E, A,B,Foole BACTERIOLOGY BY THE SAME AUTHOR. PATHOLOGY: GENERAL AND SPECIAL For Students of Medicine. Fully Illustrated. Third Edition to be published early in 1912. SERUM AND VACCINE THERAPY, BACTERIAL THERAPEUTICS AND PROPHYLAXIS, BACTERIAL DIAGNOSTIC AGENTS. Second Edition. With H2 Figures. Crown 8vo. 7*. (id. net. Edited by PROF. R. T. HEWLETT, M.D., F.R.C.P. THE CELL AS THE UNIT OF LIFE and other Lectures delivered at the Royal Institution London, 1899-1902. AN INTRODUCTION TO BIOLOGY By the late Allan Mac-i'aiivfn- m d B Sc Fullerian Professor of Physiology, Royal Institution, London. With 16 Illustrations. 8vo. 7*. c>d. net. E. A.B.POOLE Town Hal] Newark, A MANUAL OF BACTERIOLOGY E. TANNER HEWLETT, M.D., F.E.C.P., D.P.H.(Lond.) PROFESSOR OF BACTERIOLOGY IN THE UNIVERSITY OF LONDON; ,. DIRECTOR OF THE PUBLIC HEALTH DEPARTMENT, KING'S COLLEGE, LONDON ; DIRECTOR OF PATHOLOGY, SEAMEN'S HOSPITAL, GREENWICH; LECTURER ON BACTERIOLOGY, LONDON SCHOOL OF TROPICAL MEDICINE CLINICAL AND APPLIED BY FOURTH EDITION LONDON J. & A. CHUECHILL 7, GREAT MARLBOROUGH STREET 1911 PRINTED BY ADLARD AND SON LONDON AND DOKKING 9^ BOWL ■ k ' % .*-;ysisi**i* l ; ? - * ■". Y J3£_ L PREFACE TO < THE FOURTH EDITION In this Fourth Edition the text has been subjected to complete revision, some slight alterations have been made in the arrangement of the sections, and much new matter has been incorporated; otherwise the general scheme and scope of the book remain much the same as in the previous editions. Several new illustrations have been added, for the photo-micrographs of which I am indebted to my friend and colleague Mr. J. E. Barnard, F.E.M.S., Lecturer on Microscopy in King's College, London. R. T. H. King's Collegk, London; October, 1911. PREFACE In the following manual I have endeavoured to give some account of those portions of Bacteriology which are of especial interest in clinical medicine and hygiene. The preparation of tissues, methods of culture, descrip- tions of pathogenic organisms and their detection, the examination of water, etc., have therefore been given at some length. As it would be impossible in the space at my disposal to include everything relative to the subject, a selection has had to be made, and such details as the celloidin method, Loffler's stain for flagella, the strictly animal parasitic diseases (with a few exceptions), etc., have, among others, been omitted. At the end of the sections dealing with the pathogenic organisms which attack man, some directions have been given for the bacteriological clinical diagnosis and exami- nation, but these are in no way exhaustive ; in fact, it would not be possible in a short work to give a scheme of examination which would cover every case. These directions will also render the book of service in the lal (oratory, while I venture to hope that the details given in the Appendix on the use of the remedies and diag- nostic agents of bacterial origin may be of value to the practitioner. Vlii Manual of Bacteriology I have to thank Mr. Peyton Beale, Dr. Lambert Lack, and Mr. F. J. Tanner, for suggestions and criticism, and the last-named gentleman for the aid he has freely given me in the revision of the proof-sheets. I am also indebted, indirectly, in many ways to my colleagues, Dr. Macfadyen and Mr. Foulerton. My thanks are due to Mr. J. Barnard and to Mr. Frank Stratton respec- tively for the photo-micrographs and original drawings, while for the eight borrowed illustrations blocks have been kindly lent by Messrs. Baird and Tatlock, and Messrs. Swift & Son. May, 1898. CONTENTS PAGE CHAPTER i Introduction . I. The Nature, Structure, and Functions of the Bacteria : their Classification, General Biology, and Chemistry— Bac- teria and Disease . . . • • II. Methods of Cultivating and Isolating Organisms . . . • • .44 III. The Preparation of Tissues and Organisms for Staining and Mounting— Staining and Staining Methods . . . .87 IV. Methods of Investigating Microbial Diseases —The Inoculation and Dissection of Animals — Hanging- drop Cultivation — Interlamellar Films— The Microscope . 123 Y. Infection — Vegetable and Animal Parasites — The Infective Process — Anti-bodies — Anti-sera and Antitoxins — Immunity — Opsonins ...... 151 VI. Suppuration and Septic Conditions . . 233 VII. Anthrax ...... 264 VIII. Diphtheria . . . . . .277 Diphtheria in England — The Diphtheria Bacillus — The Pseudo-Diphtheria Bacillus — Clinical Diagnosis —The Xerosis Bacillus — Diphtheritic Affections of Birds and Animals. IX. "Acid-fast" Bacilli — Tuberculosis — Leprosy — The Smegma Bacillus — G-landers . . 314 X. Typhoid Fever — Para-typhoid Fever — Bacillus Enteritidis and the Gartner Group — Swine Fever — Bacillus dysenteric — Bacillus coli ..... 370 XI. Bubonic Plague — Chicken Cholera — Mouse Septicemia ..... 412 XII. Pneumonia. Influenza, and Whooping-cough 429 x Manual of Bacteriology CHAPTER pAOE XIII. Anaerobic Organisms — Tetanus— Malignant (Edema — Bacillus Botulinus — Bacillus Welchii — Bacillus cadaveris sporogenes — Black Quarter — Clostridium butyricum 1 1 J XIV. Asiatic Cholera— Spjrillum Metchnikovi — Spirillum of Finkler and Prior — Spiril- lum TYROGENUM — SPIRILLUM RUBRUM . . 457 XV. Streptothrix Infections — Actinomycosis — Mycetoma — Leptothrix buccalis — Clado- thrix dichotoma — mycosis tonsillaris . 475 XVI. The Blastomycetes . 487 The Pathogenic Blastomycetes — Sporotrichosis — Thrush — Saeeharomycetes and Torulae — Yeasts and Fermentation. XVII. The Hyphomycetes — Aspergillosis — Ring- 'worm ...... 498 XVIII. The Protozoa . . . . .508 The General Structure of the Protozoa — Pathogenic Amoeba? — Trypanosomata — Leishman-Donovan Body — Spirochaetce — Syphilis — Coccidia — Malaria. XIX. Scarlet Fever — Hydrophobia — Infantile Paralysis— Typhus Fever — Yellow Fever — Dengue — Phlebotomus Fever — Vaccina and Variola— Malignant Disease . . 