gelatine becomes liquid along the needle-track, while on agar-agar a pale yellow layer develops. This microbe, which is non-pathogenic, was isolated, by Deneke, from old cheese. S. tyrogenum is capable of withstanding a temperature of — 18° C. for several days.1 Spirillum Finkleri. — The rods are curved, and they are larger and thicker than the Spirillum cholera Asiaticce. On gelatine-plates they grow rapidly, forming small white dots with a brownish tinge; and the gelatine is liquefied very rapidly. The fluid (from the liquefaction) becomes completely turbid, whereas in S. cholerce Asiaticce the upper part remains clear. In gelatine tube cultivations, liquefaction occurs in the form of a funnel-shaped tube, and the fluid becomes turbid. On agar-agar and potatoes white films or layers are formed. S. Finkleri was discovered in the dejecta of cases of cholera nostras, and it was said to be identical with the Spirillum of Asiatic cholera; but it is quite distinct. • 1 Griffiths in Proc. Roy. Soc., Edinb., vol. xvii. p. 263 ; and Researches on Micro-Organisms, p. 176. 172 A MANUAL OF BACTERIOLOGY Spirillum Obermeieri. — This microbe is the cause of relapsing fever, and was first discovered by Ober- meier 1 in the blood of patients suffering from the disease. Carter 2 reproduced the disease in monkeys, in whose blood and organs the spirilla were found in great numbers. This microbe (16 to 40 p long), which is motile, exhibits spiral forms, and, according to Albrecht,3 produces spores. 8. Olermeieri (Fig. 33, 7) has been artificially cultivated by Koch.4 The microbe only occurs during the relapses, and is absent during the non-febrile intervals. Spirillum tenue. — This spirillum measures from 4 to 15 ^ in length, and about 2'25 p in breadth. It usually occurs in various infusions, in which it moves about with great rapidity. It occurs in swarms or united in a zoogloea. Spirillum undula. — It measures from 8 to 1 2 yu- in length, and from 1-1 to 1*4 //, in breadth (Fig. 33, 5). There is a flagellum at each end, and the microbe is actively motile ; although at times it forms a zoogloea. It occurs in bog- water and various infusions. Spirillum wlutans. — This microbe occurs in marsh water and various infusions. It measures from 20 to 30 fj, in length, and 1-5 to 2 /*, in breadth (Fig. 33, 6). The protoplasm contains a number of dark granules, and there is a flagellum at each end. Spirillum sanguineum. — This was observed by 1 Centralblattfur Med. Wissensch., 1873. 2 Lancet, vol. i. p. 84, and p. 662. 3 St. Petersb. Med. Woch., 1879. 4 Deutsche Med. Woch., vol. xix. THE BIOLOO Y OF MICROBES^ ETC. 173 Cohu and Warming in pond-water. It is said to be morphologically identical with Spirillum volutans. The cells contain numerous red bodies and many sulphur granules. According to Saville Kent,1 this microbe is not identical with Ehrenberg's Ophido- monas sanguinea : the latter being a true monad. Spirillum concentricum. — This microbe was dis- covered by Kitasato in putrefactive blood. It grows rapidly on gelatine-plates, giving rise to greyish- white round colonies, each of which has concentric markings. It does not liquefy the gelatine, and is non-pathogenic. Besides the above-mentioned spirilla, there are the following, which occur in brackish and sea water : S. violaceum, S. Rosenbergii, S. attenuatum, etc. ; but the reader is referred to the works of Warming for an account of these microbes. SPIROCILET.E. Spirocliceta plicatilis. — This microbe is of extra- ordinary length— 110 to 225 p (Fig. 33, 19). It occurs in stagnant water. The threads are arranged in wavy lines. Spirochceta gigantea. — The threads are blunt at both ends. It occurs in sea-water. YEAST-FUNGI. These fungi are not microbes (i.e. they are not Schizomycetes), but belong to an altogether different order — the Saccharomycetes. They multiply chiefly Manual of the Infusoria, p. 244. 174 A MANUAL OF BACTERIOLOGY by gemmation or budding, but they can also produce spores, especially when they are deprived of nourish- ment. These organisms occur widely distributed in air, soil, and water, and they are the cause of the alcoholic fermentation. Saccharomyces cerevisice. — This organism is some- times termed Torula cerevisice, and is the true fer- ment of beers. The cells are round or oval (8 to 0 fj, long), and are either isolated or united in small colonies. The spore-forming cells (when isolated) measure from 11 to 1 4 //, long ; and the spores mea- sure from 4 to 5 fi in diameter. This organism occurs in beers brewed by both the high or low systems of fermentation. Fig. 39, 1 and 2, represent the beer- ferments. There are two races of this species, high (1) and low (2) yeasts. The cells of high yeast are slightly larger and more round than those of low yeast. Low yeast never rises to the surface of the fermenting wort, which is kept at a temperature varying from 4 to 5° C. This low fermentation is a slow process, occupying about fourteen days. The low fermenta- tion gives rise to ' Lager ' or ' Bavarian ' beer. High yeast rises to the surface as the fermentation pro- ceeds, and the wort is kept at a temperature varying from 16° to 20° C. The fermentation is rapid, and rarely occupies more than a few hours or so. The high and low yeasts are not different species. Both high and low yeasts secrete a soluble enzyme which converts maltose and sucrose into invert sugars (dextrose and levulose) : — CI2H22On + H20 = C6HI206 + C6H1206. [maltose] [dextrose] [levulose] THE BIOLOGY OF MICROBES, ETC. 175 Saccharomyces minor. — This organism (Fig. 39, 3) consists of a spherical cell measuring 6 p in diameter. It occurs in chains of six or nine cells. The spore- forming cells each measure from 7 to 8*5 p, in dia- meter, and contain from 2 to 4 spores, each hav- ing a diameter of 3 -5 /^. Hansen and En gel state 3 n$ ® O $ ^ o^ ^ o o 00 Q O Cb m FIG. 39. YEAST-FUNGI. that this yeast is the cause of fermentation in bread. Saccharomyces ellipsoideus. — The cells are ellipti- cal (Fig. 39, 4), mostly 6 //, long, and are isolated or united in little branched colonies. Two to four 176 A MANUAL OF BACTERIOLOGY spores are found in a mother cell. It is a low yeast when grown in beer wort ; but it is really a species of wine ferment, which produces the spontaneous fermentation in must. Saccharomyces conglomeratic. — The cells are al- most round (Fig. 39, 5), measuring from 5 to 6 ^ in diameter, and united in clusters. This organism occurs in wine at the beginning of the fermentation, and on decaying grapes. Saccharomyces exiguus. — The cells are conical (5 fju x 2 -5 //,), and are united in slightly branched colonies (Fig. 39, 6). Spore-forming cells each contain from two to three spores, which lie in a row. This organism occurs in the after-fermenta- tion of beer. Saccharomyces Pastorianus. — The cells are oval or elongated (Fig. 39, 7). 'The colonies consist of primary club-shaped links (18 to 22 //, long), which build lateral, secondary, round or oval daughter-cells (5 to 6 //, long).' The spores number from two to four. This organism occurs in the after-fermentation of wine, fruit-wines, and fer- menting beer. It is very common in the air. Saccharomyces apiculatus. — The cells are lemon- shaped (Fig. 39, 8) and from 6 to 8 yu, long x from 2 to 3 fju broad, and sometimes slightly elongated. Gemmation occurs only at the pointed ends. Spore formation is unknown. It occurs in fermented wine, in spontaneous fermentations of all kinds of fruits, and in certain kinds of beer. It is a low yeast, giving rise to a feeble alcoholic fermentation, and it does not invert sucrose. When mixed with S. THE BIOLOGY OF MICROBES, ETC. 177 cerivisice it retards the action of the true beer ferment.1 Saccharomyces mycoderma. — The cells are oval, elliptical, or cylindrical (Fig. 39, 9), measuring about 7 fi long and about 2 //, thick. They are united in richly-branched colonies ; and the cells are often elongated, so as to resemble a hyphal filament. This organism forms the scum on the surface of beer, wine, sauerkraut, and fruit-juices. It has nothing to do with the alcoholic fermenta- tion; and is not identical with Bacterium aceti (Mycoderma aceti), which is the microbe of the acetic fermentation in wines and beers. Saccharomyces vini. — This organism is the true wine-producing ferment, for it is the cause of the alcoholic fermentation of grape-juice. Its cells are elliptical, slightly smaller than those of S. cerevisiw. It forms spores, and is very common in the atmo- sphere.2 It should be borne in mind that fermentation is not a chemical, but a vital process; for the researches of Pasteur and others have shown that every fermentation has its specific ferment; in all fermentations in which the presence of an organised ferment has been ascertained the ferment is neces- sary. 1 See Martinand and Reitsch's paper in the Comptes Rendus, t. 112 (1891). 2 For further information concerning the yeasts see Jorgensen's Micro-Organisms of Fermentation ; Pasteur's Etudes sur la Biere, Etudes sur la Vin ; Engel's Les Fermentes Alcooliques ; and the papers of Hansen. CHAPTEE VI INFECTIOUS DISEASES AND MICROBES, ETC. 'THE study of disease-gerrns by the new and accurate methods of bacteriology has led to a clearer and better understanding of the manner in which, at any rate, some of the infectious diseases spread. While it was understood previous to the identification of their precise cause that some spread directly from individual to individual (e.g. small-pox, scarlet fever, diphtheria), others were known to be capable of being conveyed from one individual to another indirectly, i.e. through ad- hering to dust, or being conveyed by water, milk, or by food-stuffs (e.g. cholera, typhoid fever). But we are now in a position to define and demonstrate more accurately the mode in which infection can and does take place in many of the infectious diseases. By these means we have learned to recognise that the popular distinction between strictly contagious and strictly infectious diseases — the former comprising those diseases which spread, as it were, only by contact with a diseased indi- vidual, while in the latter diseases no direct contact is required in order to produce infection, the disease 178 INFECTIOUS DISEASES AND MICROBES, ETC. 179 being conveyed to distant points by the instru- mentality of air, water, or food — is only to a very small extent correct. Take, for instance, a disease like diphtheria, which was formerly considered a good example of a strictly contagious disorder ; we know jiow that diphtheria, like typhoid fever or scarlet fever, can be, and, as a matter of fact is, often con- veyed from an infected source to great distances by the instrumentality of milk. In malignant anthrax, another disease in which the contagium is convey- able by direct contact, e.g. in the case of an abrasion or wound on the skin coming in contact with the blood of an animal dead of anthrax, we know that the spores of the anthrax bacilli can be, and, in many instances are, conveyed to an animal or a human being by the air, water, or food. The bacilli of tubercle, finding entrance through a superficial wound in the skin or mucous membrane, or through ingestion of food, or through the air, can in a susceptible human being or an animal produce tuberculosis either locally or generally. The differ- ence as regards mode of spread between different diseases resolves itself merely into the question, Which is, under natural conditions, the most common mode of entry of the disease-germ into the new host? In one set of cases, e.g. typhoid fever, cholera, the portal by which the disease-germ generally enters is the alimentary canal ; in another set an abrasion or wound of the skin is the portal, as in hydrophobia, tetanus, and septicsemia ; in another set the respiratory organs, or perhaps the alimentary canal, or both, are the paths of entrance 180 A MANUAL OF BACTERIOLOGY of the disease-germ, as in small-pox, relapsing fever, malarial fever ; and in a still further set the portal is just as often the respiratory tract as the alimentary canal, or a wound of the skin, as in anthrax, tuberculosis. But this does not mean that the virus is necessarily limited to one particular portal, or that it must be directly conveyed from its source to the individual that it is to invade. All this depends on the fact whether or not the microbe has the power to retain its vitality and virulence outside the animal or human body.' 1 It must be borne in mind that not all the diseases described in the present chapter can, at the present time, be termed true microbian diseases ; yet with the progress of science, and by following the lines already laid down, we have not the slightest doubt that in time the microbes of all the infectious diseases will be discovered and cultivated. YELLOW FEVER. Micrococci (0*6 to 0'7 //, diam.) have been found in the kidney, spleen, and liver during the course of yellow fever. They form rosaries and masses, which greatly distend the blood-vessels and give rise to hemorrhages. The yellow-fever microbe is termed Micrococcus amaril by Dr. Domingos Freire. This microbe grows on gelatine, and reproduces the disease in rabbits and guinea-pigs. If, however, the microbe is cultivated in gelatine for six genera- tions it loses the greater part of its virulence, and 1 From a lecture delivered at the Royal Institution, London (February 20, 1891), by Dr. E. Klein, F.E.S. INFECTIOUS DISEASES AND MICROBES, ETC. 181 when this attenuated virus is introduced into the body by inoculation, it produces a mild type of yellow fever,-and confers immunity against the fatal type of the disease. From 1883 to 1890 Freire l has inoculated 10,881 persons in Brazil with cultures of M. amaril. The mortality of those so vaccinated was 0'4 per cent., although the patients lived in districts infected with yellow fever, whilst the death-rate of the uninoculated during the same period was from 30 to 40 per cent. Yellow fever is distributed (within certain areas) by moist winds and human intercourse. "Water and the soil have nothing to do with the spread of the disease, although it is a disease which clings to the ground, hence one of the reasons of its endemic nature. It is always prevalent in the plains near the sea-coast, and along the courses of the great rivers. Heat (21° C.) and a certain saturation of the atmosphere are essential conditions for an epidemic of yellow fever. Frost puts an end to an epidemic at once, and storms, heavy rains, or cold weather check its progress. HYDROPHOBIA. Hydrophobia or rabies is a canine disease, which is communicated by a bite, and the inoculation of man and other animals by the saliva. The exact nature of the microbe of this disease is not yet known. According to Pasteur,2 Fol,3 Babes,4 and 1 Comptes Rendus, 1889 and 1891, 2 Comptes Rendus, 1884. 3 Ibid., 1885, p. 1276 ; Le* Microbes, 1885, p. 41. 4 Les Bacteries, 1890, p. 550. 182 A MANUAL OF BACTERIOLOGY Dowdeswell,1 the microbe appears to be a micro- coccus, and it has been observed in microscopical sections of the spinal cord of animals dead of rabies. Dr. Fol's preparations were made by hardening the spinal cord or brain by immersion, directly after death, in a solution containing 2-5 grammes of potassium bichromate, and 1 gramme of copper sulphate in 100 cc. of .water. The piece of tissue is divided so as to be able to take up Weigert's FIG. 40. MICROCOCCI IN HYDROPHOBIA. A, In cerebral matter (after Fol). B, In human saliva. x 1000 solution of hsematoxylin ; then placed in absolute alcohol, imbedded in paraffin, and cut into sections not more than -^Q mm. in thickness. The sections are finally decolorised by a solution containing 2*5 grammes of potassium ferrocyanide, 2 grammes of borax in 100 cc. of water. In these sections, Fol found small micrococci (0*2 //, in diarn.) in the lymph spaces of the neuroglia, and between the 1 Journ. Roy. Microscop. Society, 1886 ; and Lancet, 1886. INFECTIOUS DISEASES AND MICROBES, ETC. 183 axis cylinder and its medullary sheath. This microbe (Fig. 40 A) occurs in groups and as diplococci, but never in chains. According to Fol, if a cultivation (in bouillon) be made of part of the brain, there is a deposit which, on inoculation into healthy animals, produces all the features of rabies. If, however, the cultivation be more than six days old there are no marked toxic effects. Fol says that nothing can be distinctly made out by merely reducing the nervous tissue of a rabid animal to a pulp and examining it microscopically, as recom- mended by Gibier. Babes states that he has found micrococci in the brain and spinal cord of rabid animals. These measure from 0'6 to 0*8 p in diameter, i.e. from three to four times as great as the microbe described by Fol. These micrococci are stained in situ by Loffler's alkaline methylene blue solution. They are culti- vated on blood serum or agar-agar (at 37° C.), and on bouillon made with the brain of a rabbit. The micrococci grow slowly and give rise to grey spots. ' A pure culture of the second, or even of the third generation, when inoculated into animals occasion- ally produces hydrophobia, but in most cases the cultures have no pathogenic properties, and it must, therefore, be concluded that the microbe has either lost its virulence or that it is not the actual cause of the disease.' The late Mr. G. F. Dowdeswell observed a microbe, measuring about the same diameter as Babes' micrococcus, in the central canal of the 1 Comptes Rendus, 1883, p. 1701 ; 1884, pp. 55 and 531. 184 A MANUAL OF BACTERIOLOGY spinal cord and the medulla oblongata of dogs dead of rabies. The author has also observed a micrococcus (Fig. 40B) in the saliva of a woman suffering from hydrophobia.1 The micrococcus, which is deeply stained by methylene blue, measures from 0'6 to 0*8 fj, in diameter. This microbe does not occur in healthy human saliva. We cannot say that the microbe of rabies has been isolated with anything like success, for the above investigations do not fulfil Koch's canons (see Chapter i.) to ascertain the pathogenic nature of the microbe or microbes in question. It is probable that the virus of rabies will not develop in the absence of a living pabulum, and in all probability it is not possessed of powers of active resistance to those injurious influences which act upon it when exposed to the air, etc. In fact, the virus of rabies cannot survive the drying, changes of temperature, etc., it necessarily undergoes when scattered over the ground, as we often see happen by the slobbering of a rabid animal. The saliva of rabid animals does not contain a ptomaine, for when it is diluted with a small quantity of sterilised distilled water, and then heated to 90° C. for a few hours, the saliva loses its virulent power. This proves that no alkaloid was present, because it would not have been destroyed on the application of heat.2 Besides, M. Nocard 1 The saliva was kindly sent to the author by Dr. T. M. Dolan, of Halifax. 3 Griffiths' Researches on Micro-Organisms, p. 193. INFECTIOUS DISEASES AND MICROBES, ETC. 185 dialysed the pure saliva of rabid animals, and proved that its solid constituents were always virulent, and reproduced the disease when injected into healthy animals, while the fluid portion, simi- larly injected, remained inactive. If an alkaloid or ptomaine had been present it would have been found in the fluid portion, and would have given rise to toxic effects when injected into the system. Al- though a ptomaine has not been discovered in the saliva of rabid animals, Dr. Anrep1 isolated a poisonous ptomaine from the brain and medulla oblongata of rabbits suffering from rabies. This ptomaine reproduced all the characteristic symp- toms of the disease, and it is stated that a gradual habituation of the animal to small doses of the ptomaine produced a certain degree of immunity. Babies is not a disease of the blood, for the sup- posed microbe is not found in the blood system, and when the blood of a rabid animal is injected into animals it does not reproduce the disease. In fact, the virus is located in the nervous system, especially the medulla oblongata. The period of incubation of rabies is usually not less than from four to six weeks, and sometimes longer. ' At the end of this incubation period the wound, first of all, becomes slightly uncomfortable ; there is itching, and the heat becomes almost intolerable, especially as this is usually accom- panied by a sharp stinging pain; the patient becomes feverish and very thirsty; the face is pallid and has a peculiar anxious expression, the 1 British Medical Journal, 1889, p. 319. 