562 XX. Some Diseases not previously referred to, with a discussion of their causation — Micro-organisms of the Skin and Mucous Membranes ..... 585 XXI. The Bacteriology of Water, Air, and Soil, and their Bacteriological Examination — Sewage — Bacteriology of Milk and Foods 603 Some of the Commoner Organisms found in the Air, Water, and Soil. XXII. Disinfection ..... 659 Heat — Steam Disinfection — Chemical Disin- fectants— Theory of Disinfection— Methods of determining Disinfectant Power. French Weights and Measures and their English Equivalents — Solubilities .... 685 INDEX .687 LIST OF PLATES PLATE I. PHAGOCYTOSIS AND M. PYOGENES . to face p. 238 II. STREPTOCOCCUS PYOGENES . . „ 244 III. THE MENINGOCOCCUS AND GONOCOCCUS „ 256 IV. ANTHRAX ...... 264 V. ANTHRAX . . ... 268 YI. DIPHTHERIA . . . . 280 VII. THE HOPMANN BACILLUS AND VINCENT'S ANGINA . . „ 310 VIII. THE TUBERCLE BACILLUS .■■-.-.„ 318 IX. THE TUBERCLE BACILLUS . 326 X. LEPROSY AND B. SMEGMATIS . . „ 352 XI. B. MALLEI AND GLANDERS NODULE . „ 364 XII. BACILLUS TYPHOSUS . . . . „ 372 XIII. B. TYPHOSUS AND B. COLI . 402 XIV. PLAGUE 414 XV. B. PESTIS AND CHICKEN CHOLERA . „ 416 XVI. UIPLOCOCCUS PNEUMONIJE. . 4q2 xii Manual of Bacteriology PLATK XVII. B. TETANI AND B. WELCEII . . to face p. 452 XVIII. SPIRILLUM CH0LER2E AND CULTURES OF SPIRILLA . . . 460 XIX. ACTINOMYCOSIS BOVIS AND MYCETOMA „ 476 XX. ACTINOMYCOSIS HOMINIS ...» 478 XXI. TRYPANOSOMA GAMBIENSE AND SPIRO- CHAETA RECURRENTIS (OBERMEIERI) „ 524 XXII. TREPONEMA PALLIDUM 526 XXIII. TREPONEMA PALLIDUM AND COCCIDIUM OVIFORM E 528 XXIV. THE MALARIA PARASITE . • „ 550 XXV. TERTIAN "ROSETTE" AND HALTERI- DIUM D ANILE WSKYI ...» 556 XXVI. PIROPLASMA CANIS AND HiEMOGREGA- GARINE OP COBRA . » 560 A MANUAL OF BACTERIOLOGY INTRODUCTION. ^Bacteriology is that branch of Biology which deals with the study of Mici'O-organisms, particulai-ly the minute vegetable organisms known as Bacteria. The scope of bacteriology is difficult to define exactly, for the term is often used in a comprehensive sense equiva- lent to micro-pathology, or even micro-biology, and all investigations conuected with micro-organisms, animal and vegetable, may be included under it. So exten- sive, however, has the subject become that the animal micro-organisms are now being studied as a separate branch, Protozoology. Bacteriology deals with micro- organisms particularly in their relation to processes- disease, fermentation, putrefaction, and the like — while their structure, functions, and life-his-tory are to a large extent left to the botanist and zoologist. There is no space in a work ol this kind to enter into the history of the science, but the names of Leeuwenhoek (1675), Miiller (1786), Schwann (1837), Cohn, Pasteur, Lister, 1 2 Manual of Bacteriology and Koch will ever hold an honourable place in its annals. The study of micro-organisms must always be of considerable importance in general biology, for their vital phenomena arc comparatively simple, and throw much light on the more complex processes occurring in the higher orders of living beings. Weismann based his theory of heredity on the fundamental conception of the immortality of these unicellular organisms. Excluding accidents, they are immortal — they repro- duce themselves by a process of simple division, an individual dividing, and two daughter forms taking the place of the original parent one, and although the parent has disappeared yet there has been no death, no dissolution ; its protoplasm or living material is still existent in its progeny and is immortal, since this process of reproduction apparently may go on indefinitely. Moreover, the study of the mutability and possible transformation of species of micro-organisms is likely to throw light on the theory of evolution. Organisms such as bacteria multiply so rapidly that fifty or sixty generations may be developed in thirty hours, a, number which would take years to attain if even the most rapid breeder among mammals were the subject of experiment, and as they occur in vast numbers the opportunity for variation is extensive. These are some of the relations which micro-organisms have to general biology. In what may be termed the economy of nature micro-organisms are all-important ; without them there would be no putrefaction, no decay, and the dead remains of animal and vegetable life would so accumu- late as to encumber the earth, which would become sterile for the want of the organic matter originally derived from it, but of which there was no return. In Life without Bacteria fact the higher plants, and indirectly, therefore, animals also, arc dependent for their existence upon the presence of bacteria in the soil, which break up and render assimilable complex substances presented to them as manures. The question of life, animal and vegetable, without bacterial activity is an important and interesting one. It would seem from the experiments of Duclaux1 that the higher plants in ordinary circumstances are unable to obtain nutriment unless the complex compounds, pro- teins, urea, and other nitrogenous bodies, which form the important constituents of many manures, are broken down into simpler ones through the agency of bacteria. He sowed seeds in sterile soil free from nitrates, nitrites, and ammonia, which was plentifully watered with sterile milk and solutions of sugar and starch. No changes occurred in these substances, the seeds lost weight, and the seedlings dwindled and died. As regards the higher animals various views have been ex j tressed. Pasteur considered that their life also would probably be impossible without the presence of bacteria in the intestinal tract. Nenclci expressed the opinion that this idea of Pasteur's was an erroneous one, and his experiments in conjunction with Macfadyeu and Sieber2 showed that any considerable decomposition of the food by bacteria first takes place in the large intestine, and that the digestive juices alone, without the co-operation of bacteria, are able to prepare the constituents of the food for absorption. Nuttall and Thierfelder obtained unborn guinea-pigs by Csesarian section with antiseptic precautious, and afterwards kept them in a sterile environment and fed them on sterilised food. Not only did the animals live, but 1 Comp. llend.i t. 100, p. Gti. 3 Journ. of Anat, ami Physiol., kxv, p. 390. 4 Manual of Bacteriology they were even in a more thriving condition than those naturally brought up. The intestinal tract was found to be sterile on the eighth day. Ou the other hand, Schottelius found that chickens reared on sterile food were retarded in development, and experiments by Moro on turtle larvae lead to the same conclusion, viz. that intestinal bacteria are necessary for normal nutrition. Levin, however, found that the intestinal tract in many Arctic animals — the polar bear, reindeer, seal, eider duck, etc. — is generally sterile, and in these instances, therefore, bacteria are not required for normal nutrition. Commercially, micro-organisms are of the utmost importance. Without them there would be no fermen- tation, and the wine, beer, and indigo industries, the ripening of cheese and tobacco, and many like processes would be non-existent. From a financial aspect also micro-organisms cannot be ignored, for many of the so-called "diseases" of beer and wine, which often occasion great loss, are due to the entrance of adven- titious forms, while the silk industry and sheep farming in France were once threatened with extinction owing to the ravages of pebrine and of anthrax respectively, but through the genius of Pasteur were restored to their former prosperity. There is no need to emphasise the importance of micro-organisms from a medical and hygienic point of view, but the fact may be recalled that sixty years ago the mortality after operations was very high, and that 40 per cent, of these deaths were caused by pyemia, septicaemia, and hospital gangrene, conditions which are due to the entrance of micro- organisms, and which are now almost preventable, thanks to the antiseptic system introduced by Lord Lister. The theory of spontaneous generation or abiogenesis is intimately connected with the study of bacteria. Abiogenesis b The putrefaction of animal and vegetable fluids even after boiling, and the growth in them of minute living forms, were held by many to be a sure proof of the development of life from inanimate matter, of the spontaneous generation of the living from the non- living. A succession of investigators, however, showed (1) that if the fluids be boiled sufficiently long, and be then sealed up so as to prevent the access of air, they do not undergo putrefaction ; (2) that the sealing up may be dispensed with, provided the air be first filtered through cotton-wool before being admitted to the Masks; (3) that even the cotton-wool is not needed if the air be passed slowly through a long and tortuous channel, so as t > deposit its solid particles. Tyndall showed that putrescible fluids may be exposed in open vessels in a ch-sed chamber in which the air has been undisturbed for some time and its solid particles thereby deposited on the walls of the chamber, which had been smeared with glycerin ; he also proved that vegetable infusions and the like, which putrefy after having been boiled for ten minutes, do not do so if the boiling be repeated on two or three successive days, and explained this by the supposition, that while the fully developed bacteria are destroyed by the first boiling, their more resistant spores remain alive, but these on being left for twenty-four hours germinate into the less resistant bacterial forms, which are destroyed by the second boiling, and by the repetition of the process complete sterilisation may ultimately be obtained. It is this process of " discontinuous sterilisa- tion," as it is termed, which is employed by the bacterio- logist for the preparation of sterile culture media.1 1 The writer believes that this explanation is only partially true, ami would ascribe some of the sterilising effect of repeated heatings simply to the injurious action of alternate heating and cooling. 6 Manual of Bacteriology The idea, of nbiogenesis (or as he prefers to term it, " archebiosis ") has recently been revived by Bastian. He claims that certain saline solutions which have been boiled or even heated above the boiling-point in sealed tabes alter a time show the development of various living organisms, including bacteria and yeasts.1 Dunbar,2 as the result of a series of experiments conducted over a long period and with every care to prevent contamination, has come to the conclusion that the bacteria are not an independent group of organisms, but that the bacteria, yeasts, and moulds are stages in the life-history of green algse. The observations were carried out both by culture methods and by micro- scopical examination. A culture of a single-celled alga belonging to the Palmellacea was obtained, but by modifying the culture medium in which a pure culture of the alga was growing, by the addition of acid, of alkali, or of traces of copper salts, other organisms, generally bacteria, occasionally moulds and yeasts, and even spirocheetes, made their appearance. Granting that there is no flaw in the experimental methods, and every care seems to have been taken to exclude con- tamination, etc., the results are susceptible of another explanation, viz. that the secondary growths were derived by transformation of the algal cells, in fact by the phenomenon of heterogenesis which has been claimed by Bastian to occur. Undoubtedly