186 A MANUAL OF BACTERIOLOGY muscles of the face being drawn and restless, and gradually this expression amounts to one of actual terror or horror. On the second or third day the patient becomes much more excited, is restless in every sense of the word, and a very peculiar feature is that he has a characteristic habit of giving a suspicious side-glance as though constantly looking out for some hidden danger; then as the fever advances a rambling delirium supervenes ; the thirst increases, but along with this there is great difficulty in swallowing — especially fluids — and after making one or two attempts to swallow, the very sight of water suggests such horrors that, thirsty as the patient is, he is anxious to avoid it. Then muscular tremors are noted ; these become more and more marked, and violent spasms are easily stimulated, as in tetanus. A sharp sound, a touch, a bright light, or even a breath of air, may give rise to violent muscular convulsions, and eventually the patient is slowly suffocated as in tetanus' (Woodhead). Such are some of the tor- turing symptoms of hydrophobia. But it may be stated that the symptoms are varied, depending upon the nature of the region in the nervous system — encephalon or spinal cord — where the virus locates itself. The virus is found in every part of the encephalon. Although the saliva of rabid animals is virulent, it is not used by Pasteur in his prophylactic treatment, the reason being that, as saliva contains various microbes, it may give rise to septic poisoning, etc., as well as rabies. There- fore Pasteur has recourse to the central nervous INFECTIOUS DISEASES AND MICROBES, ETC. 187 system, where the virus is obtained in a pure state. This pure virus is continually being inoculated on the surface of the brain of healthy animals; the object of this is to keep up the supply of the virus, in question. The virus can be intensified or modi- fied by passing it through various animals. For instance, by passing it from the dog to the monkey, and subsequently from monkey to monkey, the virus grows weaker at each passage, until its virulence entirely disappears. Successive passages from rabbit to rabbit, and from guinea-pig to guinea-pig, increase the virulence of rabies virus. The intensified virus comes to a fixed maximum in the rabbit. If now transferred to the dog it remains intensified, and shows itself to be much more virulent than the virus of ordinary street rabies. So great is this acquired virulence, that the intensified virus injected into the blood- system of a dog unfailingly gives rise to mortal madness. These facts suggested to Pasteur that, by keeping a set of attenuated viruses of different strength, some not mortal, he could preserve the animal economy against the ill effects of more active ones, and these latter against the effects of mortal ones. The sets of attenuated viruses are not obtained by the passage of the virus through different animals, for the method now in use at the Pasteur Institute consists in suspending portions (a few centimetres in length) of the spinal cords of inoculated rabbits in a dry atmosphere (i.e. the marrows are desiccated in sterilised bottles of one litre capacity by means 188 A MANUAL OF BACTERIOLOGY of caustic potash). By this method the virulent power gradually diminishes, and finally disappears. By using attenuated viruses of varying intensities (prepared by desiccation), Pasteur has successfully treated numberless animals and human beings which are now refractory to rabies. To prepare the inoculating fluid a mad dog is killed, and the brain and medulla oblongata are carefully removed with sterilised instruments, etc. Very small pieces of the medulla oblongata and of , — — the Central canal are then placed in a sterilised glass. They are triturated with a glass rod, and when reduced to a fine jelly-like mass sterilised veal bouillon is added in quantity to about half a table-spoonful. This dilute dog- virus is used for inoculating a rabbit on the surface of the brain. A full- grown living rabbit is placed upon a dissecting board, flat on its abdomen, and its four limbs secured by strings to pegs driven in the wood (Fig. 41). After this the animal is placed under the influence of chloroform. The hair is cut away, and an inci- sion, one inch long, is made from a point midway between the eyes. The operator cuts down to the skull, which is then trepanned (Fig. 4 la), and a little circular disc of bone is removed, as far as possible without injuring the external membrane of the brain. At this point the operator takes a hypoder- FIG. 41. TREPANNING A RABBIT. INFECTIOUS DISEASES AND MICROBES, ETC. 189 mic syringe (see Fig. 20), filled with the diluted dog- virus, and inserts it under the dura mater, injecting two drops of the virus. The disc of bone is then replaced, and the skin flaps are sewed together by means of two or three sutures. ' A pad of cotton wadding, carefully purified by heat, is used to dry the skin, after which a little of the same wadding is used as a dressing ; this dressing is kept in position by a free application of flexile collodin, the two together forming an air-proof shield, through which no microbes from the external air can make their way to the wound, which, as a rule, heals up most perfectly in less than a couple of days/ After death the brain and medulla oblongata are removed, and a dilute virus is prepared from them, as in the case of the dog-virus. This is injected beneath the dura mater of a second rabbit, the operation being repeated in fresh rabbits until the shortest incubation period has been reached. This incubation period of seven days' duration is reached by the fiftieth passage, the rabbit taking ill on the seventh day, and dying on the tenth day or later, is the one used for human inoculations as well as for the purpose of perpetuating the disease in other rabbits. By dealing with a sufficiently large number of animals it is possible to have a rabbit dying every day, and thus also to put one spinal cord in a desiccating bottle every day. By the fourteenth day there will be a set of fourteen marrows under- going the desiccation. These marrows vary in virulence. The marrow of one day's desiccation is the most virulent, and the virulence of the other 190 A MANUAL OF BACTERIOLOGY marrows decreases gradually until the fourteenth day of desiccation, when a minimum is reached. At the Pasteur Institute, the marrows of more than fourteen days are thrown away as being inert and useless. A person having been bitten by a mad dog is first injected * with the weakest virus, and on each successive day or so with gradually stronger viruses until the more powerful or most powerful virus is used. After this treatment the patient very rarely dies of rabies. During the years 1886-9 no less than 7893 patients were treated at the Pasteur Institute, and out of this number there were 53 deaths, which represents a mortality of 0'67 per cent. But since 1889 the mortality has been re- duced to 0*2 per cent., due, no doubt, to the better skill in the application of the treatment.2 It may be stated in passing that at the Pasteur Institute, Paris, there are two rabbits inoculated, and, consequently, also two dying (of rabies) every day, ' for fear if one alone were used it might die from accident, and the series be interrupted. Prac- tically one animal is found to be quite sufficient, and the second one is only inoculated for prudence' sake.' ' The medulla or cord of a rabbit in which the 1 In the hypochondria (i. e. certain abdominal regions). 2 Concerning the interesting statistics of the Pasteur Insti- tutes of St. Petersburg, Odessa, Moscow, Warsaw, Charkow, Turin, Bucharest, Naples, and Havannah, the reader is referred to the latest edition of Cornil and Babes' book — Les Bactdries (1890). INFECTIOUS DISEASES AND MICROBES, ETC. 191 incubation has been seven days, when injected intra- cranially into a dog, develops rabies in the latter animal in about twelve days. The nervous matter of this dog, inoculated back by the same process into rabbits, at once reproduces the malady after an incubation of seven days, and thus the series is recovered/ Pasteur's treatment is prophylactic and not cura- tive, for it is powerless against the disease when the first symptoms have once made their appearance. Hence the necessity of early treatment. The mode of action of the Pasteurian inoculations has been explained by the two following theories : (1) Metschnikoff1 states that the white blood- corpuscles (phagocytes) absorb and digest the living microbes, and their power of absorption for microbes is trained and increased by the progressively stronger inoculations, so that finally the virus deposited by the rabid animal can also be absorbed and destroyed. The whole process is carried out, therefore, in the lymphatic system. (2) Woodhead and Wood be- lieve that the treatment consists essentially in caus- ing the tissues to acquire a tolerance before the microbe has had time to develop. ' The tissue cells are acted upon by increasingly active virus, each step of which acclimatises the cells for the next stronger virus, until at length when the virus formed by the microbes introduced at the time of the bite comes to exert its action, the tissues have been so far altered or acclimatised that they can continue their work undisturbed in its presence, and, treating 1 Fortschrift der Medicin, 1885. 192 A MANUAL OF BACTERIOLOGY the microbes themselves as foreign bodies, destroy them. When the cells are suddenly attacked by a strong dose of the poison of this virus they are so paralysed that the microbes can continue to carry on their poison-manufacturing process without let or hindrance ; but when the cells are gradually though rapidly, accustomed to the presence of the poison by the exhibition of constantly-increasing doses, they can carry on their scavenging work even in its presence, and the microbes are destroyed, possibly even before they can exert their full poison- manufacturing powers. Some such explanation as this would account for the interference with the course of the disease even after the patient has been bitten. The microbe is localised, it takes some time to form its poisonous products, and whilst this is going on the whole of the nervous and other tissues are being gradually acclimatised by the direct application of small quantities of the poison artificially introduced.'1 In concluding our remarks concerning rabies, it may be stated that the rabid marrows can be pre- served for several months in pure and neutral glycerine. Hence the use of this fluid for preserv- ing the marrows (for inoculation against rabies) during their transit from France to foreign countries. For further information the reader is referred to the undermentioned books and papers on the subject.2 1 Woodhead's Bacteria and their Products, p. 327. 2 Pasteur in Comptes Rendus, 1881-86; Dolan's Hydrophobia : M. Pasteur and his Methods ; Gamaleia in Annales de VInstitut INFECTIOUS DISEASES AND MICROBES, ETC. 193 ERYSIPELAS. This disease is due to the Micrococcus erysipela- tosiis (0*4 fj, diam.) which abounds in the lymphatic vessels of the skin at the margin of an erysipelatous zone. This microbe, which is smaller than M. vaccinice, occurs singly and in chains, as well as zooglcea. The microbe grows on nutrient gelatine, agar-agar, and solid blood-serum, as a whitish film on the surface of the nourishing medium. Orth * and Fehleisen2 have both cultivated the microbe artificially, and reproduced the disease in rabbits. But Fehleisen went a step further and reproduced the disease in man by inoculating three patients with pure cultivations of the microbe. 'These inoculations were justifiable because they were undertaken with a view to cure certain tumours. Thus one case of lupus, one case of cancer,3 one case of sarcoma, were considerably affected, and to the good of the patients.' In the human subject typical erysipelas was produced in fifteen to sixty hours after inoculation. Pasteur, 1887; Reye8 in Gac. Med. Mexico, 1889, p. 344; Dolan in Provincial Medical Journal, 1890, p. 137 ; Zagari in Giornale Inter nazionale delle Scienze Mediche, 1890 ; Hime in Lancet, 1886, p. 184 ; Griffiths' Researcftes on Micro-Organisms, p. 323. 1 Archivfur Experim. Pathol, 1874. a Die Aetiolcyie des Erysipels, 1883. 8 If cancer is due to Scheuerlein's Cancer bacillus, it is pro- bable that the M. erysipelatosus is antagonistic to its growth. 194 A MANUAL OF BACTERIOLOGY PUERPERAL FEVER. According to Heiberg,1 micrococci have been found in the form of chains and zooglcea in all organs affected in this disease. Heiberg's micrococcus has not yet been artificially cultivated, consequently we cannot say that the microbe is the real cause of this highly infectious disease.2 The infectiousness of puerperal fever is now well established, although the microbe or microbes which give rise to the different symptoms classed under the name of puerperal fever have not been isolated. Certain poisonous ptomaines have been isolated by Bourget from the viscera of a woman who died of puerperal fever, and subsequently he proved the existence of the same ptomaines in the urine of patients suffer- ing from the same disease. Pasteur and others are convinced that with the possible exception of cases where, by the presence either of internal or external abscesses, the body before confinement contains microbes, the antiseptic treatment ought to be infallible in preventing puerperal fever from declar- ing itself. It may be stated that the introduction of the antiseptic and aseptic methods has produced not only a remarkable diminution of mortality, but also of the morbidity or illness incident to the puerperal state. 1 Die Puerperalen und Pydmiochen Processe. 2 In 1889 a midwife carried the contagion to five different women, all of whom died of the disease (The Echo, Sept. 17, INFECTIOUS DISEASES AND MICROBES, ETC. 195 Prof. I. Giglioli1 gives the following statistics concerning the patients in the Maternity Hospital at Copenhagen between the years 1850 and 1874 (the antiseptic method being introduced in the year 1870):— From 1850 to 1864 the mortality was 41'6 per 1000. „ 1865 „ 1870 „ „ 19-6 „ „ 1870 ,,1874 11-4 „ And in Naples the mortality during the years 1875-78 was 0'12 per 1000 patients. Dr. W. 0. Priestley gives in his paper, which was read before the Congress of Hygiene,2 an inter- esting table showing the maternal deaths in six lying-in hospitals since the introduction of antiseptic and aseptic methods. With these he contrasted the figures of M. Le Fort before the era of anti- septics : — MORTALITY IN MATERNITY HOSPITALS FROM ALL CAUSES IN VARIOUS COUNTRIES OF EUROPE (LE FORT). Before the introduction of Antiseptics. Deliveries. Deaths. Per 1000. Total, . 888,312 30,394 34'21 1 Fermenti e Microbi, p. 157. 2 Held in London during September 1891. 196 A MANUAL OF BACTERIOLOGY After the introduction of Antiseptics. I Date. Deliveries. Deaths which would have Deaths, occurred on basis of Le Fort's figures. Vienna, . . 1881-5 15,070 106 516 Dresden, . 1883-7 5,508 57 188 Russia, ... 1886-9 76,646 290 2,622 New York, . . 1884-6 1,919 15 66 Boston, . 1883-6 1,233 27 42 London (General Lying-in Hospital), 1886-9 2,585 16 88 Total, 102,961 511 3,522 The number of lives saved out of the 102,961 deliveries since the introduction of antiseptics is the following : — Expected deaths on Le Fort's basis, . 3522 Actual deaths, . . . 51 11 Saving, . . 3011 From the above figures it will be seen that while according to M. Le Fort, the maternal deaths in European lying-in hospitals were 34'21 per 1000 under the old regime, the mortality is now reduced to somewhat less than 5 per 1000. This computa- tion, put in another way, indicates that if the former rate of mortality had been maintained 3522 maternal 1 4-363 per 1000. INFECTIOUS DISEASES AND MICROBES, ETC. 197 deaths might have been expected, whereas the actual deaths were only 511. In other words, 3011 lives of mothers were saved as the result of new and purely scientific methods of treatment.1 INFLUENZA. This is a very different disease from the catarrhal affections known by the same name. It is really an acute specific disease running a definite course like scarlatina or measles ; but very little is known of the cause or nature of this ubiquitous disease which has attacked humanity in its own violent fashion at short intervals from probably the earliest ages. The history of the recorded epidemics of La Grippe is marvellously complete for centuries. Every country and every climate in the world is subject to it, yet it appears to find a permanent home nowhere as a constant or endemic resident, but to disappear from the face of the earth for a series of years. It is, however, probable that the microbe of this disease has some undiscovered endemic source. The symptoms of epidemic influenza follow pre- cisely the type of the other infective fevers, and preserve a remarkable uniformity and individuality in successive epidemics. Sir Morell Mackenzie'2 believes that the disease is due to ' a specific poison of some kind which gains access to the body, and, 1 For further information on the subject of puerperal fever see Flessinger's paper in Gaz. Med. de Paris, 1889, p. 313 ; and Widal's Etude sur V Infection Puerperale (1889). 2 Fortnightly Review, June 1891. 198 A MANUAL OF BACTERIOLOGY having an elective affinity for the nervous system, wreaks its spite principally or entirely thereon. In some cases it seizes on that part of it which governs the machinery of respiration, in others on that which presides over the digestive functions ; in others again it seems, as it were, to run up and down the nervous key-board, jarring the delicate mechanism, and stirring up disorder and pain in different parts of the body, with what almost seems malicious caprice.' Therefore, according to Mac- kenzie, the supposed microbe resides in, or acts on, the nervous tissues of the body. There are many reasons for thinking that the contagium of influenza is borne through the air by winds rather than by human intercourse. One reason for thinking so is that it does not appear to travel along the lines of human communications, and, as is seen in the infection of ships at sea, is capable of making considerable leaps. Dr. Parsons, on the other hand, believes that the epidemic is propagated mainly by human intercourse, though not in every case necessarily from a person suffering from the disease. Concerning the germ of influenza, Klebs thought that he had discovered this in certain Flagellata found in the plasma or corpuscles of the blood during the febrile stage, but no cultivations were made. Gluber found a micrococcus (in pairs) in the blood ; and Frankel noticed the same microbe in the sputum of a patient suffering from influenza. This microbe may have been Micrococcus pneumonice, as pneumonia frequently follows an attack of the INFECTIOUS DISEASES AND MICROBES, ETC. 199 disease. Eibbert thinks that Micrococcus pyogenes is invariably present, and is the actual cause of influ- enza ; but Besser has shown that it is common in healthy men at least during the epidemic. In 1884 Seifert found a micrococcus in the sputa of influenza patients and of no others. Although the microbe of influenza has not yet been isolated, there is little doubt that influenza is a microbian disease ; for its constancy of type, the mode of its transmission, its independence of climatic and seasonal conditions, all suggest that its cause is 'specific' — i.e. having the properties of growth and multiplication which belong to a living thing.1 PNEUMONIA. In this disease large numbers of micrococci are present in the lungs. The microbe (Micrococcus pneumonice, Fig. 33, 17) was discovered by Friedlander,2 and occurs in the sputa of pneumonic patients, either singly, as diplo- cocci, short chains, and zooglcea. Sometimes the microbes are free, while at other times they are encysted in the lymphatic cells. They are oval, encapsulated microbes, and have been cultivated in blood-serum, peptonised gelatine, bouillon, and on steamed potatoes. 1 See Sisley's Epidemic Influenza (1891) ; Erodie's paper in Nature, July 23, 1891 ; Parsou's Report on Influenza to the Local Government Board (1891) ; the Hon. R. Russel's pamphlet, The Spread of Influenza (1891) ; Cantani's V Influenza (1890) ; Tala- mon's La Grippe et les Microbes (1890). See also the Appendix. 2 Virchow's Archiv, vol. Ixxxvii. 200 A MANUAL OF BACTERIOLOGY Griffini and Cambria1 observed the same micro- cocci in the blood of pneumonic patients. Salvioli and Zaslein2 cultivated these microbes, derived from the same source, in bouillon at 37° to 39° C. ; and when injected into mice and rabbits they gave rise to pneumonia. Giles3 found the same microbes in many cases of pneumonia in India ; and pure culti- vations, when injected into the subcutaneous tissues of rabbits, produced the disease. Those researches have been confirmed by Afanassiew. When the artificially - cultivated microbe is inoculated in the tissue of the lungs it produces in animals all the characteristic symptoms of pneumonia; the lungs become red, solid, and en- larged, and pieces of them sink in water. In pneumonia the blood is considerably altered, for the hsematin, globulin, and the salts are greatly reduced. According to Emmerich, the growth of the micrococcus of pleuro-pneumonia on peptonised gelatine is similar to the one derived from human pneumonic sputum; and when this microbe is injected into rabbits it produces typical pneumonia. Nolen and Poels 5 injected pure cultivations of the microbe of human pneumonia into cattle, and pro- duced pleuro-pneumonia with all its characteristic symptoms. However, it may be mentioned that 1 Gentralblatt fur d. Med. Wissemch., 1883. 2 Ibid., 1883. 3 British Medical Journal, 1883. 4 Comptes Rendus de la Socidte de Biologie (Paris), t. 5. 5 Centralblattfur d. ;Med. Wissensch., 1884. INFECTIOUS DISEASES AND MICROBES, ETC. 201 Dr. Klein1 does not accept these statements without reservation. Professor Brieger2 has shown that when M. pneumonia is grown in solutions of glucose or sucrose, acetic acid is formed along with ethyl alcohol and formic acid. The same products are formed when the microbe is grown in solutions of creatine and calcium lactate. Dr. P. F. Frankland3 has recently investigated the action of the same microbe on various carbo- hydrates, with the following results : — (a) Micrococcus pneumonice sets up a fermentive process in solutions of dextrose, sucrose, lactose, maltose, raffinose, dextrin, and mannitol. (b) It does not ferment solutions of dulcitol or glycerol, and has thus the power, like the Bacilhis ethaceticus (see p. 155), of distinguishing between the isomers, mannitol, and dulcitol. (c) The fermentation of mannitol is represented by the following equation : — 6C6HU06 + H20 = 9C2H5HO + 4CH3COOH + 10C02 -j- 8H2. In other words, the above equation represents the quantitative decomposition of mannitol into alcohol, acetic acid, carbon dioxide, and hydrogen. It would be interesting to ascertain whether acetic acid and alcohol are formed in human milk during an attack of pneumonia; for it may be stated that the lactose is reduced from 43-6 to 30-2 parts per 1000. 1 Micro-Organisms and Disease, p. 77 (3d. ed.). 2 Zeit. Physiol. Chem., vol. viii. p. 306 ; and vol. ix. p. 1. 3 Journal of Chemical Society, 1891, p. 253. 202 A MANUAL OF BACTERIOLOGY SCARLATINA. This disease is the result of the action of the Micrococcus scarlatince, which has been found in the blood, organs, the exudations and tissues of the ulcerated throat, and in the desquamating epi- demic cells of this disease. M. scarlatince has also been observed in the urine of patients suffering from scarlatina ; and this fluid contains a ptomaine represented by the formula C5H12N04. The same ptomaine has also been extracted from pure cultivations of Micrococcus scarlatince in pepto- nised gelatine. In fact, the microbe forms this ptomaine from the medium in which it lives.1 M. scarlatince^ (0.5 //, diam.) occurs singly, as diplococci, in chains, and in masses (Fig. 33, 9) ; and it grows on the surface of nutrient gelatine, as well as in the depth of that medium. It also grows on agar-agar and in beef bouillon. On nutrient gelatine with slanting surface this microbe forms greyish, circular, flat discs, which ultimately form a grey film. Gelatine tubes inoculated by stabbing show some characteristic features. After twenty- four hours' incubation the surface of the stab appears sunk, and the depression thus formed increases in breadth and depth during the next two or three days, so that by that time there is a distinct funnel-shaped depression indicating the upper end of the channel of inoculation marked as a white streak. Then, commencing at the bottom 1 See Dr. A. B. Griffiths' paper in Comptes Rendus de I'Aca- ddmie des Sciences, vol. cxiii. (Nov. 9. 1891), p. 656. INFECTIOUS DISEASES AND MICROBES, ETC. 203 of the funnel, the gelatine becomes liquefied, and this liquefaction gradually extends in breadth, and always in depth, along the line of the growth. The liquefied part of the gelatine is clear, and at the bottom of it is a whitish precipitate. In stab-cultivations, using agar-agar or solid blood serum as the medium, white dots make their appearance in the streak; but these are of a brownish colour where thickly packed together. On the surface of agar-agar or solid blood serum a continuous film is formed. In alkaline bouillon the growth forms whitish, fluffy, or loose masses at the bottom of the tube. In milk M. scarlatinas grows fairly well, and turns the milk at first thick, then quite solid ; sometimes this occurs after two or three days' incubation at 37° C., sometimes a little later. Drs. Klein and Power have proved that a certain eruptive disease of the teats and udders of cows is capable of communicating scarlatina to human beings through the medium of the milk derived from such cows. In certain extensive outbreaks of scarlatina at Hendon, Wimbledon, etc., Dr. Klein found Micro- coccus scarlatince in the blood of scarlatina patients both during life and after death ; and he found the same microbe in the tissues and organs of persons dead of scarlatina. These outbreaks of scarlatina were traced by Power and others to the milk-supply from certain farms where the cows were suffering from what is now known as cow -scarlatina. In both human and bovine scarlatina the same microbe 204 A MANUAL OF BACTERIOLOGY (M. scarlatince) is always present in the tissues, organs, and blood; and from both sources sub- cultures of the microbe, when inoculated into healthy cows, produce the disease. For instance, when pure subcultures of the microbe were inocu- lated into calves and cows, the microbe was found in the spleen, kidneys, teats, udders, lung, skin, etc. Fig. 42 represents a section through the skin of the nostril of a calf that had been experimentally FIG. 42. SECTION THROUGH SKIN OF THE NOSTRIL OF A CALF THAT HAD BEEN EXPERIMENTALLY INFECTED WITH M. SCARLATINA DERIVED FROM A HUMAN SOURCE (Klein). infected with M. scarlatince derived from a human source. In this figure it will be observed that the microbe is present in large numbers. In fact, Dr. Klein's important researches on the relationship existing between the cow - disease, already alluded to, and human scarlatina may be summarised as follows : — (a) The disease in man and in the cow alike is characterised by closely similar anatomical features INFECTIOUS DISEASES AND MICROBES, ETC. 206 (b) From the diseased tissues and organs of man and cow alike the same microbe can be separated, and artificial subcultures be made from it. (c) These subcultures, no matter whether estab- lished from man or cow, have the property, when inoculated into calves, of producing in them every manifestation of what is known as the Hendon cow-disease, except the sores or ulcers on the teats and udders — no doubt for the reason that the milk apparatus is not yet developed in calves. (d) Subcultures of the microbe made from human scarlatina and inoculated into recently calved cows produced in them, along with other manifestations of the Hendon cow-disease, the characteristic ulcers on the teats — ulcers identical in character with those observed at the Hendon farm. (e) The subcultures, established either from the human or the cow disease, have an identical pro- perty of producing in various rodents a disease similar in its pathological manifestations to the Hendon disease of cows and to scarlatina in the human subject. (/) Calves fed on subcultures established from human scarlatina obtain the Hendon disease. (g) Children fed on milk from cows suffering from the Hendon disease obtain scarlatina. Bearing on the same subject, it may be mentioned that in the parish of St. George's, London, five persons were attacked with scarlatina on the same day (in October 1886). They had used a cheap brand of condensed milk ; and in this milk Dr. Klein proved (both by cultivation and inoculation) the presence of Micrococcus scarlatinas. 