bacteria exhibit variations and muta- tions, not only in morphology (see p. 15) but also in function. Thus pathogenic organisms may become non- pathogenic, and Twort has succeeded in training B. 1 See various papers in the Proc. Roy. Soc. Loud, and The Evolution of Life, Metkuen, Jl)(»7. - See Journ. Roy. Inst. Pub. Health, vol. xv, No. U, 1907, p. (37!). Literature on Bacteria 7 typhosus to ferment; lactose, which ordinarily it does not. Some recent experiments by Horrocks1 suggest that the B. typhosus may, by symbiosis with B. coll, be converted into B. alcaligen'es. Minchin in a presidential address to the Quekett Microscopical Club points out that syngamy (sexual reproduction, e.g. conjugation) is of the greatest impor- tance in preserving differentiation of species, and that without it a species will tend to break up into races. It therefore follows that there are no true species among organisms of the bacterial grade, if it be true, as is usually held, that syngamy does not occur amongst them, and the so-called species of bacteria are to be regarded as mere races or strains capable of modifica- tion in any direction. Doubtless immense progress has been made during the last two or three decades, but a vast amount still remains to be done. We have only touched the fringe of the explanation of the difficult jn'oblems of immunity, of the extraordinary variations in virulence and effects of the same organism, and of the important question of cui'e in, and prevention of, infective diseases, while the chemistry of the products of bacterial activity is still in its infancy. The literature of Bacteriology is now becoming somewhat extensive. In the following pages a good many references to original papers have beeii introduced, so that further informa- tion may be obtained if required, the aim being so far as possible to refer to easily accessible papers which contain a more or less full bibliography on a particular subject. Kolle and Wassermann's Handbuch dtr Pathogenen Milcroorganismen is the most encyclopaedic work on pathological bacteriology yet published. 1 Seo Brit. Med. Juum., 1911, i, p. 10?:J. 8 Manual of Bacteriology CHAPTER I. THE NATURE, STRUCTURE, AND FUNCTIONS OF THE BACTEKIA : THEIR CLASSIFICATION, GENE UAL BIOLOGY, AND CHEMISTRY BACTERIA AND DISK ASK. The Bacteria or Schizotnycetes ("fission fungi") are minute vegetable organisms for the most part unicellular and devoid of chlorophyll, which multiply by simple transverse division or fission ; this distinguishes them from the yeasts, in which multiplication takes place by budding or gemmation. A certain number of filamentous forms are also included, serving to connect the unicellular ones with the multicellular true fungi. The " fission plants " may be placed in a sub-kingdom, the Schizo- phyta, which may be divided into two classes : Class I, Schizophycea?, the blue-green alga?, and Class II, Schizo- mycetes, the bacteria. The unicellular plants are sometimes termed the " Protophyta." It must be understood that there are connecting links between the different groups, and that there is no sharp line of demarcation between them. The relation of the bacteria to other lower plants is shown in the following scheme : Structure of Bacteria 9 Relation of Bacteria to Lower Plants. Thallophyta (lower plants without fibro-vascular bundles, and with no distinction between root and stem) Forms with chlorophyll Forms without chlorophyll (Algae, desmids, etc.) | Multicellular. Spores in Unicellular. Spores frequently differentiated cells or spore- absent. Spore-bearing- cells not, bearing- organs. Frequently or but slightly, differentiated, a sexual method of reproduction No form of sexual reproduction The true Fungi (Eumycetes) I I including moulds (Hypho- Eeproduction Reproduction mvcetes) by fission by budding | I The Schizomycetes The Blastomycetes or Bacteria or Yeasts The size of the bacteria is variable, but they are all microscopic, measuring from 0-3 ,u to 30-40 /.i in diameter or in length.1 Their shape likewise is very different in the different species; some are spherical, others ovoid, others rod-shaped or filamentous, while in some the rod or filament is twisted into a spiral. The bacterial cell consists of a cell-membrane enclosing the transparent, more or less structureless living matter or protoplasm, the cell-plasma or cytoplasm. Biitschli has described the bacterial plasma as having a reticular structure, but in the young cell this is probably either an artifact or a " false image " due to faulty illumina- tion ; the most that can be seen is a fine granulation. The protoplasm frequently contains larger granules composed of fatty or protein matter, pigment, and in some species of sulphur. Occasionally certain granules stain blue with iodine. One to three spherical granules are also present in each cell; these seem to take part in the division of the cell (see below), and if the latter be stained with a very dilute mixture of roseine and 1 /x = micron = 0"001 mm. Seep. 150. 