206 A MANUAL OF BACTERIOLOGY These investigations prove that cows suffer from scarlatina ; that the specific microbe circulating in the blood of the diseased animals contaminates the milk; and that such milk conveys the disease to human beings. The disease has also been directly communicated to man by inoculation with the virus from the ulcers on the teats and udders. In a particular case, recorded by Dr. J. Cameron,1 a man received the virus of scarlatina into a recent scratch upon his forefinger while milking a diseased cow. As both human beings and cows are liable to be attacked with scarlatina, and as the milk of the latter (when diseased) is capable of producing an extensive outbreak of the disease in human beings, it is advisable that milk should be boiled before use. .This destroys any microbes which may be present.2 LEPROSY. The microbe of this disease is Hensen's Bacillus leprce. It measures from 4 to 6 //, long and about 1 /i wide (Fig. 33, 2), and it occurs in masses within the large leprosy-cells of the nodules of the skin and organs, as well as of the mucous membrane of the mouth, palate, and larynx. Two types of leprosy are described — the anaesthetic and tuber- cular varieties ; the first variety is more frequently seen in the tropics, the latter in temperate climates. In the anaesthetic variety the bacillus is present in 1 Transactions of Epidemiological Society, 1885-6. 2 For full details of Klein's researches see the Reports of Medical Officer to Local Government Board, 1885-6 ; 1886-7 ; 1887-8; 1887-9. INFECTIOUS DISEASES AND MICROBES, ETC. 207 the interstitial tissue of the nerves. B. leprce is sometimes motile and produces spores. It grows on blood serum and alkaline infusions of meat- extract ; and Damsch1 has produced the disease in cats by inoculating them with leprous tissues. The microbe is absent in the blood of lepers ; therefore it probably spreads by the lymphatic vessels. Hansen's discovery2 of B. leprce lias since been con- firmed by Neisser,3 Cornil,4 Babes,5 Hillis,6 Stevens,7 Thin,8 Rake,9 Kobner,10 Bordoni-Uffreduzzi,11 and Gianturso ; 12 and during the present Leprosy Com- mission in India, Drs. Rake, Buckmaster, Thomson, and Kanthack have also succeeded in rearing B. leprce13 on blood serum ; but growths of this microbe are difficult to obtain. Bordoni-Uffreduzzi obtained ' growths from the marrow of a bone in which there were a number of free leprosy bacilli ; 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 I Virchow's Archiv, vol. xcii. 9 Ibid. vol. Ixxix. 3 Ibid. vol. Ixxxiv. 4 Union Medicate, 1881. 5 Archives der Physiologic, 1883. • Transactions of Pathological Society, 1883. 7 British Medical Journal, 1885. 8 Med.-Chir. Transactions, vol. Ixix. 9 Transactions of PathoL Soc., 1887. 10 Virchow's Archiv, vol. Ixxx. II Zeitschrift fur Hygiene, vol. iii. p. 178. 12 Centralblatt fur Bakteriologie und Parasitenkunde, vol. ii. p. 701. 13 Concerning the differences between the leprosy and tubercle bacilli, 'see Slater's paper in Quart. Journ. Micros. Science, 1891, 208 A MANUAL OF BACTERIOLOGY yellow films with irregular borders. On glycerine agar-agar they are said to have developed as small, grey, munded, 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 microbe could be grown on serum or on ordinary gelatine and agar-agar, but much more slowly than when glycerine had been added.' Leprosy, or elephantiasis grecorum, is a specific disease, characterised by the slow development of nodular growths in connection with the skin, mucous membranes, and nerves, and by the super- vention of ansesthesia, paralysis, and a tendency to ulcerative destruction and gangrene. Although prevalent in the Middle Ages, leprosy is very rare in Europe at the present day, being confined to isolated areas on the shores of Spain, Portugal, Sweden, Norway, Iceland, and in Italy, Kou mania, Hungary, and Greece, where it is still endemic. It is, however, common in Egypt, Morocco, Cape Colony, Madagascar, Southern Asia (including Japan), Brazil, United States of Colom- bia, Guiana, Argentina, New Zealand, and in certain islands of the Pacific Ocean (especially Hawaii). In the United States of Colombia leprosy first made an appearance in 1646, and was introduced into that country from Spain. It seems to have spread slowly but surely throughout a great part of the country during the succeeding two hundred years; but since 1870 the increase in the number of cases has been much more rapid, and within that INFECTIOUS DISEASES AND MICROBES, ETC. 209 period the disease has spread to districts where it was previously unknown, until now almost every district in Colombia is more or less infected. According to a medical authority residing in Bogota, it is stated that one-tenth of the inhabit- ants of Santander and Boyaca are lepers. As the population of these two states is about 1,000,000, this estimate would give 100,000 lepers in that por- tion of Colombia alone. Another authority states that there are only 30,000 lepers in the two states previously mentioned; but whichever figure is. correct, it shows that a large percentage of the inhabitants are suffering from this fell disease. Marriages constantly take place between non-lepers and lepers, and children are born of these unions ; but they generally develop the disease in a few years. The lepers also marry among themselves, and their children are almost always lepers. Very little is done in the way of isolation, consequently leprosy is bound to spread more and more through- out Colombia unless some great effort is made to arrest its progress. It is the universal opinion all over Colombia that leprosy is both contagious and hereditary ; but it is probable that the system requires to be predisposed by bad food, unsuitable climate, dirty and confined lodging, exposure to chills and damp, etc., before leprosy can be con- tracted by contagion. There is no doubt that the absence of hygienic appliances and personal clean- liness aid its development immensely.1 So far as is at present known, there is no cure for 1 See the British Consular Report from Boyota,, 1891. 210 A MANUAL OF BACTERIOLOGY leprosy;1 but no doubt, with growing experience, leprous vaccine will soon be discovered ; and it is even possible that, with the experience already gained, such a result may at once be obtained (Pasteur). SYPHILIS. Syphilis is a specific disease ; and, ' after the local introduction of the syphilitic poison, some ten to fifty days elapse before the true Hunterian chancre first appears, but at the same time indurated buboes or glands may be detected in the. groins. In a few weeks the blood becomes tainted by the peculiar virus, and this interfering with the nutrition of the blood capillaries and tissues, produces a series of morbid phenomena, divided by syphilographers into secondary and tertiary, the term primary being retained for the manifestations due to local inocu- lation. Leaving no tissue untouched,2 syphilis is well known also for the variety of its manifestations and for its propensity to attack parts of the body often respected by other forms of skin disease and blood poisoning. A proneness to leave behind much dusky, copper-coloured staining of the skin, whilst 1 It is stated that leprosy has been cured by the ' Mattei remedies ' (Report of the St. Joseph's Asylum at Mangalore, 1891), but these 'remedies' have been proved to be quack pre- parations, etc., by the medical profession. 2 Hence the reason that Byron called this disease — 'the great:'— ' I gaid the small- pox has gone out of late ; Perhaps it may be follow'd by the great. ' (Don Juan, c. i., v. 130.) INFECTIOUS DISEASES AND MICROBES, ETC. 211 its inflammatory eruptions scarcely cause itching, are features of diagnostic interest.' The Bacillus of syphilis was discovered by Dr. S. Lustgarten 1 in the nucleated cells of various syphi- litic products, e.g. ' in the discharge of the primary lesion and in hereditary affections of tertiary gummata.' He never found the microbe free between the tissue elements, but always enclosed in cells. Nevertheless, it may be stated that Eve and Lingard 2 isolated a bacillus from the blood, as well as from the diseased tissues in syphilis, which they cultivated in artificial media. Lustgarten's bacillus measures from 3 to 4 //, long and 0.8 //, wide (Fig. 33, 21); it has a swelling at each end. It is believed that this microbe produces spores, and, according to Lustgarten, it is the virus of syphilis. Doutrelepont, De Giacorni, and Schlitz have confirmed Lustgarten's observations. TETANUS. Tetanus or lockjaw is an infectious disease caused by the Bacillus of tetanus, which inhabits certain soils; for it was proved by Nicolaier3 that soil obtained from streets and fields,4 when inoculated into mice, rabbits, and guinea-pigs, gave rise to the characteristic symptoms of tetanus. The microbe of this disease forms spores. ' These spores gaining 1 Med. Jahrbucher der K. K. Gesellsch. d. Aerzte (Vienna), 1885. 2 Lancet, 1886, p. 680. 3 Dissertation (Gottingen), 1885. 4 Soils obtained from cultivated gardens and from woods do not give rise to tetanus. 212 A MANUAL OF BACTERIOLOGY access to an abrasion or wound of the skin in man or animals, are capable of germinating there and multiplying, and of producing a chemical poison, which is absorbed into the system, and sets up the acute complex nervous disorder called lockjaw.' The tetanus bacillus- (1.2 yu, long) produces spores only at one end (Fig. 43), and in the spore-bearing condition is known as the drum -stick -shaped bacillus. It is motile and anaerobic, growing on gelatine- plates (containing glucose) in an atmo- sphere of hydrogen. In tubes containing blood serum or nutrient gelatine, it grows in the depth of the me- dium, forming a kind of cloud. The medium emits a fusty smell, which is characteristic of this microbe. In obtaining culti- vations of the tetanus bacillus, other anaerobic microbes grow, and also pro- duce spores. But Kitasato found that the tetanus bacillus produced spores earlier than the other bacilli present in tetanic pus. Consequently, he devised the following method for separating the tetanus bacillus from the other microbes : — As soon as spore-formation in the tetanus bacilli had com- menced, the tubes (containing them) were heated for a considerable time at 80° C., with the result that all the bacilli were destroyed, but not the FIG. 43. THE TETANUS BACILLUS. (x 1000.) INFECTIOUS DISEASES AND MICROBES, ETC. 213 spores of the tetanus bacilli. These spores after- wards germinated (at 30° C.), and give rise to pure cultivations of the tetanus bacillus. This microbe is localised at the actual point of inoculation (i.e. in the pus and the walls of the abscess), and is never present in the internal organs. The ptomaine, which the tetanus bacillus gives rise to, is manufactured at the site at which it is actually introduced, and ' from this point it is absorbed into the body, and is carried to the special tissues on which it acts/ Professor L. Brieger1 has succeeded in isolating four ptomaines from pure cultivations of the tetanus bacillus. This first is tetanine (C13H30N204), which produces tetanus in animals ; the second is tetano- toxine (C5HnN), which produces tremor and paralysis, followed by violent convulsions; the third is spasmotoxine (formula unknown), which produces tonic and clonic convulsions; and the fourth ptomaine (which has not been named) causes tetanus, accompanied with a flow of saliva and tears.2 Tetanine has also been extracted from the limb of a patient who had died from tetanus. Brieger looks upon the poisonous substance tetano- toxine as a toxalbumin; but he may have over- looked the possibility that this proteid may contain a ptomaine closely bound to it, or in an isolated condition within its molecules. 1 Virchow's Archiv, vol. cxii. (1838), p. 549 ; vol. cxv. (1889), p. 484 ; Berliner Klinische Wochenschrift, 1888 ; and Unter- suchungen iiber Ptomaine, 1886, p. 89. 2 These tetanic ptomaines do not occur in the urine of patients suffering from tetanus. 214 A MANUAL OF BACTERIOLOGY As the tetanus bacillus is localised, there can be no doubt that tetanus is due to the above poisons (manufactured indirectly by the bacillus) producing effects after getting into the blood, by virtue of some selective action on certain parts of the motor nerve-centres. The spores of the tetanus bacillus have an ex- tremely wide distribution, being found in soils, etc., in various parts of the world. According to Bos- sano,1 soils which contain much organic matter nearly always contain tetanus bacilli, ' and that latitude, climate, and special meteorological condi- tions have far less influence on their development than defective drainage, imperfect hygienic condi- tions, and the degree of cultivation of the soil.' Dr. Kitasato 2 has recently shown how to produce immunity against tetanus, and he has cured animals suffering from this disease. Kitasato first renders an animal immune against tetanus, and then injects the blood serum of that animal into animals suffering from the disease. In order to render an animal immune or unsusceptible, the tetanus bacilli are first injected ; this injection being followed by in- jections of iodine trichloride, which are repeated at intervals of twelve hours. After four days the animal, which under ordinary circumstances would have died from tetanus, is not only cured, but ren- dered immune against the disease. The blood serum of such an animal has been found in successive 1 Comptes Rendus, tome 107, p. 1172; and Recherches Ex- pdrimentales sur VOrigine Microbienne du Tetanos (1890). 2 Deutsche Medicinische Wochenschrift, 1890, No. 49, et seq. INFECTIOUS DISEASES AND MICROBES, ETC. 215 experiments on mice and rabbits to act as a com- plete cure. Kitasato's experiments prove (a) that the blood of rabbits which have been rendered un- susceptible to tetanus possesses properties destruc- tive of the tetanus virus ; (&) that these properties are to be observed also in extra-vascular blood and serum free from cells ; (c) that these properties are of so permanent a nature that they are still mani- fested by such serum after it has been injected into other animals ; consequently, by transfusion of such blood or serum, important therapeutic actions can be obtained ; (d) that this power of destroying the tetanus poison is absent from the blood of such animals as are not immune against tetanus; and after such animals have been killed by the tetanus poison, it can be shown to be present in their blood and tissues. If animals (such as mice and rabbits) highly sus- ceptible to tetanus are cured by this treatment, we have good reason to believe that it will also cure human beings, which are far less susceptible to the disease. MALARIA. The discovery of the Bacillus malarice placed malaria among the acute specific diseases. Concern- ing the distribution of malaria, moisture and air have much to do with it, for the disease is more abundantly developed in wet than in dry years. Moisture in the soil is essential for the production of malaria, while clayey, loamy, and marshy soils 216 A MANUAL OF BACTERIOLOGY favour its development. Professor C. Tommasi- Crudeli 1 states that the following conditions are necessary for the Bacillus malaria to produce spores : (a) ' Une temperature de 20 degres centigrades environ ; (b) un degre" mode're d'humidite* perman- ente; (c) 1'action directe de 1'oxygene de Fair sur toutes les parties de la masse [that is, of the soil]. II suffit que Tune de ces trois conditions fasse deTaut, pour que le deVeloppement des sporules, et la multiplication du ferment malarique, soient ar- retes.' In marshy districts, the larger the amount of organic matter present in a soil, the greater will be the prevalence of malaria. The disease is more prevalent the lower the level of the country, although in Central Africa a height of 2500 feet is not free from it. Both air and water may convey the dis- ease, and there is little doubt that it finds an entrance into the system by means of air, potable water, and food. Bacillus malarice (2 to 7 //. long) gives rise to leptothrix filaments, and produces spores either at the ends or in the centre of the cell (Fig. 38, 18). This bacillus was found in the blood of malarial patients by Klebs and Tommasi-Crudeli,2 and they also found it in the spleen, medulla, lymphatic glands, and venous blood of persons dead of malaria. On gelatine B. malarice gives rise to a well-developed growth, and when a drop of the culture is inoculated 1 La Malaria de Rome et VAncien Drainage des Collines Romaines (Paris), 1881 ; and Atti ddla R. Accademia dei Lincei, 1879. 2 Atti ddla R. Accademia dei Lincei, 1879, 1880, and 1881 ; and Archivfur Experimental Pathologie, 1879. INFECTIOUS DISEASES AND MICROBES, ETC. 217 in rabbits it reproduces malarial fever, with all its characteristic symptoms, the threads and spores of the bacilli being found in abundance both in the spleen and the marrow. This microbe grows also on albumin, urine, and other media in the presence of air, and at a temperature of about 20° C. B. malaria was originally discovered in the soil of the Roman Campagna, and Antonio Ceci 1 obtained pure cultures of the microbe from this soil. When these pure cultures were inoculated in animals they pro- duced malaria or intermittent fever. Dr. B. Schiavuzzi2 has confirmed Klebs and Tom- rnasi-Crudeli's discovery of Bacillus malarice, and that it is the real cause (directly or indirectly) of malaria. Cohn 3 has also verified the work of the Italian bacteriologists. On the other hand, Laveran,4 Richard,5 Marchia- fava and Celli,6 Golgi,7 Evans,8 and others have dis- covered certain organisms allied to the Flagellata in the blood of patients suffering from malaria. These organisms have been called Plasmodium malarice, and they are said to give rise to intermittent fever in man after intravenous injection. The blood cor- puscles of a person so infected again contain the plasmodia ; and it is further stated that these or- ganisms alter the composition of the blood. 1 See Professor Giglioli's Fermenti e Microbi, p. 592. 2 Atti della R. Accademia dei Lincei, 1886. 3 Beitrage zur Biologic der Pflanzen, 1886, p. 245. 4 Comptes Rendus, 1881-2. 5 Ibid. 1882. 6 Annali di Agricoltura (Roma), 1886, p. 4. 7 Archivioper h Scienze Mediche, vol. x. (1886), p. 109. 8 Proceedings of Royal Society, 1891. 218 A MANUAL OF BACTERIOLOGY In a paper read before the Accademia del Lincei on May 2, 1886, Professor Tommasi-Crudeli1 says that he does not accept the statement that the plas- modia found in the blood of malarial patients are the cause of malaria. In fact, he says ' la grande extensione dell' infezione malarica : le varie forme, ora lente e latenti, ora rapide e intense, nelle quali questa infezione si manifesta : la lunga persistenza, anche allo stato latente, della malaria in un terreno : son tutti forti argomenti contrari alia ipotesi che la malaria sia dovuta ad un parassita di natura animale ; e favorevoli all 'opinione che i germi malarici siano Schizomiceti, simili a quelli delle tuberculosi, e di altre persistenti infezioni.' The alteration in the composition of the blood in patients suffering from malaria (previously alluded to) may be due to a soluble enzyme secreted by B. malarias (Schiavuzzi), and certainly this is not im- probable, for Dr. Lauder Brunton, F.RS.2 has shown that many microbes have the power of ' manufac- turing a ferment suited to their needs.' Bacillus malaria is inhaled into the blood by way of the lungs, and perhaps it may enter through the stomach and skin also. It flourishes in marshy districts, in deltas, on alluvial soils, and on the banks of tropical rivers — in fact, a proper degree of porosity, of temperature, and of humidity of soil favour the growth of this microbe : hence the reason 1 This eminent savant has been obliged to give up his im- portant investigations. He wrote to the author as follows : ' I have been compelled to give up microscopical researches since 1886, because my eyes are almost ruined.' 2 Proceedings of Royal Society, vol. xlvi. p. 542. INFECTIOUS DISEASES AND MICROBES, ETC. 219 that B. malaria has been called 'an earth-born poison.' 1 This microbe is said to be heavier than most gases, ' and scarcely floats six feet above the ground ; it may be wafted some distance by winds, but mountains hold it back, and belts of trees, especially the eucalyptus, destroy its efficacy.' Gubler2 and many others have shown that the eucalyptus or ' fever-destroying ' tree has consider- able power in destroying the microbe of malaria, this being due to the action of the aromatic gases given off by the tree. One instance may be cited of the fever-destroying properties of the eucalyptus. ' In a desolate part of the Campagna there stands an old monastic institution upon a spot consecrated by tradition as that whereon St. Paul was martyred. For centuries this part of the Campagna [Tre Fon- tane 3] was a stronghold of pestilential fever, and prolonged residence in the monastic institution in question surely led to death. Some few years ago a band of Trappist monks planted the eucalyptus in its cloisters, and the trees have since grown to a great height. What is more important, however, is that the place is now once more habitable, and fever, it is said, reigns there no more.' 4 There are also plantations of the eucalyptus in Corsica, Algeria, Italy, California, Australia, and other parts of the world ; and there is little doubb that these trees are antagonistic to the spread of malaria, because the 1 Felkin in Proc. Roy. Soc. of Edinburgh, vol. xvi. p. 269. - Journal de Pharmacie et de Chimie, 1871. 3 Known anciently as Aquae Salvise. 4 Kingzett's Nature's Hygiene (3rd ed.), page 266; see also Giglioli's Fermenti e Microbi, pp. 247-257. 220 A MANUAL OF BACTERIOLOGY essential oil secreted by the trees contains a hydro- carbon— C10H16 ; and as this is vapourised, it is re- solved in the presence of atmospheric oxygen and moisture into camphoric peroxide, camphoric acid, and hydrogen dioxide l : — (a) 2 010H16+502 = 2C10H1404+2H20, (/9) C10H1404+2H20 = C10H1604+H202; and it is the hydrogen dioxide, so produced, which destroys the microbia of malaria. In the treatment of malaria certain medicinal substances are used. (1) Tommasi-Crudeli 2 recom- mends arsenious acid in small doses ; and, according to many English authorities, Fowler's solution (con- taining 1 part of arsenious acid in 120 parts of water) should be prescribed in 5 to 10 minim doses three times a day. (2) Quinine salts, in large doses, have also been recommended, especially by travellers who have had to pass through malarial districts. TYPHOID FEVER. The microbe of this disease has been found in Peyer's glands, the spleen, larynx, lungs, liver, and 1 Mr. C. T. Kingzett, F.C.S., manufactures these substances on a large scale. He decomposes the essential oils (principally turpentine oil), in the presence of water, by passing a current of air into them, the products being sold as ' Sanitas ' fluid and oil, both of which are powerful germicides. Kingzett imitates the decomposition of the essential oils by a similar process as the one which goes on naturally in the eucalyptus, pine, and camphor forests. It may be stated that 0'4 gramme of ' Sani- tas' oil completely destroyed Micrococcus prodigiosus, Bac- terium allii, Bacillus tuberculosis, and Bacillus subtilis when grown in various media as tube-cultivations. " Atti della R. Accademia del Lincei, 1885. INFECTIOUS DISEASES AND MICROBES, ETC. 221 in the lymphoid follicles of the intestine in fatal cases. Sometimes the microbe is present in "the kidneys and urine. Bacilhis typhosus measures from 2 to 3 ft long, and from 0*3 to 0'5 //, wide ; and it forms filaments which sometimes measure 50 //, in length. It (Fig. 33, 4) has rounded ends ; and it has been stated that spore -formation takes place at the extremities of the rods. This statement is, however, doubted by some bacteriologists; because the so-called spores have never been observed to germinate, etc. B. typhosus grows on bouillon, nutrient gelatine,1 steamed potatoes (at 37°C.), and blood serum ; and it can grow either in the presence or in the absence of free oxygen. On gelatine- plates, the microbe gives rise to greyish colonies with irregular margins, without liquefying the gela- tine. In tube-cultivations, a growth appears as a bluish-grey film on the surface, whilst 'in 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 sterilised potatoes. It is characteristic in that, even when there is a most luxuriant growth of the typhoid bacillus, it cannot be recognised by the naked eye, even at the end of three or four days, except by a peculiar moist ap- pearance 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 microbe from all others. It will be remembered, however, that the potato is slightly acid ; and it 1 Gaffky in Mitth. aus dem. k. Getundheitsamte, 1886. 222 A MANUAL OF BACTERIOLOGY 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.' Fraerikel and Simmonds1 state that this microbe is the cause of typhoid fever, for they have pro- duced the disease in monkeys, mice, and rabbits, by inoculation, from a pure cultivation of the microbe. Many other microbes (especially micrococci2) 'appear in the intestines when the disease is ap- proaching its end, but the bacillus in question is the only one found in the blood and internal organs [as well as in the roseolous eruption], so that it is really characteristic of the disease.' According to Janowski,3 the action of light is detrimental to the growth of B. typhosus; and he has also proved that a temperature of 55°C., con- tinued for ten minutes, destroyed the microbe. Although destroyed at 55° C., B. typhosus has been found alive in ice which had remained continuously frozen for a period of 103 days;4 and Dornil has discovered that ice is often a medium for transmit- ting infectious diseases — especially typhoid fever. But if ice is a means of transmitting typhoid fever, potable water is a much more dangerous source of infection. ' The remarkable instance which occurred at the Caterham Waterworks (1879), where by the 1 Die Aetioloyische Bedeutung des Typhus-bacillus, 1886. 2 Klein, Reports of Medical Officer of the Privy Council, 1875. 3 Centralblattfur BaJcteriologie, Bel. 8 (1890). 4 F. Davis's Handbook on Potable Water (1891). INFECTIOUS DISEASES AND MICROBES, ETC. 223 merest accident of one workman suffering from typhoid fever, who went down into the well and worked there a few hours, and defiled the well, thus contaminating hundreds of millions of gallons of water which were pumped out and distributed to the townspeople round about, four hundred cases of typhoid fever followed the next week, and seventy or eighty deaths occurred in consequence ' (Hogg). Certainly this instance proves that water is a source of infection ; but potable water is more frequently contaminated by the excreta of patients suffering from typhoid fever ; and when such is the case, an epidemic of typhoid fever is the result of drinking such water. In 1874, an epidemic of this disease broke out at Over-Darwen, when 2035 persons were attacked, which terminated in 104 deaths. The outbreak was traced to the water supply. In 1884, a similar epidemic broke out at Zurich j1 the origin of which was traced to the water of the river Limmat having been polluted with sewage contain- ing typhoid-fever dejecta. Epidemics of typhoid fever have also occurred at Florence,2 Vienna, Home, Naples, etc., which have been traced to potable waters having been contami- nated with the evacuations of typhoid- fever patients.3 1 Revue d* Hygiene, 1885. 8 Tommasi-Crudeli in Istituto di Anat. Patologico (Turin), 1882, p. 154. 3 See also Thome's Reports to Medical Officer of Local Govern- ment Board, 1880, et seq. ; Cassedebat in Comptes Rendus de VAcademie des Sciences, vol. ex., and Annales de VImtitut Pasteur, 1890 ; Giglioli's Fermenti e Microbi, pp. 268-282 ; Dr. E. Frankland's Experimental Researches in Pure, Applied, and 224 A MANUAL OF BACTERIOLOGY As the stools or dejecta of typhoid-fever patients contain the typhoid bacilli, they are highly infec- tious ; consequently they should always be disin- fected before being thrown away. This would greatly interfere with the spread of the disease. Several authors have recommended carbolic acid or mercuric chloride for disinfecting the stools ; but iron sulphate, according to Jalan de la Croix, is far more powerful than carbolic acid, and is only slightly inferior to mercuric chloride : besides, iron sulphate is a cheap disinfectant, non-poisonous and inodorous, and therefore may safely be recom- mended for the purpose of disinfecting the stools of patients suffering from typhoid fever and other infectious diseases. The author1 has proved the high value of iron sulphate as a germicidal and fungicidal agent ; and this compound readily de- stroys Bacillus typliosus. It may be stated that Dr. Proust 2 has used, for a number of years, iron sulphate to disinfect the stools in cases of typhoid fever. Bacillus typhosus forms a ptomaine, which has been extracted from pure cultures of the microbe, ill glycerine-bouillon (3: 100), by Brieger.3 This Physical Chemistry, p. 605 ; and S. T. Griffiths in the Tarn- worth Herald, August 15 and 22, 1891. 1 Proceedings of fioyal Society of Edinburgh, vol. xv. ; Journal of Chemical Society, 1883-87 ; Chemical News, vols. xlvii.-lvi. ; Bulletin de la Societd Chimique de Paris, 1889, p. 667 ; The Diseases of Crops (G. Bell & Sons). s Traitd tf Hygiene. 3 Untersuchungen ilber Ptomaine, 1886, p. 85 ; and Virchow's Archiv, 1889, p. 488. See also Gautier's Chimie Bioloyitjiie (1892), p. 269. INFECTIOUS DISEASES AND MICROBES, ETC. 225 base, which has been called typhotoxin (C7H17N02), dilates the pupil, produces diarrhoea, and rapidly kills animals. Luff l has also extracted a ptomaine from the urine of typhoid fever patients ; but no formula has been given to this base (i.e. it has not been submitted to quantitative analysis). Dr. Lauder Brunton says, in regard to typhoid fever, that 'the symptoms do not point so much to the c formation of a poison affecting the body generally, as to the local action of the microbes upon the intestines, although in some epidemics of typhoid fever the intestinal symptoms are but slightly marked, while bronchial irritation is due to the action of a microbe or to a ptomaine pro- duced by it on the bronchial mucous membrane/ CHOLERA. Since the great epidemic of 1832, cholera lias had a peculiar fascination for those interested in the subject; for the disease has always been shrouded in mystery until recent times. ' Before the three last epidemics (1865, 1873, 1884) cholera usually came to Europe by what may be called the Continental 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 1 British Medici Jwmal, 1889, p. 193. P 226 A MANUAL OF BACTERIOLOGY whole of Europe.' The native habitat or the endemic area of this terrible disease is in India — especially in the delta of the Ganges. ' 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 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 dis- charges of choleraic patients, without, however, being able to assign to them or prove for them any specific rdle in the causation of the disease.' x Since 1848, many scientists have been at work trying to establish a specific cause of cholera ; but it was not until 1884 that Dr. R Koch2 discovered the comma bacillus in choleraic dejecta, etc. Although many distinguished pathologists have not accepted Koch's evidence of the bacillary nature of Asiatic cholera, there can be no doubt, after the important and extensive researches of Drs. Macleod and Milles,3 that the comma bacillus of Koch is the cause (directly or indirectly) of Asiatic cholera. The comma bacillus or Spirillum cholerce Asiaticce measures from 1'5 to 2-5 //, long and O6 p broad (Fig. 33, 3). It occurs singly, in pairs often S~ shaped, in filaments which are screw-shaped, and 1 Woodhead's Bacteria and their Products, p. 151 (W. Scott). 2 Deutsch. Med. Woch., 1884; Berlin Klin. Woch., 1885. s Proceedings of Royal Society of Edinburgh, vol. xvi. p. 18. INFECTIOUS DISEASES AND MICROBES, ETC. 227 in zooglcea, and it is motile and aerobic. Numbers of this microbe are found in the ' rice-water ' stools formed by the desquamation of the mucous mem- brane of the intestines. They also occur in the in- testinal follicles, and in the sub-epithelial spaces, and probably in the kidneys and urine. There are several other comma-shaped bacilli, but these differ in many respects from the microbe which Koch has so frequently found in choleraic dejecta. The following is the list of the other comma-shaped bacilli, with the names of their dis- coverers : — (a) Finkler and Prior's bacillus (Spirillum Fink- leri), found in cholera nostras. It is thicker than Koch's bacillus ; and the colonies on gelatine plates are much larger than those of the comma bacillus of the same age. (6) Lewis's Spirillum sputigenum was found in the saliva ; it is thicker than Koch's bacillus, and is quite distinct from the latter microbe, (c) Miller's bacillus was found in some cases of caries of the teeth ; it is similar to Tinkler's bacillus. (d) Kuisl's bacillus, found in human faeces, is also similar to Tinkler's bacillus, (e) Spirillum tyrogenum (see Fig. 24) of Deneke is smaller than Koch's bacillus. It occurs in old cheeses, and, unlike the comma bacillus, it will not grow on steamed potatoes. (/) Klein's bacillus was found in some cases of diarrhoea, especially in monkeys. It grows differently in gelatine, giving rise to an offensive smell, (g) Ermengem and others have found comma-shaped bacilli in the intestines of guinea-pigs, pigs, rabbits, horses, etc., 228 A MANUAL OF BACTERIOLOGY but, unlike Koch's bacillus, they will not grow in 10 per cent, gelatine, (h) Lingard found two kinds of comma-shaped bacilli in a case of noma, the smaller of which is said to have been similar to the choleraic one. (i) Gamaleia's bacillus was found in a fatal fowl disease which was prevalent at Odessa, (j) Weibel found various forms in mucus, but their mode of growth is distinct. Koch's Spirillum cholerce Asiatics is always present in Asiatic or malignant cholera, and it has not been found apart from this disease, and dis- appears from the body with the disease. Its habitat is the intestinal canal, and the detection of this bacillus enables the physician more readily to diagnose the earliest cases in an epidemic of cholera. Ermengem,1 Watson Cheyne,2 Koch,3 Nicati and Kietsch,4 Macleod and Milles,5 and others, have produced the disease in dogs and guinea-pigs by inoculation with pure sub-cultures of Koch's comma bacillus. The last two investigators have arrived at the following conclusions concerning cholera and its microbe : — (a) The comma bacillus (Koch's) is always present and associated with certain changes in the small intestine in cases of Asiatic cholera. (&) There is no evidence to show that it is a normal inhabitant of the human alimentary canal, and 1 Recherches sur le Microbe du GhoUra Asiatique (1885). 2 British Medical Journal, 1885. 3 ' Etiology of Cholera ' in Laycock's Microparasites and Disease. 4 Revue d' Hygiene, 1885 ; Archives de Physiologic, 1885. 5 Loc. cit., pp. 18-35. INFECTIOUS DISEASES AND MICROBES, ETC. 229 therefore no proof of the assertion that it is a result of the disease, (c) The means used to introduce the comma bacillus into, and those used to lessen the peristalsis of, the small intestine of the guinea- pig, cannot be regarded as causing appearances like those of Asiatic cholera, or as causing the death of the animal, far less a mortality of over 60 per cent. (d) Pure cultivations of the microbe are pathogenic to the guinea-pig, (e) The contents of the ileum from those animals killed by injections of pure cultivations of the bacilli act in the same manner as pure cultivations of that microbe. (/) The microbe multiplies in the small intestine of the animal, and there is associated therewith changes similar to those in man in Asiatic cholera, (g) As there are conditions which favour the passage alive of the comma bacillus through the stomach of the guinea-pig, and also conditions which favour its multiplication in the small intestine of that animal; so in man, as there cannot be a doubt that the microbe finds conditions favourable to its multipli- cation in his small intestine, it must have found conditions favourable to its entrance alive through, in all probability, the mouth and the stomach (Macleod and Milles). The comma bacillus grows in neutral bouillon, gelatine, agar-agar, milk, and on steamed potatoes. It grows best if the medium is slightly alkaline, and at a temperature ranging from 16° to 40° C. On gelatine plates the colonies (Fig. 44) are evident in about twenty-four hours, and appear, under a low power, as small, somewhat irregular pale masses. 230 A MANUAL OF BACTERIOLOGY These gradually increase in size, and, where near the surface of the gelatine, a small depression forms over them, so that, on looking from the side at the surface of such a cultivation, it presents numerous little depressions instead of the original smooth surface of the gelatine, each depression correspond- ing to a colony of these bacilli. As the colony in- creases in size it becomes less compact, and the gelatine in the immedi- ate vicinity becomes fluid.1 At this stage zooglcea are formed. The colony goes on in- creasing in size for a few days, but ultimately ceases to extend, or extends only very slow- ly. Tube-cultivations are also characteristic. In twenty-four hours, at a temperature of 18° C" gr0wth is evident along the needle- track as a whitish line, broader at the upper part, and gradually tapering to the lower. At the upper part of the gelatine there is a slight depression, and during the next twenty-four hours the growth becomes more marked, the depression increasing in size so as to look like 1 Dr. Lander Brunton has shown that this liquefaction is due to a [ferment (enzyme) secreted by the comma bacillus (Proc. Roy. Soc., vol. xlvi. p. 542). FIG. 44. COLONIES OF CHOLERA-BACILLI ON GELATINE-PLATE. (X80.) INFECTIOUS DISEASES AND MICROBES, ETC. 231 an air-bubble at the top of the track. In the following days the gelatine at the top becomes liquid, and this liquidity extends gradually to the bottom of the track, thus there is a funnel-shaped appearance from the greater amount of the fluid at the top than at the bottom. At the same time, the mass of bacilli falls to the bottom of the fluid and assumes a somewhat rosy colour, so that there is a rose-coloured convoluted string running down the lower part of the track. The fluid at the upper part, which in about a week has extended to the sides of the tube, becomes clear, except a very thin layer at the top, which remains opalescent, the top itself being often covered with a very fine scum. Scattered over the solid gelatine forming the sides of the funnel are seen numerous small irregular • refracting particles. These are FlG. 45. TUBE-CULTIVATION the small zooglcea masses which OF CHOLERA-BACILLI. . . ,, ^ . , , (After Watson Cheyne.) have fallen to the sides and bottom of the funnel-shaped cavity (Fig. 45), and which Dr. Watson Cheyne considers the most typi- cal appearance during the growth of the comma bacillus in tube cultivations. On agar-agar the comma bacillus grows fairly well, but it does not liquefy this medium. On blood serum (at 37° C.) this microbe grows most luxuriantly. It also grows in milk, but gives rise to no noticeable alterations ; 232 A MANUAL OF BACTERIOLOGY ' it may, therefore, be readily understood how deadly the cholera microbe may become if it once finds a resting-place in milk.' 1 Brunton, Lewis, and Cunningham, Klebs and Cantani, and others, have all obtained indications of a poison or ptomaine in cholera dejecta. Pouchet, Brieger, and Yilliers have extracted several ptomaines from cholera dejecta, as well as from pure cultivations of the comma bacillus (Brieger). Dr. Lauder Brunton 2 says, c The symptoms occur- ring in cholera are probably due to the action on the tissues of a poison [or poisons] generated by the microbe, and not of the microbe itself, just as intoxication is due to the alcohol produced by the yeast plant, and not to the action of the plant itself on the nervous system and blood.' Besides the ptomaines produced by the comma bacillus, this microbe secretes a soluble enzyme.3 Cholera follows the course of rivers. Moisture in the atmosphere and the soil is needed for its distribution. Moist winds spread it, but the great factor in the distribution of cholera, as already stated, is human intercourse. Although human intercourse is the chief factor in distributing this 1 Hence milk adulterated with water from districts in which there are persons suffering from cholora may be the means of causing an epidemic of the disease. The same may be said of typhoid fever. 2 Disorders of Digestion (1888), p. 41 ; see also pp. 292 and 263 ; and Practitioner, 1884, et seq. 3 See Dr. Brunton's paper in Proc. Roy. Soc. , vol. xlvi. p. 542 ; and Dr. G. E. C. Wood's paper in Proc. Roy. Soc. , Edinburgh, vol. xvii. p. 29. INFECTIOUS DISEASES AND MICROBES, ETC. 233 disease, potable water is one of the most convenient vehicles for the distribution of the comma bacillus. If the dejecta of one or more choleraic patients contaminate a water supply, the water becomes a medium for spreading the disease. Such are the conclusions of Koch, Macnamara,1 and many other observers. 'In India, in the regions in which 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 consider- able 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 recep- tion of sewage and excreta of all kinds, especially in times of illness, when neither patients nor nurses have strength or time to see these are properly removed.' The recent epidemics of cholera in India, Spain, Japan,2 and other countries, have been traced to the water supply ; 3 and it is stated that the epidemic of 1884 killed 80,000 persons in Spain alone.4 But it may be stated ' that with all the improvements that have been made in the drainage system and water supply of Lower Bengal, cholera 1 British Medical Journal, 1884, p. 502. 2 An epidemic of cholera or korera-byo (as the Japanese call it) occurred in Japan in 1890, and there were 13,141 deaths out of 21,116 cases (vide Sir Edwin Arnold's Seas and Lands [1891], p. 474). 3 Lancet, 1885, et seq. 4 Giglioli's Fermenti e Microbi, p. 300 *eq. 234 A MANUAL OF BACTERIOLOGY 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/ It should be borne in mind that in cases of cholera, isolation and disinfection are absolutely necessary to prevent the disease spreading.1 For further information on the subject of cholera and its microbe, the reader is referred to the under- mentioned works.2 GLANDERS. This contagious infective disease is caused by the Bacillus mallei (Fig. 33, 12), which has been found in the lungs, liver, spleen, and nasal membranes of horses and sheep dead or dying from glanders. The same microbe has been found in human glanders or farcy ; and the death of Dr. Hoffmann, of Vienna, in 1889 is a standing proof of the patho- genic nature of this microbe, and its being the cause of the disease known as glanders.3 In man, this 1 Cameron's The Cholera Microbe and How to meet it (Bailliere & Co.). 2 Klein's Bacteria in Asiatic Cholera ; Brunton's Disorders of Digestion (1888), p. 262; Thome's 'Sea-Borne Cholera' in British Medical Journal, 1887 ; Straus, Roux, Nocard, and Thuillier in Comptes Rendus de la Sotiete de Biologie, 1883 ; and Bellews's History of Cholera in India (1885). Griffiths' Researches on Micro-Organisms, p. 15. INFECTIOUS DISEASES AND MICROBES, ETC. 235 microbe has been found in the blood and pus of the ulcers. According to Loftier, glanders is essentially a disease of hot countries, ' where the comparatively high temperature appears to be extremely favour- able 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 glanders per annum per 100,000 horses in the following countries : — Norway, 6 ; Denmark, 8'5 ; Great Britain, 14; Sweden, 57; Wurtemburg, 77; Russia, 78; Servia, 95; Belgium, 138; the French Army, 1130; and the Algerian Army, 1548.' B. mallei measures from 2*5 to 5 p long, and about one-fifth of its own length broad. It grows on blood serum (at 38° C.), sterilised potatoes (at 37° C.), in neutral solutions of extract of beef (at 37° C.), and in various vegetable infusions. Horses, asses, cats, rabbits, mice, and guinea-pigs, inoculated with a few drops of a pure cultivation of this microbe, have died with the characteristic lesions of glanders (glanderous ulcers and modules in the internal organs, and on the nasal septum). Stables, in which glanders has occurred, should be thoroughly washed out with a 2-per cent, solu- tion of carbolic acid or some other equally powerful disinfectant. DIPHTHERIA. Diphtheria is an extremely infectious disease which attacks man and certain animals. 