1,1 Manual of Bacteriology methylene blue are coloured pink, contrasting with the rest of the protoplasm, which is stained blue. Unless the roseine-staining granules, jusl mentioned, be regarded as such, no nuclear structure is present in the cell, nor does the suggestion that the bacteria are to be regarded as primitive nuclei almost devoid of pro- toplasm seem to be tenable. Another and more likely view is that i he bulk of the cytoplasm consists of a mixture of nuclear material with non-chromatic substance. The cell-membrane is usually invisible, but if the cell is treated with salt-solution (2"5 per cent.) phis- molysis takes place, the protoplasm shrinking away from the membrane, which then becomes visible. It can also be stained in vivo with very dilute solutions of roseine. The cell-membrane sometimes becomes thickened, swollen, and gelatinous on its outer surface, forming a layer or so-called "capsule" around the organism. The clear spaces frequently seen around bacteria in dried and stained preparations, especially in those from blood and lymph, are generally artifacts and not true capsules. In Cltidothruv and some other forms the cell-membrane becomes hardened, leading to the production of a firm sheath. When bacteria assume the resting stage groups of them adhere to- gether in a kind of jelly-like matrix, forming what is known as a "zoogloea." The chemical composition of bacteria varies much, not only in different species, but even in the same species when grown on different nutrient media. All bacteria contain proteins, lipoid substances, and salts. Bacterial protein according to Nencki, differs from ordinary protein matter in not being precipitated by alcohol and in not containing sulphur; it was termed by him « myko-protem." This does not appear t0 be the case with the proteins obtained by grinding Fertility of Bacteria 11 bacterial cells, which seem to agree with other proteins in heat-coagulation, etc. The proteins are mainly globulins ami nucleo-proteins. The cell wall is relatively insoluble, and generally consists chiefly of a material like chitin, and not of cellulose; in this respect bacteria resemble animal rather than vegetable cells. Carbohydrates are gene- rally scanty. Spores differ from the parent cells in con- taining a much larger proportion of solids and less water. All species of bacteria, but especially the smaller ones, when suspended in a fluid exhibit what is known as Brownian movement, consisting of an oscillation with some amount of rotation about a fixed point, but there is little actual movement of translation, unless due to flotation. This Brownian movement is physical and not vital in origin, and occurs with all fine particles suspended in a fluid, and must be clearly distinguished from a true vital motility. Some bacteria are always motionless, others are more or less motile, but these, too, have a resting stage. For motility to occur the cells must be young, and the conditions favourable to growth and development. Motility is due to delicate protoplasmic threads termed " flagella" connected with the outer layer of the cell protoplasm; these vibrate to and fro and propel the organism through the medium. A cell will, however, move indifferently in either direction ; if a motile organism be watched it will often be seen to proceed rapidly in one direction, stop, and then return without turning round. The flagella are not visible in the living state, unless dark ground illumination be used, nor by the ordinary methods of staining, unless previously treated with a mordant, and are extremely liable to be broken off. They vary considerably in number and in length; some organisms have but a single fiagelluna at one 12 Manual of Bacteriology pole (monotrichic) , e.g. Bacillus pyocyaneus, others have two or more flagella forming a brush or tuft (lopho- trichic), e.g. Spirillum rubrnm, while others may be almost entirely covered with them (paritrichic) , eg. B. typhosus ; in some the flagella are short and straight, and in others long and twisted. The motility of organisms does not necessarily depend directly upon the number of flagella they possess, an organism with a few flagella often being more active than another possessing many, and some are apparently non-motile, though well-marked flagella can be demonstrated. Generally, however, an organism with several flagella will be more motile than a similar form with a few. I See also p. 1 86.) Darwin says : " In looking at Nature it is most necessary never to forget that every single organic being may be said to be striving to the utmost to increase in numbers," and in no group perhaps of the animal and vegetable kingdoms is (his more marked than among the bacteria. Reproduction is entirely non-sexual, and takes place in two ways— by simple division or fission and by spore formation. Repro- duction by transverse Bssion is common to all bacteria; the bacterial cell becomes constricted at its middle and finally separates into two parts, and thus two young cells take the place of the parent one; repro- duction by fission is therefore also an increase m numbers. The fission is always transverse, never longitudinal,1 the rule being in cell-division that the new membrane is formed in the most economical manner. Longitudinal division, on the other hand, seems to be very common among the Protozoa. i Longitudinal division has been described in "a f ew species, but ifcs occu°rence is so rare that a doubt must arise as to whether these forms are true bacteria. Reproduction of Bacteria 13 Previous to division the rod-forms become elongated and the spherical ones ellipsoidal, and there is an increase in the number of the roseine-staining granules, partly by division of pre-existing ones and partly by new formation. The constriction in the majority of cases involves and passes through one of the grannies. In the monotrichous and lophotrichous bacteria it is always the non-flagellated end of the dividing cell which bears the flagella of the new cell. Under favourable conditions reproduction may be very rapid, fission occurring every twenty or thirty minutes (Klein), so that, the increase being in a geometrical ratio, the number of individuals which might arise from a single bacterium in three or four days is almost inconceivable, and would en masse weigh thousands of tons; fortunately there are many checks to such a rapid multiplication. Frequently, although the protoplasm divides, the division of the cell-membrane is incomplete, resulting in a loose union of the cells with the formation of a pseudo-filament. These filaments often become much