236 A MANUAL OF BACTERIOLOGY Two microbes were originally isolated by Klebs and Loffler from human diphtheritic membranes; but Dr. Klein1 has shown that the Klebs-Loffler bacillus No. 1 is not constant in diphtheritic mem- branes, does not act pathogenically on animals ; and does not grow on solid gelatine at 20° C. In fact, this microbe has been termed the pseudo-diphtheria bacillus. The other species, Klebs-Loffler bacillus No. 2, is always present in diphtheritic membranes — in fact, it is present even in the deeper layers of \ ,' FIG. 46. BACILLUS DIPHTHERIA (Klein). A, The Bacillus x 1000. B, Section through the mucous membrane of pharynx of a child dead of diphtheria. C, Colonies from a plate-cultiva- tion of B. diphtheria. the membranes in great masses, and almost in pure culture. This microbe acts virulently on animals, and grows on gelatine at 19-20° C. Klein considers this bacillus to be the true microbe of diphtheria (Fig. 46 A and B). On the slanting surface of gelatine in tubes, 1 'Etiology of Diphtheria' in Reports to Local Government Board, 1889-90, p. 143; Proc. Roy. Soc., 1890; Centralblatt fur Bakteriologie, Bd. vii. (1890). INFECTIOUS DISEASES AND MICROBES, ETC. 237 Bacillus diphtherias (No. 2) gives rise to greyish dots after 36-48 hours' incubation at 20° C. After three or four days, these appear as white round convex droplets, which ultimately aggregate together forming yellowish -brown colonies. Colonies are also formed when the microbe is grown as a plate- cultivation (Fig. 46 C). In alkaline bouillon, B. diphtherice gives rise to a turbidity in twenty-four hours after inoculation; and afterwards a greyish- white precipitate is produced at the bottom of the tube. In milk kept at 18-20° C., this microbe grows very rapidly. The milk always remains fluid ; but in two or three days after inoculation, flakes of casein separate. B. diphtherice (from 3 to 6 /z, long) does not pro- duce spores ; but it gives rise to a soluble enzyme and one or more ptomaines. Drs. Eoux and Yersin l isolated an enzyme, from a pure cultivation of the microbe in question, which produces all the symp- toms of diphtheria. This is a true enzyme, for boiling water destroys its action. The author2 extracted a ptomaine (C14H17N206) from urine in cases of diphtheria; and the same ptomaine was also obtained from pure cultures of B. diphtherice on peptonised gelatine. This ptomaine is not present in normal urine. Brieger and Fraenkel3 have also isolated a toxalbumin from 1 Annales de VInstitut Pasteur, 1888. 2 Griffiths in Comptes Rendus, vol. cxiii. p. 656 ; Nature, vol. xlv. p. 72. 3 Berlin Klin. Woch., Bd. xxvii. pp. 241 and 1133. 238 A MANUAL OF BACTERIOLOGY pure cultivations of the microbe. This substance is said to have produced toxic effects when injected into animals. ' These observers, however, did not separate from the albumoses that were formed any enzymes that might be present, consequently they were working with a mixture of substances. The products that they obtained gave most of the re- actions of albumoses ; they were certainly toxic, but they probably contained both enzymes and albumoses' (Woodhead). B. diphtherice (No. 2), which is identical with those of Koux and Yersin, Zarniko, Escherich, and Loffler, acts very virulently on guinea-pigs on sub- cutaneous inoculation : at the seat of the injection a tumour is produced, which in its pathology and in microscopic sections, completely resembles the diphtheritic tissue of the human subject. In human diphtheria B. diphtherias is present only in the diphtheritic membrane, but neither in the blood nor in the diseased viscera ; the same holds good of the experimental guinea-pigs. In sub- cutaneous inoculation with artificial culture, though it causes in these animals acute disease and death — the lungs, intestine, and kidney are greatly con- gested— the diphtheria bacillus remains limited to the seat of inoculation (Klein). Klein has shown that this microbe also attacks the cat and cows, as well as man and the guinea- pig. But, unlike human diphtheria, the disease locates itself in the lungs of the cat (Fig. 47), i.e. the lung is the organ in which the diphtheritic process in the cat has its seat. The domestic cat INFECTIOUS DISEASES AND MICROBES, ETC. 239 is, therefore, a means of introducing diphtheria into a household. Klein has also shown that a definite disease can be produced in the cow by the B. diphtherice, con- sisting of a diphtheritic tumour at the seat of inoculation with copious multiplication of the bacilli, a severe pneumonia, and necrotic change in the liver; the contagious nature of the vesicular eruption on the udder and excretion of the bacilli in the milk prove that in the cow the bacilli are absorbed as such into the system. The mor- phological characters and the pathogenic ac- tion of these bacilli from milk were exactly the Same as those from FjG 47 BACILLUS DIPHTHERIA human diphtheria. Ac- (Klein.) COrding tO the Same ^presents a cover-glass preparation of fresh lung exudation from a cat that authority, 1 litre (T76 died of naturally acquired diphtheria in •m'nftA nf -millr Prmrmnorl a house ^herein diphtheria afterwards Q attacked the children of the household. between 30,000 and (x 1000.) 40,000 bacilli; therefore, there is little doubt that cows suffering from diph- theria are capable of transmitting the disease to human beings by means of the milk ; and human beings suffering from the same disease may also infect a milk-supply, and so spread the disease among the consumers of such milk. Dr. G. Turner1 states that fowls, turkeys, and 1 Reports to Local Governme.nt Board, 1886-7, p. 3. 240 A MANUAL OF BACTERIOLOGY pheasants also suffer from diphtheria, for he found the characteristic diphtheritic membranes in these birds; and he has also seen fowls and pigeons which had also been inoculated with diphtheritic membrane from a child's throat attacked with a disease which in all respects resembled what Turner regards as natural fowl-diphtheria. Similar accounts have been received from foreign bacteriologists,1 so that the identity and transmissibility of the dis- ease from fowls to men seems very probable. It may be stated, en passant, that Power 2 traced the outbreaks of diphtheria in 1886 at York and Camberley to the infectiousness of the milk- supplies ; and there is no doubt that milk is a medium in which other diseases besides diphtheria may be spread over a wide area. For some years, there has been a serious increase of diphtheria in this country, which Dr. Thome3 attributes to the increasing aggregation of children in elementary schools ; and Dr. Seaton,4 to the pre- sent systems of water-supply and sewerage. B. diphtherias is possessed of great tenacity of life. If it is dried and kept at 33° C. it is still alive after three months ; but at 45° C., this microbe is killed in four days. ' If a fragment of the false membrane containing bacilli be removed, wrapped in sterilised paper, or linen, and be carefully pro- tected from the action of light, cultivations may be 1 British Medical Journal, 1884; Journal d* Hygiene, 1884. 2 Report to Local Government Board, 1886, p. 311. 3 Diphtheria : its Natural History and Prevention (1891). 4 Report of International Congress of Hygiene, 1891. INFECTIOUS DISEASES AND MICROBES, ETC. 241 made from it at any time during a period of five months. If, however, instead of keeping it dry and in the dark, fragments of these membranes are exposed to the light and moistened and desiccated alternately, the virus is destroyed much more rapidly. From all this, and from the fact that the bacillus is destroyed by moist heat at 58° C., it is evident that by far the best method of disinfecting clothes, the floor, the walls, and furniture, is by the use of a liberal supply of boiling water ; for although a temperature of 98° C. (dry), continued for an hour, is necessary to destroy the vitality of the bacillus, moist heat at a very much lower degree (acting only for a minute or two, according to the temperature), is sufficient to disinfect everything on which it is allowed to act ' (Woodhead). Drs. Behring and Kitasato 1 have recently dis- covered a method of producing immunity against diphtheria. As this is similar to Kitasato's method of treating tetanus, which has been already described (see p. 214), no further remarks are needed. 'Antiseptic throat washes,2 not merely gargles, 1 Deutsche Medicinische Wochenschrift, 1890, p. 1113 ; and Zeitschrift fur Hygiene, 1890-1. 2 The following is an excellent antiseptic throat wash : — IJ. Potass, chlor. pulv. , 3 ij. Acid hydroch. fort, 3 j. Let stand mixed for 10 minutes, then add water gradually shaking each time to . f . 5 vi. I Syrup, . . f.5j. { To be used with a spray apparatus or syringe. This fluid not only loosens the diphtheritic membrane, but also destroys the bacilli. 242 A MANUAL OF BACTERIOLOGY plenty of fresh air, and good nourishing food, are what are required in the treatment of diphtheria. Kill the germs as far as possible by means of the antiseptics [germicides], and strengthen the tissue cells by plenty of oxygen, and by promoting the excretion of effete products, by food and exercise, so that the cells shall be able to form their protec- tive products, and shall also be able to play their part as phagocytes when called upon to do so.' It should be borne in mind that in diphtheria the bacilli are localised in the throat ; but the poisonous products (ptomaines and enzyme), which the bacilli form, pass into the system. If the bacilli are destroyed by germicides,1 these poisonous products cannot increase in the system ; and if they have not already accumulated in too large a quantity, they are readily excreted. ' Another important point is that the disappearance of the bacilli from the mouth is not simultaneous with the removal of the false membrane, and Roux and Yersin have found that the specific bacillus may persist in the mouth for several days (in one case fourteen days) after all traces of the membrane have disappeared, and they give the good practical advice that diphtheritic patients who are becoming convalescent should not be allowed to associate with their school-fellows, play-mates, or families, for at least a fortnight after the membrane has disappeared ; and that it is quite as important to wash out the throat freely three or 1 Dr. Wagner (Jour, fur PraJct. Chemie, vol. xi. ) has success- fully used a solution of salicylic acid in the treatment of diphtheria. INFECTIOUS DISEASES AND MICROBES, ETC. 243 four times a day with disinfecting lotions as that the clothes and bed linen should be thoroughly disinfected/ TUBERCULOSIS. Tuberculosis, in its varied protean guises, is one of the most widespread and deadly diseases in these northern latitudes. It has been stated that at any given time there are 200,000 persons in this country suffering from phthisis pulmonalis — the commonest form of the disease — and in each year nearly 70,000 persons die from it. The following tables show the death-rates per million from tuberculosis at different ages :— (a) From Phthisis. Age 10. Age 15. Age 20. Age 25. Age 35. Age 65. Age 75. Males, . . . 628 2093 3687 3941 4089 2152 752 Females, . . 1077 3019 3809 4175 3842 1364 546 (b) From other Tubercular Diseases. Age 5. Age 10. Age 35. Age 75. Males, .... 5008 641 103 94 Females, 3942 515 98 89 Tuberculosis is known by various names, according to the parts of the body the disease may happen to attack, or according to the kind of lesions it pro- 244 A MANUAL OF BACTERIOLOGY duces, or, finally, according to its general effect on the body. Thus phthisis or consumption, lupus, caseous pneumonia, cheesy inflammation of the lungs, consumption of the intestines, tabes mesenterica, tubercular pleurisy, ceseous broncho-pneumonia, scrofula, tubercular meningitis, etc., are all forms of the same disease, which is produced by a microbe — Bacillus tuberculosis — discovered by Pro- fessor R Koch1 in 1882. This bacillus lives in the blood and tissues, and gives rise to tubercles, which are small abnormal nodules of newly-formed tissue studding the diseased organ or organs. Each tubercle is made up of nucleated cells and tubercle bacilli, the latter being located chiefly in the giant cells. As the tubercles are continually being thrown off from the diseased person or animal, tuberculosis is an infectious disease. B. tuberculosis attacks other animals besides man ; among these may be mentioned cows, fowls, rodents, pigs, etc. Although tuberculosis is essentially the result of the action of Koch's bacillus, there are certain factors which render man and animals liable to contract the disease, and thereby receive the poison. These factors are deficiency of oxygen by bad venti- lation, foods (from tuberculous animals), certain diseases,2 starvation, inheritance, predisposition, etc. The last-named factor may be acquired through the system being of a lower standard than usual, or may be inherited. 1 Berliner Klin. Wochenschrift, Bd. xv. p. 221. 2 Among the diseases which render man liable to contract tuberculosis are syphilis, diabetes, measles, whooping-cough, etc. INFECTIOUS DISEASES AND MICROBES, ETC. 245 Tuberculosis, or that form of the disease known as phthisis (consumption), runs through certain families. There are two theories which account for the inheritance of phthisis — (a) that the tissues of children born of phthisical parents are especially favourable to nourish the tubercle bacilli; i.e. the tissues form a fertile soil for the subsequent growth of the microbes; (b) that the tubercle bacilli are actually contained in the ovum or among the sper- matozoa, and so become a constituent part of the embryo and foetus which develops within the uterus. Baumgarten records the fact that he has observed the tubercle bacilli in the ovum of the rabbit, and many observers have frequently seen the bacilli mingled with active spermatozoa. Pro- fessor Johne, of Dresden, discovered numerous tubercles in the lungs of a foetal calf of seven months intra-uterine growth. This proves that if the ovum had not been inoculated, it received the virus (i.e. the tubercle bacilli) through the placenta, which amounts practically to the same thing. Similar intra-uterine inoculation has been shown to be more than probable in the human being ; and Professor Burdon Sanderson1 believes that many cases of phthisis are congenital, i.e. dependent on causes which have operated before birth. Besides being hereditary, tuberculosis is also infectious, i.e. the disease is capable of being trans- mitted by direct or indirect infection from one host to another. There are four modes in which the tubercle 1 Report of International Congress of Hygiene, 1891. 246 A MANUAL OF BACTERIOLOGY bacilli enter the body, viz., by pulmonary inhala- tion (atmospheric infection), swallowing (enteric infection), direct inoculation, and heredity, (a) Inhalaton is the commonest mode of infection. Koch and numerous other observers have proved that animals, after a few inhalations of phthisical sputum, disseminated in a spray, readily become infected with tuberculosis. Eansome l has isolated the tubercle bacilli from the breath of patients suffering from advanced phthisis ; and the author 2 has confirmed Kansome's investigations ; therefore it will be seen that tuberculosis may pass from husband to wife, and vice versd ; and it may also affect members of the same family, not because of any hereditary taint, but through the simple fact of close companionship.3 The sputa or expectorations of phthisical patients are highly infectious, even after being desiccated for several months. Bacillus tuberculosis is often to be found in places lived in by consumptives ; and Prausnitz has lately collected the dust in various compartments of trains which often convey patients from Berlin to Meran, and inoculated a number of guinea-pigs with it. Two, out of five compartments so examined, were found to contain the bacillus; the dust of one rendered three out of four guinea-pigs tuberculous, while that of the other compartment infected two of these 1 Proc. Roy. Soc., 1882. 2 Proc. Roy. Soc. Edinburgh, vol. xvii. p. 268. 3 See Weber's book, The Gommunicability of Consumption from Husband to Wife ; and Heron's Evidences of the Communi- cability of Consumption. INFECTIOUS DISEASES AND MICROBES, ETC. 247 animals. The animals were killed after several months, and their organs had developed tubercles containing the characteristic bacilli. (b) Swallowing, or enteric infection, is a means of introducing the tubercular virus into the animal economy. Babbits, guinea-pigs, fowls, pigs, etc., become tubercular when fed upon tubercular tissues, sputum, saliva, milk, pure cultivations of the tubercle bacilli, etc. Klebs, Arloing, Chauveau, Villemin, Gerlach, Baumgarten, and others have shown, by direct experiment, that the milk, flesh, etc., ' from cattle affected with tuber- culosis would, when introduced alone or along with other food into the alimentary canal of rabbits, etc., give rise to tuberculosis in the pharynx, in the lymphatic glands of the neck, the stomach, intestine, omentum, liver, and spleen, and then, later, in other organs.' Many authorities state that the flesh of tuberculous animals (cattle, fowls, pigs, etc.) give rise to tuberculosis in human beings. On the other hand, there are authorities which state that there is not much danger of human beings contracting tuberculosis from eating meat from tuberculous cattle; but it is a unanimous opinion among all competent authorities that the milk of tuberculous cows is a source of great danger to human beings — often giving rise to tuberculosis, especially in chil- dren. It should be borne in mind that 'boiling always destroys the virulence, even when the milk contains bacilli, which is the case when the udder of the affected cow is itself tuberculous ;' and the risk of in- fection is greatly diminished, if not abolished, when meat from tuberculous cattle is thoroughly cooked. 248 A MANUAL OF BACTERIOLOGY The experiments of Galtier, Bang, and others have proved that the various products derived from milk — butter, cheese, and butter-milk — may all contain the tubercle bacilli, and that these retain their vitality in such products for a period of from four- teen to thirty days. The majority of these bacilli may be separated from milk if the cream is first removed by means of a centrifugal machine, but if the milk is very rich in bacilli a few usually remain in the milk, and even in the cream. In order to do away with this danger, it is necessary to expose the milk or the cream before churning to a temperature high enough to kill the tubercle bacilli (85° C. for about five minutes), (c) Direct inoculation is the third mode of infection. When tubercular matter or pure cultivations of the tubercle bacilli are intro- duced beneath the skin of susceptible animals, such as rabbits, guinea-pigs, cats, etc., they always produce, in four or more weeks, the typical tubercular lesions — swollen lymphatic glands, tubercles in the spleen, liver, and lungs, and en- largement and caseation of the bronchial glands. Besides, there are instances recorded in which sores on the udder of cows have infected with tuberculosis the hands of the persons milking them ; and it is not improbable that the common house-fly may disseminate the virus of phthisis by inoculating open sores on the hands and face (Spillman and Haushalter1). Bacillus tuberculosis measures from 2 to 8 yu, long and about 0*2 //, broad. It occurs in phthisical 1 Comptes Rendus, vol. cv. INFECTIOUS DISEASES AND MICROBES, ETC. 249 sputum (Fig. 48), in the cells of tubercles, and in the blood,1 tissues, urine,2 faeces, saliva,3 and sweat 4 of tuberculous patients. Watson Cheyne5 and other observers believe that the microbe is a spore- producing bacillus ; but this assertion is doubted by Lankester6 and others. B. tuberculosis has been cultivated artificially, and it has been proved that the strength of its virulence is not lessened by suc- cessive cultivations. When inoculated into various animals it always produces tuberculosis. The pre- sence of this microbe in the sputa of patients sup- posed to be suffering from phthisis is a ft-^-* certain diagnosis ; and it may be men- tioned that the mi- crobes are most numerous in the small caseous dots contained in the sputa. These dots should be searched for, then crushed between two cover glasses, dried, stained, and examined with high powers. B. tuberculosis grows on solid blood serum at 37° C. (i.e. the temperature of the body), and in eight 1 Weichselbaum in Wiener Med. Blatter, 1884. 2 Bates in Centralblatt fur d. Med. Wissemch., 1883, p. 145. 3 Griffiths in Proc. Roy. Soc., Edinburgh, vol. xv. p. 44. 4 Griffiths' Researches on Micro-Organisms, p. 268. 5 The Practitioner, 1883, p. 248. « Nature, 1884. FIG. 48. BACILLUS TUBERCULOSIS. A, From human sputum, a, Bacilli, b, Nuclei, x 1500. B, Bacilli, x 435. 250 A MANUAL OF BACTERIOLOGY or ten days after inoculation gives rise to whitish or yellowish drops or ' scales.' There is no lique- faction of the medium if the culture is perfectly pure. The bacillus also grows on the surface of bouillon (containing glycerine), forming a delicate thin film. Pawlowsky1 has grown the tubercle bacillus on sterilised potatoes ; but to succeed with this medium a considerable quantity of moisture must be kept in contact with the growing microbe. Nocard and Koux 2 have shown that most luxuriant growths of the tubercle bacillus are readily obtained when the microbe is grown on agar-agar and blood serum to which 6-8 per cent, of glycerine has been added ; but after many successive cultivations on these glycerine media, the virulence of the microbe becomes distinctly diminished. B. tuberculosis forms cellulose in the organs and blood of tuberculous persons ;3 and it has been recently stated that the microbe, when growing in glycerine bouillon, produces an albumose.4 The tubercle bacillus has great tenacity of life, for the author 6 has shown that it is capable of being dried up for three or four months at a temperature of 32° C. without losing its vitality : and Cornil was able to demonstrate that at the ordinary temperature of the room the tubercle bacillus, kept in water from the Seine, still retained its vitality after seventy 1 Annales de I'Institut Pasteur, 1888-9. 2 Annales de I'Institut Pasteur, 1887, p. 19. 3 See the author's Researches on Micro-Organisms, p. 155. 4 Crookshank and Herroun in British Medical Journal, 1891, p. 401. 5 Proc. Roy. Soc. Edinburgh, vol. xv. p. 42. Y ^r W* AttJB UFI7BESITT1 INFECTIOUS DISEASES AtfD MICROBES, ETC. 251 days' immersion in that medium. As already stated the best temperature for the growth of this bacillus is 37° C. ; at 40° C. its activity is diminished ; and at a temperature ranging from 50° to 60° C. it is killed. Boiling or strongly heating cultivations of all microbes destroys them, or, in other words, the media so treated become sterilised. Goethe knew nothing about microbes, yet, with the genius of a great poet, he makes Mephisto say : — * Der Luft, dem Wasser, wie der Erden Entwinden tausend Keime sich, Im Trocknen, Feuchten, Warmen, Kalten ! Hatt' ich mir nicht die Flamme vorbehalten, Ich hatte nichts Aparts f iir mich. ' In addition to the action of heat, sulphuretted hydrogen, ozone, a solution of salicylic acid, and the electric current (E.M.F. of 2'16 volts), all destroy the vitality of Bacillus tuberculosis.1 Although it is out of place to discuss the methods used in the treatment of infectious diseases in a manual devoted to general bacteriology, we give a very brief account of what is known as 'Koch's cure' for tuberculosis. Ever since Dr. Koch dis- covered the tubercle bacillus (in 1882) he has been endeavouring to obtain an inoculating fluid which would kill the bacilli, and bring about a sufficiently strong and healthy reaction to expel them from the body without, at the same time, destroying healthy organs. Such a fluid Koch believes he has dis- covered in his tuberculin,2 which is a glycerine 1 See the author's book, loc. cit., pp. 176, 182, 184, and 227. 2 Deutsche Medizinische Wochenschrift, Nov. 14, 1890, and Jan. 15, 1891. 252 A MANUAL OF BACTERIOLOGY extract from pure cultivations of destroyed tubercle bacilli. This so-called lymph contains mineral salts, colouring substances, unknown extractive matter, besides the dead bacilli. According to Koch, some of these substances can be removed from the 'lymph' tolerably easily. The effective substance is mainly insoluble in absolute alcohol, and can be precipitated by it, not, indeed, in a pure condition, but still com- bined with the other extractive matter, which is also soluble in alcohol. The colouring matter may also be removed, so that it is possible to obtain from the extract a colourless dry sub- stance, which contains the effective substance in a much more con- centrated form than the original glycerine solution. The effective FIG. 49. INJECTING KOCH'S ' LYMPH.' Substance appears to be a derivative from albu- minous compounds, and is closely allied to them. It is not a ptomaine ; but appears to be an enzyme ; and tuberculin contains less than 1 per cent, of this enzyme.1 The treatment consists in injecting, subcutane- 1 See also Hunter's paper in British Medical Journal, 1891 (ii), p. 169. INFECTIOUS DISEASES AND MICROBES, ETC. 253 ously, small doses l of diluted (with water) tuber- culin into the back of patients (Fig. 49) suffering from certain forms of tuberculosis; and as the treatment progresses the doses are slowly increased 'as long as there may be bacilli in the body.' Koch's ' lymph ' does not kill the tubercle bacilli, but destroys the tuberculous tissues, and thereby starves the bacilli contained in such tissues. It also sets up a localised reaction in the vicinity of the bacilli, by means of which the cells are so strengthened that they are able to prevent the extension of the bacilli into the surrounding parts ; in fact there is a battle between the cells and the bacilli, and if the former are strengthened, it is possible for them to destroy the latter ; and this is what Koch's ' lymph ' is believed to do. • As to the value of Koch's treatment, there is no decided opinion among those best capable of judg- ing ; for some authorities are against, while others are in favour of, the ' lymph ' as a diagnostic and curative agent. Professor K. Virchow 2 (the greatest living pathologist) ' has found, in a number of cases that have come under his observations,' — a compar- ative 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 localisation of the disease is not by any means perfect, that there is a tendency of tubercu- lous material that should be thrown off to continue 1 0-0005 to o-oi cc. 2 Berliner Klinische Wochenschrifl, Jan. 21, 1891, p. 49. 254 A MANUAL OF BACTERIOLOGY 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 tubercu- lous focus.' According to Dr. Cornil, tuberculous affections of the skin are ameliorated by Koch's remedy, but it should be sparingly employed in the incipient stages of phthisis ; and it is useless, and even dangerous, in advanced and acute cases of phthisis. Nevertheless, Professor Koch has made a great advance in the therapeutic treatment of infec- tious diseases.1 ANTHRAX. The disease known as anthrax, splenic fever, splenic apoplexy, or malignant pustule, is a disease affecting man and animals. ' In some countries the losses to agriculturists and farmers owing to the fatal character of the disease in sheep and cattle is enormous. In man it is chiefly known among wool- sorters and those engaged in the handling of hides. This disease has been definitely proved to be due to the Bacillus anthracis, which, after its entry into the system of an animal or human being, multiplies very rapidly in the blood and spleen, and, as a rule, pro- duces a fatal result, at any rate in sheep and cattle.' 1 Various methods for treating phthisis are detailed in the author's book: Researches on Micro -Organisms, pp. 286-3] 9 (Bailliere & Co.) ; and see also Dr. Drewitt's paper in Trans. Clin. Soc., 1887. Drewitt treated a child suffering from lupus partly by scraping and partly by salicylic acid. INFECTIOUS DISEASES AND MICROBES, ETC. 255 Bacillus anthracis measures from 5 to 20 /i long, and from 1 to 1*25 //, broad (Fig. 50), and often occurs in masses of filamentous threads. It pro- duces oval spores, and when either the bacillus or its spores are injected into mice, guinea-pigs, sheep, rabbits, etc., they die with all the characteristic B FIG. 50. BACILLUS ANTHRACIS. A, Bacilli (a) forming spores ( x 1200). B, Convolutions of bacillary threads (x 320). lesions, etc., of anthrax. Even the inhalation of the spores is capable of giving rise to anthrax in man and susceptible animals. B. anthracis has been found in the blood, spleen, and other organs, also in the urine and faeces of animals suffering from or 256 A MANUAL OF BACTERIOLOGY dead of anthrax. This microbe grows in nutrient gelatine, agar-agar, neutral bouillon, and on steamed potatoes at all temperatures between 15° and 43° C., best between 25° and 40° C. Free access of air is essential for B. anthracis to produce spores. Suc- cessive cultivations of this microbe do not weaken its virulence. On gelatine plates it gives rise to small white colonies after two or three days' incuba- .tion. When these colonies are examined under low power they appear as masses of twisted threads, but in cover-glass preparations (Fig. 50 B) these thread-like filaments are readily observed. In tube- cultivations the bacillus presents a characteristic appearance. Along the track of the needle there appear lateral growths which give the culture a peculiar feather-like appearance. But after a time the gelatine liquefies, and the growth sinks to the bottom of the tube, where the bacilli undergo de- generation. On agar-agar a similar appearance is presented, but there is no liquefaction of the medium. B. anthracis grows on steamed potatoes as a creamy- white granular mass. It has been stated that the anthrax bacillus pro- duces a ptomaine called anthracin and an albumose l from the medium on which it lives. Klein and Parsons2 have shown that anthrax bacilli without spores are destroyed in five minutes when exposed to a temperature of 103° C. (dry heat), but the spores are not destroyed until they have 1 Hankin in Proc. Roy. Soc., 1890, p. 93; and Martin in Nature, vol. xlii. p. 118. 2 Report to Medical Officer of Local Government Board , 1884. IXFECTIOUS DISEASES AND MICROBES, ETC. 257 been exposed to a temperature of 104°C. for four hours (dry heat). However, boiling in water for only one minute was sufficient to render inert the spores of B. anthracis. According to MM. Chamberllent and Moussons,1 anthrax bacilli have been discovered in the milk of cows affected with the disease, and not only is milk a means of giving rise to an outbreak of anthrax, but polluted drinking water derived from wells may also spread the disease. As already stated, successive cultivations do not weaken the virulence of Bacillus anthracis, but if the microbe is cultivated in neutral bouillon at 42° or 43° C. for twenty days an attenuated virus is obtained. Pasteur's premier vaccin protects animals against the disease ; but to make them perfectly refractory, they are inoculated a second time with a vaccine (deuodeme vaccin) of less strength. Attenu- ated viruses for the protective inoculation against anthrax have also been obtained by exposing the bacilli to a temperature of 55° C., or to an aqueous solution of carbolic acid (0*5 to 1 per cent.), or sul- phuric acid in a diluted form, as well as other chemicals. According to Hankin,2 immunity against anthrax is obtained by inoculation with the albu- mose derived from pure cultivations of the bacilli, and he has also cured animals suffering from anthrax by injecting the albumose into their bodies. 1 Comptes Rendus, vol. cvii. p. 142. 2 Report of British Association, 1890 ; and British Medical Journal, 1890. 258 A MANUAL OF BACTERIOLOGY ACTINOMYCOSIS. This disease attacks cattle and occasionally man himself. It is caused by the ray-fungus or Actino- myces. 'In cattle the disease manifests itself by firm tumours in the jaw, in the alveoli of the teeth, and particularly by a great enlargement and indura- tion of the tongue — 'wooden tongue' Occasionally these tumours occur in the skin and lungs. The ray-fungus has been cultivated on solid ox-serum, and when pure cultures are injected into animals they give rise to actinomycosis. THRUSH. This disease is caused by the fungus O'idium albi- cans. It is found on the mucous membrane of the mouth of infants, causing white patches on the tongue, gums, and soft palate. Like the higher fungi, this plant is composed of hyphse and spores, which take root in the mucous lining of the mouth. The spores are produced by the division of the ter- minal cells, or sometimes by endogenous formation within the hyphse. In concluding the present chapter we may say that most infectious diseases have a microbian origin, but there are some (e.g. typhus fever, whoop- ing-cough, mumps, etc.) in which no microbes have been isolated and cultivated apart from the body ; and there are other infectious diseases which owe their origin to small animal organisms, known as INFECTIOUS DISEASES AND MICROBES, ETC. 259 Protozoa. Dysentery and tropical abscess of the liver are due to Amcebce,1 and in India a fatal dis- ease (surra), which attacks horses, mules, and camels, is caused by one of the Flagellata.1 1 See Dr. A. B. Griffiths' book, The Physiology of the Inverte- brata (Reeve and Co.). CHAPTEK VII THE MICROBES OF THE AIR 'THE solid matter floating in the atmosphere is every day becoming of greater and greater interest as we are gradually realising the important part it plays in the economy of nature, whether viewed as to its physical, physiological, or meteorological aspects. One fundamental point on which we have at present very little information of anything like a definite character is as to the number of solid par- ticles present in the atmosphere. We know that they are very numerous, and it seems probable that the number varies under different conditions of weather, but what number of particles are really present under any conditions, and how the number varies, we have at present very little idea. In this field of research the physiologists are far in advance of the physicists, as they have devised means of counting the number of live germs floating in the atmosphere, and already we have a good deal of information as to how the number varies under different conditions.' Before describing the living particles in the atmosphere we allude to some recent investigations 260 THE MICROBES OF THE AIR 261 on the number of dead or inorganic particles con- tained in the air. Mr. J. Aitken, F.R.S.,1 has in- vented an ingenious apparatus by which the number of dust particles in the atmosphere may be readily estimated. Among the results obtained are the fol- lowing : — No. of Dust Particles in Air. Source of air. No. per cc. No. per cubic in. Outside (raining) Outside (fair) . Room Room near ceiling Bunsen flame . 32,000 130,000 1,860,000 5,420,190 30,000,000 521,000 1,119,000 30,318,000 88,346,000 489,000,000 These results indicate that ' there is most dust in the air during dry weather, and perhaps during anti-cyclonic conditions, and least during wet weather, and perhaps in cyclonic areas.' Aitken has also ascertained the minimum and maximum number of dust particles per cubic centi- metre (cc.) in the air of various towns, etc. Among these results are the following : — At Hyeres (near Toulon), . from 5000 to 46,000 I , Cannes, 1550 , 150,000 Lucerne (mountain air), 210 , 2350 Paris, . 92,000 , 210,000 London, 48,000 , 150,000 Ben Nevis (mountain air), 335 473 Dumfries, 395 , 11,500 Mentone, 1200 , 7200 1 Transactions of Royal Society of Edinburgh, vol. xxxv. p. 1 ; Proceedings of Royal Socie.ty of Edinburgh, vol. xvii. p. 193, and vol. xviii. pp. 39 and 259. 262 A MANUAL OF BACTERIOLOGY Aitken concludes (1) that the earth's atmosphere is greatly polluted with dust produced by human agency ; (2) that this dust is carried to considerable elevations by the hot- air rising over cities, by the hot and moist air rising from sun-heated areas of the earth's surface, and by winds driving the dusty air up the slopes of hills ; (3) that none of the tests made of the Mediterranean sea air show it to be very free from dust ; and (4) that the amount of dust in the atmosphere of pure country districts varies with the velocity and the direction of the wind : fall of wind being accompanied by an increase in dust. Winds blowing from populous districts generally bring dusty air. It is stated that a man in the town inhales about 37,500 germs every twenty-four hours, and no fewer than 2,250,000 inorganic particles every minute.1 ' Most of these are merely annoying, though a few are real messengers of disease and death. If the lungs are warm and moist, they can repel the particles ; but with cold and dry lungs the suffering from the clogging must soon begin/ Besides the inorganic or dead particles, the air is more or less laden with living particles. The majority of these are of the non-pathogenic or harm- less kind, but there is plenty of evidence to show that pathogenic microbes lurk about in the atmo- sphere, and that many infectious diseases are propa- gated by means of air-carried microbes. Hence the reason that the study of aerial microbes is peculiarly 1 A cigarette smoker sends no fewer than 4,000,000,000 of particles (more or less) into the air with every puff he makes. THE MICROBES OF THE AIR 263 interesting and attractive. The investigations of Burdon Sanderson, Tyndall, Lister, and Lankester have all thrown considerable light upon the condi- tions of life of these lower organisms ; but Pasteur was the first investigator who made a systematic Fio. 51. MIQUEL AND DE FREUDENREICH'S FILTER. A, Filter-tube (A, enlargement of same, with cap at d). B, Aspirator. C, Gasometer. study of the presence and distribution of microbes in the atmosphere. It was not, however, until 1879 that Drs. Miquel 264 A MANUAL OF BACTERIOLOGY and De Freudenreich attempted the quantitative estimation of aerial microbes. Their method con- sists in aspirating a known volume of air through a tube containing previously sterilised plugs of glass- wool (Fig. 51). The solid particles, including any microbes, are arrested ; and the plugs of glass-wool are then thoroughly mixed with a known volume of sterilised water. The mixture is now sub-divided into such a number of equal parts that each part shall contain not more than one microbe. Each of these sub-divisions is then introduced into a cultiva- tion tube or flask (see Fig. 17) containing sterilised bouillon. These tubes or flasks are placed in an incubator, and any that have received a living microbe will, in a short time, exhibit the fact by suffering visible alteration. As an example, sup- posing the plug through which twenty litres of air were drawn, by the aspirator (Fig. 51 5), was mixed with 25 cc. of sterilised water, and twenty-five tubes of bouillon were then each inoculated with 1 cc. of this mixture, and if, after a suitable incuba- tion, it was found that only sixteen of them suffered alteration, it would be concluded that only sixteen microbes were present in the 25 cc. of water distri- buted among the twenty-five tubes, or, in other words, that the twenty litres of air contained sixteen living microbes. Miquel and De Freudenreich have since substi- tuted soluble media (powdered sugar or de-hydrated sodium sulphate) for the insoluble glass-wool. By the use of soluble filtering media, there is no chance of any microbes becoming imprisoned, as is the case THE MICROBES OF THE AIR 265 when glass-wool is used. Drs. Miquel,1 Fol,2 Gautier,3 and other French bacteriologists use soluble filtering media ; and bouillon as the medium for the growth of microbes. In England and Germany solid cultivation media have been substituted for the liquid bouillon ; and when the microbian mixture is introduced into melted nutrient gelatine, it ' can be evenly dis- persed throughout the medium by gentle agitation, and by subsequently allowing it to solidify, the microbes are not only isolated, but rigidly confined to one spot. Thus each individual microbe becomes a centre round which extensive multiplication takes place, and in a few days definite points of growth are visible to the naked eye, which are appropriately described as colonies. Although each colony con- sists of many thousands, or even millions of in- dividual microbes, yet, as in the first instance, they owe their origin to a single organism or indivisible group of organisms, it is correct to regard the number of colonies as representing the number of microbes/ One of the best methods for estimating the num- ber of microbes in a known volume of air, is that devised by Dr. W. Hesse.4 Hesse's method con- sists in slowly aspirating a known volume of air through a glass tube (28 X If in.) which has pre- viously been coated internally with a film of sterilised nutrient gelatine. The microbes suspended in the 1 Annuaire, de VObservatcrire de Afontsourix, 1880-92. 2 La Nature, 1885. 3 Rente Scientijique, 1886. 4 Mittheilungen am dem Icaiserliche.n Oesundheitnamte, vol. ii. 266 A MANUAL OF BACTERIOLOGY air are rapidly deposited within the tube, and on the surface of the gelatine give rise to colonies. Fig. 52 represents Hesse's aeroscope. At D is an india- rubber stopper, perforated to admit a small glass Fig. 52. HESSE'S AEROSCOPE. tube, plugged with cotton-wool ; and at the opposite end is a perforated indiarubber cap, which is covered by an imperforated cap (C) of the same material. The aspirator consists of two like flasks (A B); one of which is filled with water. These flasks are reversible ; but the one containing the THE MICROBES OF THE AIR 267 water is always fixed uppermost when the air is passing through the tube. The down-flow of water causes the air to pass slowly through the tube when the outer cap (C) has been removed ; and as the flasks are of known capacity, two, five, ten, or more litres of air may be aspirated through the tube. After this the cap is replaced, and the tube is then removed to a warm situation for several days, in order that colonies may develop. Before introducing the nutrient gelatine, the tube, caps, and plug are sterilised by means of a solution of mercuric chloride, and finally with FIG. 53. GRIFFITHS' MODIFICATION OF HESSE'S AEROSCOPE. alcohol. After this treatment, 50 cc. of melted nutrient gelatine are poured into the tube, which is then sterilised in a steamer by the discontinuous method. The author has made a modification of Hesse's apparatus (Fig. 53), by substituting a small exhaust pump of known capacity for the aspirator. This modification is far handier and occupies less space than Hesse's aeroscope ; while it gives results which agree with those obtained with the original apparatus. The late Dr. T. Carnelley1 also modified Hesse's 1 Report of British Association, 1887, p. 654. 268 A MANUAL OF BACTERIOLOGY aeroscope by substituting a flat-bottom flask for the tube. Dr. P. F. Frankland1 has devised a method by FIG. 54. COLONIES IN FLASK. (After Frankland.) which a known volume of air is drawn, by means of an air-pump, through a short glass tube (4 x i in.) 1 Philosophical Transactions, vol. clxxviii. p. 113. THE MICROBES OF THE AIR 269 containing two small porous plugs placed one in front of the other. The first plug consists of glass- wool coated with sugar, whilst the second contains, in addition, a layer, £ inch in thickness, of fine sugar-powder. The microbes, suspended in the aspirated air, are deposited on these plugs, which are introduced into separate flasks, each containing a a small quantity of melted nutrient gelatine. Each flask is then agitated until the plug is disintegrated, and since the sugar-coating of the glass-wool dis- solves in the liquid gelatine, the microbes become immediately detached. The gelatine is now allowed to solidify, forming a thin film over the inner sur- faces of the flasks. The flasks are finally placed in an incubator ; and in a few days colonies derived from the microbes, which were collected by the plugs make their appearance and can be counted and further studied (Fig. 54). The author1 has examined the air of Lincoln, Paris, and London. The methods used for estimating the number of microbian colonies in a known volume of air were those of Hesse and Frankland. Before August 6th, 1888, Hesse's method was used, while after that date Frankland's method was substituted for that of Hesse. The average number of colonies in three gallons (fifteen litres) of air are given in the following tables : — 1 Proceedings of Royal Society of Edinburgh, vol. xvii. p. 265 ; and Researches on Micro-Organisms, p. 59. 270 A MANUAL OF BACTERIOLOGY THE Am OF LINCOLN. \ EAR 188 7. PLACE. 4 EC • -J 2 *n f>^ % ">* tiD -*f ^5 ^ •? £ * A * * ^ •5 1* o fe i (1) Top of hill 3 6 14 16 19 25 34 30 28 12 4 (near Cathedral), (2) Base of hill 18 26 30 41 50 62 65 59 57 19 17 (Broadgate), THE AIR OF PARIS. Place or Part of Paris. Situation in Paris. August, 1887. (1) Cimetiere du Pere la Chaise, E. 96 (2) Boulevard Saint -Germain, . Centre 104 (3) Forest of Ville d'Avray, S.W. 81 (4) Rue de Rennes, . . . . Centre 99 (5) Palais du Trocadero, . ' *. W. 50 (6) Park of Versailles, S.W. 78 (7) St. Cloud, , ..';.. S.W. 82 (8) Boulevard Voltaire, E. 100 (9) Cimetiere Montparnasse, S. 98 (10) Cimetiere Montmartre, N. 95 (11) Parcdes Buttes Chaumont, . N.E. 80 THE AIR OF LONDON. Place or Part of London. July, 1888. August, 1888. (1) Forest Gate (Essex). 64 79 (2) City (near Bank), . 85 110 (3) West End (Piccadilly), . 80 96 (4) East End (near Mint), . 88 106 THE MICROBES OF THE AIR 271 The conclusion drawn from these investigations are the following : — (a) There are a larger number of microbes in the V atmosphere during the summer than either the spring or winter. They appear to reach a maximum during the month of August. (&) The number of microbes found in the atmosphere decreases, the higher one ascends. Hence near the Lincoln Cathe- dral there are fewer microbes in the atmosphere (on any given day) than in the valley of the Witham. The same remark also applies to the number of microbes found in the atmosphere at the top of the Trocadero Palace, Paris, where there are fewer microbes than in a low-lying but crowded thorough- fare like the Boulevard Saint-Germain, (c) There are a" larger number of microbes in the atmosphere of crowded centres than in less densely-populated districts, (d) By gradually passing from a large city towards the country the number of aerial microbes decreases ; e.g., there are fewer microbes in the atmosphere of the Forest of Ville d'Avray, the Park of Versailles, and the village of St. Cloud, than in the principal thoroughfares of Paris and London. Dr. P. Miquel l (who is the greatest authority on aerial microbes) has published elaborate tables con- cerning the number of microbes in the air of certain parts of Paris. During the year 1888, Miquel obtained the following mean number of microbes in the air (per cubic meter) at Montsouris, and in the vicinity of the Hotel de Ville, Paris : — 1 Annuaire de T 'Obner vatoire de Montsouris, 1877-92. 272 A MANUAL OF BACTERIOLOGY SEASONS. Montsouris. Hotel de Ville. Winter, .... 171 2870 Spring, .... 210 8920 Summer, .... 400 12280 Autumn, .... 185 6800 Annual Means, 242 7720 The mean annual results (for eight years, 1881-88) of the number of microbes contained in one cubic metre of the air at Montsouris and in the vicinity of the Hotel de Ville (i.e. in the centre of Paris) are given in the following table : — Montsouris. Hotel de Ville. January, . .... 228 2310 February, 170 3140 March, .... 255 3420 April, 358 4340 May, 379 5950 June, 448 5070 July, 676 5200 August, .... 628 5640 September, .... 470 5510 October, .... 332 4335 November, .... 239 3700 December, .... 189 2885 From the above results it will be seen (a) that there are a larger number of microbes in the atmo- sphere in the centre of Paris than at Montsouris ; THE MICROBES OF THE AIR 273 (b) that there are a larger number of microbes in the atmosphere during the summer than any other period of the year. Miquel has also shown that as the number of microbes in the atmosphere increases, so does the mortality from zymotic or infectious diseases. The investigations of Dr. P. F. Frankland on aerial microbes have added considerably to our knowledge of this interesting subject. Frankland has not only examined the air so as to ascertain the number of microbes present in a known volume, but he has discovered many new forms.1 Frankland has obtained the following results concerning the number of microbes present in ten litres, or two gallons, of air at different places : — Primrose Hill (top), ... 