curved and twisted, forming tangled masses, owing to fission talcing place in the cells in the middle of the filament as well as at the ends, so that the filaments have to become curved to make room for the new cells. Reproduction by spore formation is met with in some species, and is generally described as being of two kinds. In the first, " endogenous " spore formation, a bright refractile round or ovoid body is formed within the bacterial cell, the development of which can be watched under the microscope. Rowland describes the process of spore formation as follows : Refractile, oily- looking droplets, which do not stain with roseine, appear and ultimately coalesce, forming the spore. The cell- plasma at the same time diminishes and retracts from the cell-membrane. The roseine-staining granules increase 1 I- Manual of Bacteriology in number and aggregate into two spherical masses, which dispose themselves one al each end of the cell. The cell-membrane collapses somewhat, and, when the spore is fully formed, ruptures transversely, leaving two cup-shaped receptacles, in which the granules and remains of the plasma are still recognisable. Only one spore develops in each cell, and the spores seem to fulfil the purpose of perpetuating the race when it is threatened with extinction from adverse circum- stances. Each spore consists of a little mass of proto- plasm enclosed within a very membrane, which tends to preserve its vitality even under unfavourable conditions; for spores resist the action of desiccation and germicidal agents to a much greater degree than the fully developed organisms. Spores vary much in size and in the position they occupy within the bacterial cell in the different species ; their diameter is usually about the same as that of the cell in which they are developed, but may be much greater, and in position they may be central or ter- minal, and sometimes the spore-bearing cells are swollen or club-shaped ; these are termed " Clostridia." Endo- spores are still unknown in a large number of species. The second variety of spoliation, " arthrospore " formation, is of doubtful occurrence, but is stated to take place as follows: Some of the elements formed by fission are slightly larger, more refractile, and more resisting than their fellows, and are stated to have the properties of spores. Placed in favourable circum- stances the spore in either case germinates, it becomes swollen and granular, and loses its refractile appear- ance • a slio-ht protuberance forms, this increases in size, and an organism similar to the parent one is finally reproduced ; the empty spore membrane al 6rsi frequently encloses one extremity, and is afterwards Classification of Bacteria 15 cast off. In certain instances the spore germinates without casting its membrane, the spore membrane becoming- the cell-wall of the young organism. The ellipsoidal spores of the l>. anthraci? sprout from the end, those of B. subtilis from the side ("polar" and " equatoi-ial " germination respectively). On the Morphology, etc., of the Bacteria see Rowland, Tnnix. Jenner Inst. Prev. Med., ii, 1899, p. 143 ; Fischer, The Structure and Functions of Bacteria, 1900; Migula, System der Bakterien, i ; Dobell, Quart. Journ. Micr. Sci., 1911; Penau, Gomp. Rend., clii, 1911, p. 53. Classification of the Bacteria. Many classifications of the bacteria have been pro- posed, but none up to the present can be said to be strictly scientific, or even satisfactory from the point of view of convenience. In tlie first place, the bacteria are said to bo devoid of chlorophyll, but there are many forms intermediate between those unicellular organisms with and those without chlorophyll, so that a hard and fast line cannot be drawn. In the next place, the bacterial cells are so minute, and their vital pheno- mena so simple, that only a few broad distinctions can be observed in their morphology and reproductive processes. One of the most prominent of the older classifications was that of Cohn. He divided the bacteria into four main groups : I. The SpliEerobacteria or spherical forms. II. The Microbacteria or short rod-forms. III. The Desmobactjeria or long rod-forms. IV. The Spirobacteria or spiral forms. -Zopfs classification (1885) has many points to com- mend it, but is largely based on 'the doctrine of Isomorphism. By pleomorphism is meant a, variation m the form of an organism during its life-cycle, a 16 Manual of Bacteriology coccus, for example, growing into a rod, or a straighl rod becoming a spiral. In a peach-coloured bacterium examined by Lankester, cocci, rod, filamentous and spiral forms occurred, and the doctrine of pleomorphism received considerable support from his work, though it maybe questioned whether he was working with pure cultures. Be that as it may, a certain amount of pleo- morphism undoubtedly occurs in some organisms. In the colon, typhoid, and plague bacilli, for example, the rods may sometimes be so short as to be almost cocci, while at others they are well-marked rods and even filaments (see also p. 6). The following is an outline of Zopf's classification, the bacteria being divided into four main groups or families, which again are subdivided into smaller groups or genera : Family I. CoccacEjE. — Spherical forms only ; division occurs in one or more directions. Genus 1. Micrococcus (Staphylococcus). — Division in one direction only, but irregular, so that the cocci after division form irregular clusters. Genus 2. Streptococcus. — Division in one plane, but regular, so that the cocci form chains. Genus 3. Mkrismopedia. — Division in two direc- tions at right angles to each other, but in the same plane, so that lamella? or plates are formed. Genus 4. Sarcina. — Division in three directions at right angles to each other and in two planes, so that cubical masses are formed. Genus 5. Ascococcus. — Cocci which develop in a gelatinous matrix. Family II. Bacteriaceje.