9 „ (bottom), . . 24 Norwich Cathedral (top of spire, 300 ft. ), . 7 (tower, 180ft.), 9 ,, ,, (in the close), . . 18 St. Paul's Cathedral (Golden Gallery), . 11 (Stone Gallery), . 34 ' „ „ (Churchyard),. 70 >> £ f Reigate Hill, . J Heath near Norwich, q g I Garden at Reigate, . . 25 ° I Garden near Norwich, . . 31 § g [ Kensington Gardens, . . .13 -2^ { Hyde Park, . . 18 ^ j I Exhibition Road, . . 554 Frankland has also shown that within doors the 1 Philosophical Transactions, vol. clxxviii. p. 257. 274 A MANUAL OF BACTERIOLOGY number of microbes suspended in the air depends upon the number of people present and the amount of disturbance of the air which is taking place. Dr. Fischer1 has proved that sea air is almost free from microbes. Carnelley2 and Pe'tri3 have also shown that the air of sewers is remarkably free from microbes. This is due to the moisture on the walls of these subterranean channels. The following microbes are always present (more or less) in the atmosphere : — Micrococcus citreus conglomerate. Micrococcus violaceus. Micrococcus rosaceus. Bacterium indicum. Micrococcus prodigiosus. Bacterium aceti. Bacterium lactis. Micrococcus cyaneus. Bacterium xanthinum. Bacillus figurans. Micrococcus carnicolor. Micrococcus candlcans. Micrococcus albus. Sarcina lutea. Surcina aurantica. Bacillus Jluorescus. Micrococcus liquefaciens. Sarcina liquefaciens. Micrococcus gigas. Micrococcus chryseus. Bacillus aurescens. Bacillus aureus. Bacillus citreus. Bacillus plicatus. Bacillus chlor'mus. Bacillus polymorphic. Bacillus profusus. Bacillus pestifer. Bacillus Icevis. Bacillus cereus. Bacillus subtilis. Besides other microbes, there are always present in the atmosphere an abundance of moulds and yeast-fungi. Although the microbes connected with the com- mon infectious diseases have not been discovered in 1 Zeitschrift fur Hygiene, vol. i. 2 Philosophical Transactions, vol. clxxviii. p. 61. * Zeitschrift fur Hygiene, vol. iii. THE MICROBES OF THE AIR 275 air, ' yet there can be no doubt that, in the imme- diate vicinity of the foci of disease, such microbes are present, and that their distribution and convey- ance in the air will take place in just the same manner as in the case of non-pathogenic microbes. The investigations on aerial microbia, so far as they have as yet been carried, are of service in indicating how, we may escape from all microbes, whether harmful or harmless ; and secondly, how we may avoid the conveyance of microbes into the atmo- sphere from places where pathogenic forms are known or likely to be present. This acquaintance with the distribution of microbes in general, and the power of controlling their dissemination which it confers, is really of far wider practical importance than discovering whether some particular pathogenic form is present in some particular sample of air. It is this knowledge which has led to the vast improvements in the construction and arrangement of hospital wards and of sick-rooms generally, and which has directed attention to the importance of avoiding all circumstances tending to disturb and distribute dust. It is, moreover, this knowledge of the distribution of microbes in our surroundings which has formed one of the foundations for the antiseptic treatment of wounds — that great step in surgery with which the name of Sir Joseph Lister is associated.'1 1 For further information see Frankland's papers in Journal of Society of Arts, vol. xxxv. p. 485; Proc. Roy. Soc., 1885-86; Miquel's Les Organismes Vivants de I' Atmosphere; Prudden's Dust and its Dangers ; and Griffiths' Researches on Micro - Organisms. CHAPTEE VIII THE MICROBES OF THE SOIL SOIL is very rich in microbes, and these insignificant plants play a most important part in the processes of putrefaction and nitrification, which are always at work for man's benefit and welfare. Among the microbes present (more or less) in soil are the following : — Bacillus typhosus. Bacillus radicicola. Bacterium septicumagrigenum. Bacillus cedematis maligni. Streptococcus septicus. Bacillus subtilis. Bacillus toruliformis. Bacillus floccus. Bacillus septicus. Bacterium termo. Bacterium allii (?). The Nitrous Bacillus. The Nitric Micrococcus. Bacillus tar deer escens. Bacterium urece. Bacillus fiuorescens. Micrococcus cereus. Bacillus of Mouse Septiccemia. Bacillus mycoides. Bacillus anthracis. Bacillus of tetanus. Bacillus of malarice. Spirillum cholerce Asiaticce. In addition to the above, the spores, etc., of many of V the higher fungi are present in soil. Some of these are detrimental to the growth of vegetation, for they become internal or external parasites, and thereby produce disease.1 Not only are the higher plants attacked by parasites present in soil, but man and animals suffer from diseases, like tetanus, malaria, etc., which are caused by soil-microbes. 1 Griffiths' Diseases of Crops. 276 THE MICROBES OF THE SOIL 277 To study the microbes present in soil, both solid and liquid media are used, but the employment of the former is much more satisfactory. The follow- ing methods are used by bacteriologists to ascertain the number of microbes in a known weight, etc., of soil : — (a) A sample of the dried soil is triturated with sterilised distilled water, and then a small quantity of this water is sprinkled on the surface of a gelatine plate, (b) The soil is introduced into a test-tube containing liquefied gelatine. After a thorough shaking the mixture is poured out upon a glass plate, so as to form a plate- cultivation, (c) When bouillon is used the soil is first triturated with water, and then a drop of the water is transferred to a flask containing sterilised bouillon. Among the results obtained of the number of microbes present in various soils are the following : (A) GRIFFITHS' ANALYSES. Samples of Soil from— Number of Microbes in 1 gram. Lincoln (Monk's Road), ,, (Castle grounds), Manchester (Infirmary grounds), . ,, (Plymouth Grove), London (Forest Gate), .... ,, (Hyde Park), .... Paris (Forest of Ville d'Avray), . „ (Near Sevres) ,, (Pare Monceaux), Dieppe (near Church of St. Jacques), . ,, (near the Casino), New Zealand (after 14 weeks' desiccation), 611,000 720,000 1,230,000 550,000 430,000 820,000 780,000 880,000 754,000 1,360,000 1,200,000 240,000 278 A MANUAL OF BACTERIOLOGY (B) MIQUEL'S ANALYSES. Samples of Soil from — Number of Microbes in 1 gram. Paris (Rue de Rennes), .... 2,100,000 1 300 000 ,, (Pare du Montsouris), .... 750,000 Dr. C. N. Dowd 1 has recently ascertained the number of microbes in soil derived from various streets in New York. The soil in each case was obtained during the upturning of the streets for relaying gas and water-pipes, etc. (c) DOWD'S ANALYSES. Samples of Soil from — East Fifty-ninth Street, near Third Avenue, . East Fifty-nine Street, near Park Avenue, East Fifty-ninth Street, near Fifth Avenue, . East Fifty-ninth Street, near Madison Avenue, Eighth Avenue and Fifty-seventh Street, . . Tenth Avenue and Sixty-fifth Street, . . . Eighth Avenue, near Fifty -sixth Street, . . Eighth Avenue, near Fifty-fifth Street, . . Fifty-ninth Street and Sixth Avenue, . . . Fiftieth Street and Eighth Avenue, .... Sixth Avenue, near Fifty-eighth Street, * . Seventh Avenue and Fifty-fourth Street, . . Seventy -first Street, near Eighth Avenue, . . Forty-ninth Street and Eleventh Avenue, . . Third Avenue, near Forty-second Street, . . Third Avenue, near Forty -second Street, . . Number of Microbes per cc. 17,675 17,950 157,200 131,100 29,700 29,250 8585 3800 10,650 287 33,150 15,250 20,150 24,900 28,850 67,500 American Medical Record, 1890. THE MICROBES OF THE SOIL 279 To ascertain the number of microbes in a given sample of soil, the colonies produced on the gela- tine-plates are accurately counted. This is per- formed by the apparatus represented in Fig. 55, which will be described in the next chapter. To ascertain the characteristics of the microbes, further cultivations must be made ; the microbes must be transplanted into various media, and exposed to different temperatures ; and they must be inocu- lated into different kinds of animals. The labour of separating each species and studying it in detail FIG. 55. WOLFFHUGEL'S APPARATUS. (For estimating the number of Colonies in a Plate-cultivation.) would be extremely great ; hence microbian soil examinations have largely been confined to the determination of the number of microbes, and not to the peculiar species. In determining the signifi- cance of such examinations, we must bear in mind the following facts: — (1) The number of microbes present in a soil does not necessarily indicate the number of pathogenic forms. (2) Small samples of soil may show marked variations in the number of microbes ; this is owing to minor local influences. (3) Surface soil always contains a larger number of 280 A MANUAL OF BACTERIOLOGY microbes than sub-soil; and at a depth of 8 or 10 feet there are hardly any present. (4) Most of the microbes of soil are harmless when introduced into the human or animal body ; but the bacilli of tetanus, anthrax,1 typhoid fever, malaria, and cholera have been found in soil. Dowd's investigations have proved that the ex- posure of so much soil in the upturning of streets is detrimental to the health of the surrounding community. However, it should be remembered that so long as the soil is wet it cannot spread the microbes in the air; but the soil does not long remain wet. It dries beside the trenches, it adheres to the implements, the clothes and boots of the workmen, and, in fact, to everything which comes in contact with the trenches ; and, finally, much of it is left on the surface when the pavement is relaid. In all these conditions it may be carried away as dust. The microbes go with the dust, and access to the body is then made easy. The amount of dust in the air is much increased by these trenches ; but, on the other hand, the deeper layers of soil or earth from which this dust is derived do not contain nearly so many microbes as the surface layer. An important method for dealing with the dust of streets, especially during epidemics, is to water them with some germicidal substance, by means of the Strawsonizer or pneumatic distributor.2 This machine is capable of distributing one or more 1 Pasteur in Bulletin de VAcademie de Medecine, 1880. 2 Obtainable at Messrs. Strawson and Co., Newbury, Berk- shire. THE MICROBES OF THE SOIL 281 gallons of any fluid over an acre of land, and to a width of 23 feet. Therefore, it would be advan- tageous to use this machine for watering streets, cattle markets, etc., with a weak solution of ' sanitas,' carbolic acid, or any other cheap disinfectant. Concerning cultivated soils, nitrogen is a most important element in the growth of crops. Berthe- lot1 has shown that a fixation of atmospheric nitrogen takes place in certain vegetable soils by the action of microbes and other fungi. Hellriegel and Wilfrath have proved that legu- minous plants obtain their great supplies of nitrogen from the air. This power of absorbing free nitrogen is due to the roots of leguminous plants becoming inoculated with the microbes present in soil. The microbes, which give rise to tubercles on the roots and rootlets, enter into a partnership or symbiotic relationship with the leguminous plant for mutual advantage. These microbes have the power of bringing the free nitrogen into organic combination. Perhaps the chief soil-microbe which enters into symbiosis with leguminous plants is Dr Beyerinck's Bacillus radicicola. This microbe has been isolated from cultivated soils as well as from the tubercles on the roots of Vicia faba (the field bean) ; and Beyerinck has inoculated the roots of seedling- beans with this microbe, and in each case it multiplied within the roots, ultimately giving rise to tubercles. The process of nitrification or the conversion of organic and ammoniacal nitrogen into nitrates was 1 Comptes Rendut, vol. cviii. 282 A MANUAL OF BACTERIOLOGY first shown by Mlintz and Schloesing 1 to be due to the action of microbes in the soil. Although these savants had previously described a microbe causing nitrification, it was not until 1890 that Dr. P. F. Frankland, F.RS.,2 M. Winogradsky,3 and Mr. R. Warington, F.RS.,4 simultaneously described the true cause of nitrification. The nitrifying microbes were isolated by the fractional dilution method. (1) Frankland' s researches. — Dr. and Mrs. Frank- land have isolated a nitrifying microbe from soil. ' Nitrification having been in the first instance in- duced in a particular ammoniacal solution by means of a small quantity of garden soil, was carried on through twenty-four generations, a minute quantity on the point of a sterilised needle being introduced from one nitrifying solution to the other. From several of these generations gelatine-plates were poured, and the resulting colonies inoculated into identical ammoniacal solutions, to see if nitrification would ensue ; but although these experiments were repeated many times, on no occasion were they successful.' In other words, the microbe in ques- tion refused to grow on gelatine. The ammoniacal solution, already referred to, contained :— 1000 cc. of distilled water, 100 cc. of salt solution,5 0'5 1 Comptes Rendus, vol. xlviii. p. 301 ; vol. Ixxxv. p. 1018 ; vol. Ixxxix. pp. 891 and 1074. 2 Philosophical Transactions, vol. clxxxi. pp. 107-128. 3 Annales de FInstitut Pasteur, 1890, p. 213 seq. 4 Journal of Chemical Society, 1891, pp. 484-529 ; Chemical News, vol. Ixi. (1890), p. 135. 5 This solution contained 1 gramme of potassium phosphate, 0*2 gramme of crystallised magnesium sulphate, and O'l gramme of calcium chloride (fused) in 1000 cc. of water. THE MICROBES OF THE SOIL 283 gramme of ammonium chloride, and 5 grammes of carbonate of lime (pure) ; and in this solution the microbe grew and multiplied. As this solution con- tains no organic matter, it will be seen that nitrifica- tion can take place in purely mineral solutions. This power of growing in mineral solutions prevented the development of other microbes (present in the soil used for inoculation) which require organic matter for their growth. After proving that the microbe refused to grow on gelatine, 'experiments were commenced to endeavour to isolate the microbe by the dilution method. For this purpose a number of series of dilutions were made by the addition, to sterilised distilled water, of a very small quantity of an ammoniacal solution which had nitrified. It was hoped that the attenuation would be so perfect that ultimately the nitrifying microbe alone would be introduced. After a very large number of experiments had been made in this direction, the authors at length succeeded in obtaining an at- tenuation consisting of about one-millionth of the original nitrifying solution employed, which not only nitrified,1 but, on inoculation into gelatine- peptone, refused to grow, and was seen, tinder the microscope, to consist of numerous characteristic bacilli hardly longer than broad, which may be described as bacillococci.' The chief characters of the Frankland Bacillus of nitrification (Fig. 56 A) are the following : — (a) The solutions in which the isolated microbe 1 The presence of nitrous acid was ascertained by both diphenylainine and sulphanilic acid. 284 A MANUAL OF BACTERIOLOGY grows remain perfectly clear, (b) The microbe has the remarkable capacity of indefinite growth in a medium devoid of organic matter, (c) It is O8 fju in length, and hardly longer than broad, hence it has been called a bacillococcus. It occurs both isolated, in pairs, and in small irregular groups, (d) In the living state it exhibits a vibratory movement only, (e) The microbe cultivated in ammoniacal solutions converts the ammoniacal into nitrous nitrogen, and not into nitric nitrogen. (/) The same microbe ap- pears to grow in broth or bouillon, but not on solid gelatine-peptone. (2) Winogradsky 's re- searches.— Winogradsky has also obtained a similar bacillus to that of Frankland, which grows in an inorganic ammoniacal solution, but not on gelatine - peptone ; and he has shown that this microbe grows (and may be isolated) on the surface of gelatinous silica containing the inorganic ammoniacal salts already mentioned. This nitrifying microbe gives rise to very charac- teristic colonies on gelatinous silica. Winogradsky's bacillus measures from 1-1 to 1-8 ^ long, and does not exceed 1 //, broad. This microbe occurs singly, in pairs, rarely in chains of three to four individuals, and as zooglcea. It converts ammoniacal into FIG. 58. MICROBES OF NITRIFICATION. A, Frankland's nitrous bacillus. B, Warington's nitric micrococcus. THE MICROBES OF THE SOIL 285 nitrous nitrogen, and can grow in ammoniacal solu- tions devoid of organic matter. There is little doubt that Frankland's and Winogradsky's microbes are the same. Both sets of experiments prove that the nitrous bacillus of the soil converts ammoniacal into nitrous nitrogen, and not into nitric nitrogen. (3) Waringtoris researches. — Mr. Warington has also isolated, by the dilution method, a microbe which converts ammonia into nitrous acid only ; and confirms the investigations of Frankland and Winogradsky. In addition to this, Warington has apparently isolated a microbe from soil which con- verts nitrites into nitrates. This microbe produces neither nitrites nor nitrates in ammoniacal solu- tions; in fact, it cannot oxidise ammonia. The nitric microbe (Fig. 56 B) is a micrococcus, and grows in a solution of potassium nitrite. 'The nitrification effected by soil is thus ex- plained as performed by two microbes, one of which oxidises ammonia to nitrates, while the other oxidises nitrites to nitrates. The first microbe is easily separated from the second by successive cultivations in solutions of ammonium carbonate. The second is (probably) separated as easily from the first by successive cultivations in solutions of potassium nitrite containing monosodium car- bonate.' ' In soil the nitric microbe is equally active as the nitrous, since soil never contains any but ex- tremely weak solutions of ammonia, andsuper- carbonates are always present.' CHAPTEK IX THE MICROBES OF WATER THE organisms present in water have long been observed by the aid of the microscope, but it is only during the last decade that bacteriological methods have been introduced for the systematic examination of potable and other waters. Water is one of the most convenient vehicles for the distribution of microbes, and unfiltered water abounds in these small specks of animated matter. This need not cause any surprise, because, as we have already seen, the atmosphere and soils are laden with microbes. In fact, every shower of rain diminishes the number of microbes suspended in air. These are then found in puddles, pools, ponds, rivers, etc., and consequently are carried into well and other potable waters. Although the majority of these microbes are harmless, it is always advis- able to filter water before use. One of the best filters for this purpose is Maignen's * Filtre Eapide/ Among the various microbes found in water are the following : — ILllr* « - '§ 1 il IlllllrilliJ ^j -K> -K> ?* r* ^ ^ 5$ -»o <«> -»o *» ?^ »d *a "?? *5? ^^f*» S »d »d w 9 •ZJL 288 A MANUAL OF BACTERIOLOGY In addition to the Schizomycetes, various Protozoa, etc., are always present (more or less) in water, and Fig. 57 represents certain animal and vegetal forms found in some potable waters. Although the majority of Schizomycetes and Proto- zoa found in waters are harmless, it has been proved FIG. 57. INFUSORIA, ETC., IN WATER. 1, Daphnia. 2, Chilodon. 3, Paramcecium. 4, Acineria. 5, Paranema. 6, Cercomonas. 7, Actinophrys. 8, Amoebae. 9, Amoeba diffluens. 10, Protococcus. 11, Diatoms. 12, Desmids 13, Confervse. 14, Spores of fungi. 15, Pieces of vegetable tissue. 16, Amoeba (more highly magnified). 17, Cyclops. 18, Cypris. 19, Anguillula. that certain outbreaks of typhoid fever, cholera, etc., have been traced to water supplies; and the microbes of typhoid fever, cholera, tetanus, etc., have all been found in drinking waters contami- nated with sewage. Dysentery and tropical abscess of the liver are due to certain species of Amcebce, THE MICROBES OF WATER 289 which enter the system through the medium of water. In view of these facts the bacteriological analysis of water is a subject of great importance ; but the primary object in such analyses is not the search for pathogenic microbes. Such an investi- gation is generally fraught with insuperable diffi- culties, and, for sanitary purposes, is practically worthless. ' It is obvious that, even if the typhoid bacillus, or any other pathogenic microbe could be detected with unerring certainty in any water in which it was present, a search for this bacillus in the ordinary course of water examination would still have only a very subsidiary interest. Waters are surely not only to be condemned for drinking purposes when they contain the germs of zymotic disease at the time of analysis, but in all cases when they are subject to contaminations which may at any time contain such germs. Sewage- contaminated waters must on this account be in- variably proscribed, quite irrespectively of whether the sewage is, at the time that the water is sub- mitted to examination, derived from healthy or from diseased persons. . . . The real value of these bacteriological investigations, if judiciously applied, consists in their power of furnishing us with in- formation as to the probable fate of dangerous organisms, should they gain access to drinking water. It is by their means that we have learnt that many such organisms can preserve their vitality, nay, in some cases can actually undergo multiplication in ordinary drinking water ; that they are destroyed by maintaining the water at the 290 A MANUAL OF BACTERIOLOGY boiling point for a short time; and that they are more or less perfectly removed by some processes of nitration and precipitation, whilst other pro- cesses of the same nature are worthless, or even worse' (Frankland). Before describing the methods for the bacterio- logical examination of waters, we must allude to (a) the collection of the samples, and (b) the trans- port of the same. To collect the samples of water accurately stop- pered bottles (70 cc. capacity) are used. These must be perfectly clean, and rinsed out with dis- tilled water. Each bottle is put into a small tin canister, and the canisters (containing the bottles) are heated in a steriliser to about 180° C. for at least three hours. * The bottles thus sterilised can be easily transported without suffering contamina- tion by dust to the place where the sample is to be collected. In collecting the sample of water the outside of the bottle should be rinsed in the water before removing the stopper, and when the bottle is opened the water is at once allowed to enter and fill the bottle to the extent of four-fifths, the stopper being immediately replaced and tightly screwed in, so that the exposure to the air is reduced to a minimum. The bottle is replaced in the tin canister, and the lid closed. In collecting samples of water from rivers, reservoirs, lakes, or ponds, it is better not to remove the stopper until the bottle is completely immersed in the water, and to replace it while still beneath the surface.' After collection the sample of water should be examined as soon as THE MICROBES OF WATER 291 possible, for it has been proved by Dr. T. Leone,1 Dr. P. F. Frankland,2 and others, that microbes multiply very rapidly in water. For instance, Leone gives the following figures, which show the rapid increase of microbes in a sample of water kept for only five days : — Number of Microbes in 1 cc. of water (at 14° to 18° (7.). Water on day of collection, . » 5 after 1 day's standing . . „ 100 „ 2 days' „ , . 10,500 „ 3 „ ... 67,000 „ 4 „ „ . . . 315,000 „ 5 „ „ ; . . 