— Rods, straight or curved, at some period of the life-history, though cocci and other forms may occur. Classification of Bacteria 17 Genus 1. Bacterium. — Straight rods; endospore formation does not occur. Genus 2. Bacillus. — Straight rods; endospore for- mation occurs. Genu* 3. Leuconostoc. — Cocci and rods ; arthro- spore formation occurs in the coccoid forms. Genus 4. Clostkidium. — The same as bacillus, but the spore-bearing rods are enlarged and club- shaped. Genus 5. Spirillum. — Spiral rods; spore formation does not occur. Genus 6. Vibrio. — Spiral rods; spore formation occurs. Family III. Leptotkiche.*:. — These are unbranching thread forms. Family IV. Cladotriche^. — These are thread forms showing true but not dichotomous branching. There are many features in this classification which are of practical value. The distinction made between a bacterium and a bacillus, for example, is very con- venient. Formerly it was the custom to term a short rod a bacterium, and a long rod a bacillus, but such a division is an arbitrary one, and at one stage of its hfe-history an organism might have to be termed a bacterium and at another a bacillus. The term "bacterium " is now but little used, and any straight rod is termed a bacillus. The term " staphylococcus " is one frequently met with; it is practically synony- mous with micrococcus, and refers to cocci which are aggregated into groups or clusters. Of the twisted rods, a simple curved rod is now known as a vibrio, a definitely corkscrew form of three or a few turns is 2 18 Manual of Bacteriology a spirillum, a long and flexible bwisted filament is a spirochaeta.1 A I ater system of classification is tliat proposed by M igula.L The bacteria are divided into two orders : the Eubacteria — bacteria proper — the cells of w hich contain neither sulphur granules nor a colouring mailer, bacterio-purpurin ; and the Thiobacteria, the cells of which contain sulphur grannies and may be coloured with bacterio-purpurin. ■ The Eubacteria are divided into five families : (1) Coccaceas, (2) Bacteriaceaj, (:>) SpiriHaceas, (4) Chlarnydo-bacteriaceaa, and (■'•) Beggia- toaceas. These, again, are .subdivided into many genera, based partly on the mode of division and partly on the number and on the arrangement of the Ilagella. upon the organisms. The Coccaceaa— globular cells — contain the genera Streptococcus, Micrococcus, Sarcina (non-motile), and Planococcus and Planosarcina (motile) ; the Bacteriaceaa are defined as long or short cylindrical rods, straight and never spiral ; division in one direction only after elongation of the rods; and this family has three genera.: (a) Bacterium— -non-flagellated cells, often with endospore formation; {b) Bacillus— cells possessing both lateral and polar Ilagella, often with endospore formation j (c) Pseudomonas— cells with polar Ilagella only, rarely endospore formation. The Spiril- lacece are curved or spiral rods, and include (a) Spiro- soma, non-motile forms, (&) Microspira, motile forms with one polar Magellan), (c) Spirillum, motile forms with two or more polar Ilagella. The nomenclature of bacterial species is at present m a chaotic condition. In botanical and zoological nomenclature 1 Many of the so-called spirochaetse arc probably protozoa and not bacteria. , • . , . 2 System tier Bakterien, 1897. Abstract in Centr.f. Bakt. ( 1 1 A hi.). xxii, 1897 (September), p. 3 to Conditions of Life 11) every species has a binomial name, the first being the genei'ic, the second the specific, name. Many bacterial species have received trinomial names, which should be inadmissible. The specific name first given to an organism must stand unless it has been used for some other species. Conditions of Life of Bacteria. Bacteria, being living organisms, must be supplied with suitable nutritive substances in order that their life-processes — nutrition, reproduction, and the like — may be carried on in a normal manner. Being- devoid of chlorophyll they are mainly dependent upon complex organic compounds for the carbon, hydrogen, and nitrogen which cider into I heir composition, these elements being derived for the most part from proteins and carbohydrates. Some bacteria, however, are able to obtain the requisite nitrogen from such comparatively simple compounds as ammonia, ammonium carbonate, or nitrates, and one group can make direct use of the atmospheric nitrogen. Certain inorganic salts, sul- phates, phosphates, and sodium chloride, also seem to be necessary for normal development. These nutrient substances must be presented to the bacteria in asso- ciation with water, for without water bacterial activity ceases, though in the dry state many forms, and espe- cially their spores, may retain their vitality for a con- siderable time; absolute desiccation, however, is rapidly fatal to many. Temperature is also an important factor. Though the growth of many species occurs through a wide ■■auge there is for almost all an optimum at which growth .s best, and of a range not exceeding 5° or 10° growth usually censes below 10° C, but cold does not ' ,'mv b/cterial m'"; after expos,,,,, to the ,. ilc.se cold produced by the evaporation of liquid oxygen 20 Manual of Bacteriology ( — 170° C.) for weeks, or of liquid hydrogen ( — 252° C.) for ten hours, bacteria and their spores will grow and germinate, and their chrornogenic and pathogenic pro- perties seem to be unaltered.1 On the other hand, bacterial growth, usually ceases when the temperature exceeds 40° C. or thereabouts, and most bacteria with- out spores are destroyed within half an hour by a temperature of 65° C. The spores are far more resistant : some mav even be boiled for a short time without