500,000 If the water has to be transmitted a considerable distance, occupying several days in transit, Dr. P. Miquel 3 recommends the use of a glaciere, or box, in which the bottle is surrounded with ice. There are two principal methods in use for the bacteriological examination of water. The first is the plate-cultivation process, which consists in taking a known quantity (say 1 cc.) of the water, and mixing it with melted nutrient gelatine con- tained in a test-tube. After shaking, the contents of the tube are rapidly poured out upon a sterilised glass plate, then allowed to solidify, and finally placed in a damp chamber, kept at about 22° C. After a few days' incubation colonies make their appearance on and in the layer of gelatine. The colonies are counted by means of the eye or lens, 1 Gazzetta C/iimica Italiana, voL xv. (1885), p. 385. 3 Proceedings of Royal Society, 1886. * Manuel Pratique d' Analyse Bactiriologique des Eaux (1891), p. 26. 292 A MANUAL OF BACTERIOLOGY with the aid of Wolffhugel's counting apparatus (see Fig. 55), which consists of a glass plate, ruled with vertical and horizontal lines into centimetre squares, which are often sub-divided. The cultiva- tion-plate is placed on a black background, and the ruled glass plate placed over the former, without touching the colonies. 'If the colonies are very numerous the number in some small divisions is counted ; if less, in some large ones ; and an average is obtained from which the number of colonies on the entire surface is calculated.' The second method is largely used in France, and is known as ' fractionnement dans le bouillon.' The sample is first diluted with sterilised water of known volume. After this one gramme (1 cc.) of the water is taken up by means of a sterilised capil- lary pipette, which is dipped four times into the water at different points of the liquid mass to obtain the above-mentioned quantity. By this means a fair sample of the water is obtained. In the laboratory of Dr. P. Miquel thirty-six small flasks (each 15 cc. capacity) are each half filled with sterilised bouillon. These flasks, having each a glass cap containing a sterilised cotton-wool plug, are placed in a divided box. Each flask receives one, two, or three drops of the sample of water, as the case may be; all the flasks are placed in an incubator at 30°-36° C. during a period of at least fifteen days, when the microbian colonies are counted. Before introducing the small quantity of water into either a solid or a liquid medium, the original THE MICROBES OF WATER 293 sample should be violently shaken to ensure an even distribution of the microbes throughout the water. By using Koch's or the plate-cultivation method, the author l obtained the following average number of microbes (colonies) in 1 cc.of a sample of water from theriverWitham(at Lincoln) during the year 1887 : — January, . . 2,016 February, . 3,488 March, . . 10,287 April, . . 11,692 May, . . 11,923 June, . . 12,000 July, . . 10,184 August, . September, . 4,110 October, . . 9,621 November, . 10,211 December, . 9,787 These figures (monthly means) give a yearly mean of 8665 microbes in 1 cc., or quarterly means as follows : Spring, . . - . . . . . 11,300 Summer, .-V . ... . 11,092 Autumn, . . . . . . . 7,980 Winter, . ... .-. ... ... . 5,097 From these results the greater number of microbes in the Witham were during the spring and summer. Another series of experiments with water from certain rivers gave the following results : — Witham, 11,860; Irwell, 9230; Thames, 25,745; and the Seine, 56,219 microbes per cubic centimetre. Dr. P. F. Frankland 2 has made periodical exami- nations of the river and well waters from which the water-supply of London is derived ; and during the year 1886, he obtained the following number of colonies (on gelatine-plates) per 1 cc. of water : — 1 Griffiths' Researches on Micro-Organisms, p. 77. 2 Journal of Society of Chemical Industry, vol. iv. (1885), and vol. vi. (1887); Transactions of Sanitary Institute, vols. viii. and ix. ; Proc. Roy. Soc., 1885; and Proc. Inst. of Civil Engineers, 1886. 294 MANUAL OF BACTERIOLOGY ^ »O «O •«* "* O i« U} •**< CO * . iO CO O* » IO W rH O 1^ r-t W » "3 1 §CO IO O (N O O CM CO I-H rH r-1 CO -^J* O lO OO < ! ! .a * - • 8 s g O » i-T i-T |*S ' S 8 1 irT s 5 Ci 1 1 S 1 1 1 S « *" of CO CO . 3* „ „ „ » 6,170 » 4J „ „ „ „ 1,580 >. 6 „ „ „ „ 0 „ It has been already stated that Bacillus tetani has been found in soil ; and Mace 2 has recently found B. typhosus in various samples of soil. B. tuberculosis and B. coli communis have also been found in soil. On the other hand, De Giaxa 3 has shown that soil is a bad medium for the preservation of B. cholerce Asiaticce, this being due to the large number of saprophytic species present, whose struggle for existence interferes with the vitality of the cholera microbe. In fact, this is an important example of the survival of the fittest, for De Giaxa has proved that soil per se has no detrimental action on the cholera microbe. If soil is a bad medium for the preservation of some forms, water (especially when polluted) is a better medium for others. At the recent Congres d' Hygiene Ouvriere4 M. Gautier exhibited illustrations of the typhoid, carbuncle, cholera, and diphtheritic microbes, with many others, in Seine water ; 5 and the dis- tinguished chemist said to householders and others, 1 Zeitschrift fiir Hygiene, vol. vii. - Comptes Rendus, vol. cvi. p. 1564 ; and his Traite Pratique de Bacteriologie (1-891), p. 717. 3 Annales de Microyraphie (1890) vol. ii. 4 Held in Paris during April 1892. 5 Paris drinking-water. 340 A MANUAL OF BACTERIOLOGY 1 Do not fear these foes. If they hurt you, it is because you drink unfiltered water and eat ill-baked bread. Filter your water or boil it, and if your bread seems ill-baked, toast it well or let it stay some time in a hot oven.' If householders, corporations, and others would attend a little more to the ordinary rules of health — such as filtering water, boiling milk, destroying unsound food, removing refuse, isolating infectious persons, disinfecting articles of an infectious nature, etc. — there would be a considerable decrease in the number of infectious cases, especially during the time of epidemic diseases. In fact, these rules would go a long way towards the prevention of such diseases. There is no doubt that many of the epidemics of cholera and other infectious diseases have been largely due to bad or imperfect sanitation. In densely- populated centres it is imperative that the most perfect rules of sanitation should be practised by corporations, sanitary authorities, householders, and others. One cannot help but believe that the visita- tions of epidemic diseases in the past have been blessings in disguise, because they have taught us that cleanliness in all things (in person, food, drink, home, and city) tends directly to prevent and combat the attacks of such diseases as cholera, typhoid fever, scarlatina, etc. In past times town authorities and householders did not heed the voice of the cholera fiend, as is sung in Mackay's lyric, 'The Cholera Chant'— ' They will not hear the warning voice. The cholera comes, — rejoice ! rejoice ! He shall be lord of the swarming town ! And mow them down, and mow them down ! ' Although there is still room for improvement in sanitary matters, yet no one can be blind to the fact APPENDIX 341 that, in every direction, sanitation has made rapid progress in Great Britain. l If, by observing such rules as those specified, we can keep in check the obnoxious microbes in water and food, it is not such an easy matter to deal with those present in the atmosphere.2 But even aereal microbes (those spirits of the air) may be, to a large extent, kept in check by the use of disinfectants. XI. STATISTICS CONCERNING ZYMOTIC DISEASES. The Quarterly Report of the Registrar-General, relating to the deaths in England and Wales from zymotic diseases, gives the following figures : — 5202 deaths from whooping-cough. 2769 measles. 1306 1078 1361 890 76 diphtheria. scarlatina. diarrhoea. 'fever '(chiefly enteric). small-pox. The above figures give a total of 12,682 deaths from zymotic diseases during the first three months of 1892. 1 For those interested in sanitary matters, the author recom- mends Dr. A. C. May bury 's excellent Epitome of the Public Health Act, 1891 (H. Kimpton, 82 Holborn, London). 2 As microbes are always present in air, soil, and water, it may well be asked, ' Where do they come from ? ' We know not where ; perhaps from the djinnistan of the Persians. Y2 LIST OF FIRMS WHERE BACTERIOLGICAL APPARATUS, ETC., CAN BE OBTAINED. Microscopes, etc. C. Zeiss, Jena, Germany ; or Zeiss's agent, C. Baker, 244 High Holborn, London. Incubators, Sterilisers, etc. F. E. Becker & Co., 33 Hatton Wall, London; K. Muencke, 58 Luisenstrasse, Berlin ; R. Kanthack, Imperial Mansions, Oxford Street, London. Chemical Apparatus and Chemicals. J. Orme & Co., 65 Barbican, London. Staining Solutions, etc. F. E. Becker & Co., 33 Hatton Wall, London; R. Kanthack, Imperial Mansions, Oxford Street, London. Agar-agar and Gelatine. Christy & Co., 25 Lime Street, London ; J. F. Shew & Co., 89 Newman Street, Oxford Street, London; R. Kanthack, Imperial Mansions, Oxford Street, London. Microtomes. Cambridge Scientific Instrument Co., Cambridge ; R. Kanthack, Imperial Mansions, Oxford Street, London. Dissecting Knives, etc. C. Baker, 244 High Holborn, London. Mr. Kanthack furnishes estimates of the requirements of a completely fitted bacteriological laboratory. INDEX. Abbe's condenser, 21. Actinomyces, 82, 258. Actinomycosis, 258. Aerobic microbes, 110. Aeroscopes, 263-269. Agar-agar, 57. Agents, cementing, 91. ,, clearing, 91. ,, dehydrating, 90. ,, mounting, 91. „ washing, 90. Air, microbes of, 260-275, 330. Air, number of microbes in, 269-275. Albumin, egg, 55. Albumoses, 321-324. Amoeba, 259. Anaerobic microbes, 110. Anthracin, 256. Anthrax, 255-257. Antiseptics, 325-330. Apochromatic lenses, 17. Apparatus, microphotographic, Appendix, 332-341. Aspergillus, 52. Autoclaves, 32, 52. Bacilli, 149-170. • Bacillus alvei, 150. ,, antkracis, 255. ,, arachnoidea, 169. ,, beribericus, 149. Bacillus butyricus, 82, 156. ,, cavicida, 164. ,, cholera Asiatica, 64, 80, 339. ,, cyanogenus, 159. ,, diphtheria;, 79, 236. ,, diphtheria colum- barum, 163. „ diphtheria vitidorum, 163. ,, epidermidis, 166. ,, erythrospoi^us, 159. ,, ethaceticu-s, 155, 201, 336. ,, ethacetosuccinicu$, 336. ,, figurans, 170, 274. ,, Hansenii, 170. ,, ianthinus, 159. „ lepra, 76, 78, 206. ,, leptomitiformis, 169. ,, malaria, 215. „ mallei, 77, 234. ,, megaterium, 166. ,, cedematis maligni, 110, 160. „ of cancer, 193, 334. „ of conjunctivitis, 169. ,, of grouse disease, 154. ,, of indigo fermenta- tion, 160. „ of influenza, 333. ,, of measles, 332. „ of nitrous fermenta- tion, 166, 282. ,, of rabbit diphtheria, 164. ,, of rhinoscleroma, 160. 343 344 INDEX Bacillus of swine erysipelas, 165. ,, of swine plague, 165. ,, of symptomatic an- thrax, 157. ,, of syphilis, 211. ,, of iilcerative stoma- titis, 165. ,, of whooping-cough, 335. ,, pellucida, 169. ,, phiviatilis, 333. , , putrificus coli, 1 66. ,, pyocyaneusj 161. » pyogenesfcetidus, 164. ,, radicicola, 281. ,, septiccemice, 162-3. ,, septicus, 169. ,, spinosus, 110. ,, subtilis, 52, 65, 108, 110, 154, 274. „ tetani, 211, 339. ,, tuberculosis, 50, 66. 76, 244-254, 339. ,, typhosus, 79, 221, 339. ,, ulna, 157. ,, violaceus, 159. Bacteria, 133-149. Bacteriological laboratory, 8- 48. Bacteriology, definition of, 1. Bacterium aceti, 136, 274. allii, 134, 135. ,, Balticum, 143. ,, brunneum, 146. ,, cholerce gallinarum, 137. ,, chlorinum, 144. ,, coli commune, 139, 339. ,, crassum sputigenum, 145. ,, decalvans, 137. ,, Fischeri, 143. ,, foetidum, 139. ,, indicum, 141, 274. lactis, 137, 274. ,, lineola, 134. ,, luminosum, 144. Bacteriummerismopedioides, 141. ,, Neapoianum, 139. , , oxytocum pernicio- sum, 141. ,, Pflilgeri, 142. „ phosphor -escens, 142. ,, photometricum, 145. ,, pneumonicum agile, 145. ,, pseudo • pneumoni- cum, 138. ,^ ,, septicus agrigenum, 139. ,, septicum sputigenum, 140. ,, terrao, 133, 276. ,, violaceum, 145. ,, xanthinum, 139, 274. ^o#/w, 141. Begyiatoa alba, 168. ,, mirabilis, 168. ,, m'vea, 168. ,, roseo-persicina, 167. Berberis vulgaris, 101. Beri-beri, 149. Biology of microbes, 114, 177. Blood serum, liquid, 51. ,, ,, solid, 51. Bouillon, 49. Bread-paste, 58. Buffon's theory, 98. Camera lucida, 23. Canada balsam, 70-95. Cancer bacillus, 193, 334. Canons, Koch's, 2. Capillary pipettes, 53. Cementing agents, 91. Chamberlaiid's filter, 47. Chemical separators, 76. Cholera, 225-234, 340. Classification of microbes, 1 10, 112. Clearing agents, 91. Clip, mounting, 94. Comma-shaped bacilli, 227. INDEX 345 Concluding remarks, 330. Condenser, Abbess, 21. Cover-glass preparations, 68, 76. Cover -glass testers, 89. Cultivating microbes, methods of, 49-68. Cultivations, fractional, 59, 62. Cultivations, plate, 59. Cultivation tubes, 41-47. Cultures, drop, 65. Cutting, section, 29-30, 88. Damp chambers, 56. Definition of bacteriology, 1. Dehydrating agents, 90. Dilution method, 59, 63. Diphtheria, 235-243. Diplococcus, a, 109. Discontinuous heating, method of, 51. Disinfectants, 214, 325, 341. Diseases, microbes and, 178- 259. Dissecting instruments, 23, 24, 25. Dissecting microscope, 25. Dissecting, mode of, 26. Division of microbes, 109-110. Drawing by hand, 23. Dust in air, the, 261. Dysentery, 259. E Edinburgh laboratory, the, 8- Enzymes, 2, 18, 324. Equivocal generation, 100. Erysipelas, 193, 338. Estimating microbes in air, methods of, 263-272. Estimating microbes in soil, methods of, 277. Estimating microbes in water, methods of, 291-292. Eucalyptus, the, 219. Fermentation, 1, 174. Fermentations, pure, 336. Filter, hot-water, 50. Fission, 108, 109. Flagellata, 198, 217, 259. Flasks, cultivation, 42-47. Fluids, examination of, 67. Fluids, staining, 69-70. Formation of spores, 108-9. Forms of microbes, 107. Foul-brood, 150. Fractional cultivations, 59, 62. Fresh tissues, examination of, 67. Fresh tissues, mounting, 91. Gelatine, nutrient, 57. Germicides, 325-330. Glanders, 234, 324. Ground rice, 58. Grouse disease, bacillus of, 154. Hardening agents, 84, 86. Hay-fever, bacillus of, 154. Hydrophobia, 181-192. Identification of microbes, 1 13. Imbedding mixtures, 86-88. Incubators, 37-41. Infectious diseases and mi- crobes, 178-259. Influenza, 197-199. bacillus of, 333. Infusions, various, 52. Injection syringes, 53. Inoculating media, modes of, 58-59. Inoculating needles, 53. Instruments, dissecting, 23-25. Introduction, the, 1-7. 346 INDEX K Kakke, 149. Klein's bacillus, 227. Koch's canons, 2. ,, lymph, 252. Kuisl's bacillus, 227. Laboratory, the bacteriological, 8-48. Leprosy, 206-210. Leptothrix buccalis, 166. ,, innominata, 167. ,, parasitica, 167. Lifters, 93. List of firms, 342. Living animals, methods of in- troducing microbes into, 94. M Malaria, 215-220. Measles bacillus, 332. Measurement, unit of, 95-97. Media, cultivation, 49-68. „ fluid, 49-54. „ solid, 54-61. Merismopedia, a, 109. Methods of cultivating mi- crobes, 49-68. ,, of mounting mi- crobes, 83-97. ,, of staining mi- crobes, 68-83. Microbes and diseases, 178-259. division of, 109. of air, 260-275. of soil, 276-285. of water, 286-304. properties of, 4, 5. reproductive power of, 6, 7. size of, 5. weight of, 5. which excrete albu- moses, 323. Micrococci, 114-132. ,, in pyaemia, 132. Micrococci in rabies, 182-184. ,, in septicaemia, 132. Micrococcus amaril, 180. ,, aurantiacus, 116. ,, bombycis, 129. ,, candicans,UO,274. ,, cereus Jlavus, 118. ,, chlorinus, 55, 116, 274. ,, cinnabareus, 117. ,, citreus conglomcra- tus, 118, 274. ,, cyaneus, 117, 274. , , endocarditicus, 126. ,, erysipelatosus, 193. ,, Jlavus liquefaciens, 118. , , Jlavus decidens, 118. , , Jlavus tardigradus, 118. ,, fulvus, 117. ,, gonorrhoea, 80, 127. ,, hcematodes, 117. ,, in gangrene, 130. ,, luteus, 116, 274. ,, in measles, 126. ,, in pernicious an- aemia, 132. ,, in purpura, 123. ,, insectorum, 131. , , intracelhdaris me- ningitidis, 128. ,, inwhooping-cough, 131. , , of cattle - plague, 129. , , of foot-and-mouth disease, 130. , , of nitric fermenta- tion, 132, 285. „ of tissue necrosis, 131. ,, ovatus, 127. ,, perniciosus, 131. 199. prodigiosus, 1 14,274. pyogems, 119, 199. pyogenes alb us, 120. INDEX 347 Micrococcus pyogenes auretis, 119. » pyogenes citreus, radiatus, 119. rosaceus, 117, 274. scarlatina, 82, 202. septicus, 130. subflavus, 119. tetragenus concen- tricm, 332. tetragonus, 128. urece, 120. variolas et vaccinia, 124. versicolor, 118. violaceus, 117, 274. Microphotographic apparatus, 21. Microscope, dissecting, 25. the, 14-22. Microtomes, 27-29. Milk, 51. Miller's bacillus, 227. Mounting agents, 91. „ clip, 94. Mycoprotein, 4. N Needles, inoculating, 53. Nitric microbe, 285. Nitrification, 1, 281-285. Nitrous microbe, 284. Number of microbes : in air, 269-275. in soil, 277-281. in water, 281-287. Objectives, 16, 18, 20. Oculars, 17, 19. Oldium albicans, 258. Origin of microbes, 98-107. Pasteur Institute, 12-14, 190. Phargocytes, 191. Phthisis, 243. Pigments, 319, 324. Plate-cultivations, 59. Pleomorphism, 102-107. Pneumonia, 199-201. Potatoes, cooked, 56. Preparations, cover-glass, 68. Properties of microbes, 4. Proteus mirabilis, 148. ,, vulgaris, 146-148. „ Zenkeri, 149. Protozoa, 259, 288. Ptomaines, 305-320, 324, 337. ,, extraction of, 307- 310, 338. ,, properties of, 307. Puccinia graminis, 101. Puerperal fever, 194-197. Putrefaction, 1. Pyocyanin, 161, 319, 324. R Rabies, 181-192. Regulators, 39-41. Reproduction of microbes, 6, 108. Rice, ground, 58. Rules of sanitation, 340. 3 Saccharomyces apiculatus, 176. cerevisice, 174. conglomerate*, 176. ellipsoideus, 175. exiguus, 176. minor, 175. mycoderma, 177. Pastorianu s,176. vini, 177. Saccharomycetes, 173. Sanitas, 220, 329. Sanitation, rules of, 340. Sarcina, 109. Scarlatina, 202-206. Schizomycetes, 99, 107, 110, 173,288. 348 INDEX Separators, chemical, 76. Serum inspissator, 36. Serum steriliser, 35. Size of microbes, 5. Soil, microbes of, 276-285, 339. ,, number of microbes in, 277-281. Spasmotoxine, 213. Spirilla, 171-173. Spirillum attenuatum, 173. ,, cholerce Asiaticce, 171, 226. concentricum, 173. FinMeri, 171, 227. Obermeieri, 81, 172. Rosenbergii, 173. sanguineum, 172. sputigenum, 227. lenue, 172. tyrogenum, 61, 171, 227. undula, 172. violaceum, 173. vohttans, 172. Spirochsetse, 173. Spirochceta gigantea, 173. ,, plicatilis, 173. Spontaneous generation, 99. Staining fluids, 69, 70. Staining microbes, methods of, 68-83. Statistics concerning diseases, 341. Sterilisers, 31-37. Streptococcus, 109. Surra, 259. Syphilis, 210. Testers, cover-glass, 89. Tetanine, 213. Tetanotoxine, 213. Tetanus, 211, 215. Throat washes, 241. Thrush, 258. Tissues, examination of fresh, 67. Torula cerevisice, 174. Torulse, 52. Tuberculosis, 243-254. Tuberculous milk, bacilli in, 75. Tubes, cultivation, 41-47. Turn-tables, 92. Typhoid fever, 220-225. U Unit of microscopical measure- ment, 95-97. Vibriones, 170-171. Vibrio rugula, 170. ,, serpens, 170. Vivisection, 3, 4. W Washing agents, 90. Water and epidemics, 223, 233. ,, filtration of, 298. ,, microbes of, 286-304, 339. ,, standard of purity of, 303. ,, sterilisation of, 300-304. ,, storage of, 298. Waters, examination of, 290. Weight of microbes, 5. Yeasts, 52, 173-177. Yellow fever, 180. Zeiss's microscopes, 15. ^&^? - ---SB* Printed by T. and A. CONSTABLE, Printers to Her Majesty, at the Edinburgh University Press. wdctjrapbic Hti&reas : Sunlocks, London* 21 BEDFORD STREET, w.c. August 2893. A LIST OF MR. WILLIAM HEINEMANN'S PUBLICATIONS AND FORTHCOMING WORKS The Books mentioned in this List can be obtained to order by any Book- seller if not in stock, or will be sent ly the Publisher post free on receipt MR. WILLIAM HEINEMANWS LIST. 3 iDCJ Of PAGE Butboro. PAGE g Lee .... Leland Lie .... 4 • 7, 8 Bendall . 16 1 Lowry Bowen . . . 5 74 Brown Brown and Griffiths . Buchanan . . . Butler . . 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By JESSIE FOTHERGILL, Author of " The First Violin," &c. RELICS. By FRANCES MACNAB. A BATTLE AND A BOY. By BLANCHE WILLIS HOWARD, Author of " Guenn," &c. APPASSIONATA: The Story of a Musician, By ELSA D'ESTERRE KEELING. MR. BAILEY MARTIN. By PERCY WHITR. 12 MR. WILLIAM HEINEMANN'S LIST. fbeinemann's Jnternatfonal SLfbrarp. EDITED BY EDMUND GOSSE. New Review. — " If you have any pernicious remnants of literary chauvinism I hope it will not survive the series of foreign classics of which Mr. William Heinemann, aided by Mr. Edmund Gosse, is publishing translations to the great contentment of all lovers of literature." Each Volume has an Introduction specially -written by the Editor. Price, in paper covers, zs. 6d. each, or cloth, 3$. 6d. IN GOD'S WAY. From the Norwegian of BJORNSTJERNE BjORNSON. Athenceum. — "Without doubt the most important and the most interesting work published during the twelve months." PIERRE AND JEAN. From the French of GUY DE MAU- PASSANT. 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From the Spanish of JUAN VALERA. New Review (Mr. George Saintsbury) : — "There is no douht at all that it is one of the best stories that have appeared in any country in Europe for the last twenty years." THE COMMODORE'S DAUGHTERS. From the Nor- wegian of JONAS LIE. Athenceum. — " Everything that Jonas Lie writes is attractive and pleasant ; the plot of deeply human interest, and the art noble." THE HERITAGE OF THE KURTS. From the Norwegian of BJORNSTJERNE BJORNSON. National Observer. — " It is a book to read and a book to think about, for, incontestably, it is the work of a man of genius." LOU. From the German of BARON ALEXANDER VON ROBERTS. DONA LUZ. From the Spanish of JUAN VALERA. THE JEW. From the Polish of JOSEPH IGNATIUS KRASZEWSKI. In the Press. UNDER THE YOKE. From ihe Bulgaiian of IVAN VAZOFF. MR. WILLIAM HEINEMANN'S LIST. 13 popular 36* 6&, Novels. CAPT'N DAVY'S HONEYMOON, The Blind Mother, and The Last Confession. 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By Count LYOF TOLSTOY. Translated from the Russian by E. ]. DILLON. With Introduction by A. W. PINERO. Small 410, with Portrait, 5^. THE DRAMA, ADDRESSES. By HENRY IRVING. 8vo. With Portrait by ]. McN. Whistler. Second Edition. Fcap. 3*. (xt. SOME INTERESTING FALLACIES OF THE Modern Stage. An Address delivered to the Playgoers' Club at St. James's Hall, on Sunday, 6th December, 1891. By HERBERT BEERBOHM TREE. Crown 8vo, sewed, 6V/. THE PLAYS OF ARTHUR W. PINERO. With Intro- ductory Notes by MALCOLM C. SALAMAN. i6mo, Paper Covers, is. 6d. or Cloth, zs. 6d. each. I. THE TIMES : A Comedy in Four Acts. With a Preface by the Author. II. THE PROFLIGATE : A Play in Four Acts. With Portrait of the Author, after J. MORDECAI. III. THE CABINET MINISTER: A Farce in Four Acts. IV. THE HOBBY HORSE : A Comedy in -Three Acts. V. LADY BOUNTIFUL: A Play in Four Act>. VI. THE MAGISTRATE : A Farce in Three Acts. VII. DANDY DICK : A Farce in Three Acts. VIII. SWEET LAVENDER. To be followed by The Schoolmistress, The Weaker Sex, Lords and Commons, and The Squire, 16 MR. WILLIAM HEINEMANN'S LIST. poetry TENNYSON'S GRAVE. By ST. CLAIR BADDELEY. 8vo, paper, w. LOVE SONGS OF ENGLISH POETS, 1500—1800. With Notes by RALPH H. CAINE. Fcap. 8vo, rough edges, 3^. 6d. %* Large Paper Edition, limited to 100 Copies, ioy. 6d. Net. IVY AND PASSION FLOWER: Poems. By GERARD BENDALL, Author of " Estelle," &c. &c. 12010, cloth, 3*. 6d. Scotsman. — " Will be read with pleasure." Musical World. — " The poems are delicate specimens of art, graceful and polished." VERSES. By GERTRUDE HALL. I2mo, cloth, -$s. 6d. Manchester Guardian. — " Will be welcome to every lover of poetry who takes it up." IDYLLS OF WOMANHOOD. By C. AMY DAWSON. Fcap. 8vo, gilt top, 5*. IbeinemamVs Scientific MANUAL OF BACTERIOLOGY. By A. B. GRIFFITHS, Ph.D., F.R.S. (Edin.), F.C.S. Crown 8yo, cloth, Illustrated. 7s. 6d. 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