losing their vitality, but prolonged boiling is fatal to both bacteria and their spores. There is, however, a group of so-called thermophilic bacteria, which thrive best at a temperature of 60° to 70° C. They occur in the soil and in water, and are probably of considerable importance in the natural fermentations accompanied by the evolution of heat, such as are met with in manure heaps, the heating of hay, and firing of moist cotton.2 Free oxygen is essential to the growth of some organisms ; these are termed strictly aerobic. Others will not develop in its presence, strictly anaerobic ; others, again, while preferably aerobic or anaerobic, will grow in the absence, or in the presence, of oxygen, and are respectively termed facultative anaerobic or facultative aerobic. Some organisms are strictly para- sitic on animals or plants ; others live in water, soil, decaying matter, etc. — these are termed saprophytes ; and many are able to exist either as parasites or as saprophytes (see also p. 151). Bacterial development is much influenced by the presence of foreign substances in the nutrient medium. 1 Macfadyen and Rowland, Proc. Boy. Soc. Lond., February 1st, 1900 ; April 5th, 1900 ; May 31st, 1900. 2 Macfadyen and Blaxall, Journ. of Path, and Bad., November, 1894, and Trans. Jenner Inst, of Prev. Med., vol. ii, 1899, p. 162. Symbiosis 21 A number of metallic and other salts, chlorine, bromine, and iodine, carbolic acid, salicylic acid, etc., Lave an injurious effect upon bacterial life, inhibiting or stopping growth, or killing the organisms outright; these are of considerable practical importance and are known as germicides, antiseptics, and disinfectants. The pro- ducts produced in the nutrient medium by the bacteria themselves also sooner or later inhibit or stop further growth: a familiar instance of this is seen in the alco- holic fermentation of sugar by yeast, which ceases when the amount of alcohol reaches 12 or 14 per cent. The same reason probably accounts for the fact that growths of bacteria in culture tubes do not spread all over the surface of the nutrient medium, and why our cultures sometimes die out more rapidly than might be expected. Another point affecting bacterial life is the presence of a mixture of organisms in the same nutrient medium. If there be a very vigorous form, it may ultimately grow and multiply to such an extent as to crowd out and finally kill the other forms with which it is asso- ciated, and if the nutrient medium equally favour two species, that one which is in an excess at the beginning may outgrow the other. The occurrence of what has been termed symbiosis is of considerable interest in the life of micro-organisms, and too little attention has hitherto been paid to it. This is the co-existence of two or more species which together bring about certain changes. For example, in the well-known ginger-beer plant, Marshall Ward1 isolated several yeasts, bacteria, and moulds; of these, one of the yeasts and one of the bacteria together induce the particular changes in a saccharine fluid to which ginger has been added, which render the mixture like ginger-beer, and these changes do not occur unless both species develop together. 1 Phil. Trans. Roy. Soc. Loud., vol. clxxxiii, 1892, p. 125. 22 Manual of Bacteriology Another extraordinary feature exhibited by bacteria is the selective action exerted on certain substances which contain isomerides or right- and left-hander! modifications of a substance. The Barillas fthaceticus attacks mannitol but not dulcitol, two alcohols which are very similar in taste and properties and possess the same simple chemical formula. By a series of most brilliant researches Eniil Fischer has succeeded in determining the constitution of the various sugars, and, what, is more, has produced thorn artificially in the laboratory. The natural sugars are all compounds with dissymmetric molecules, powerfully affecting the beam of polarised light, but when prepared artificially they are without action on polarised light, because the artificial product consists of equal numbers of left-handed and right-handed molecules, and the molecules of the one neutralise the moleriiles of the other, thus giving rise to a, mixture which does not affeci the polarised beam. By the action of micro-organisms, however, on such an inactive mixture the one set of molecules is sought out by the microbes and decomposed, leaving the other set of molecules untouched, and the latter now exhibit their specific action on polarised light, an active sugar being thus obtained. Fructose was one of the principal artificial sugars prepared by Fischer; it is inactive, but consists of an equal number of molecules of oppositely active sugars termed " hevulose." One set of these hevulose mole- cules turns the plane of polarisation to the right, another set to the left— right- and left-handed hevulose. The left-handed hevulose occurs in nature, while (he rio'ht-handed hevulose, so far as is known, does not. Now, on putting brewer's yeast into a, solid ion ol fructose, the inactive artificial product, the yeast Effect of Physical Agents 23 organisms attack (lie left-handed lasvulose molecules and convert them into alcohol and C02, while the right-handed lasvulose is left untouched. Pressure, unless very great, has little effect on bacteria. Roger investigated the effects of high pressure on certain organisms in bouillon cultures. Pressures of 200 to 250 kilos, per square centimetre had no effect ; by raising the pressure to 3000 kilos, per square centimetre one third of streptococci were killed, and of anthrax without spores a good many ; while sporing anthrax, Micrococcus 'pyogenes, var. aureus, and the colon bacillus were unaffected.1 Oar countrymen Downes and Blunt first; called attention to the injurious effect of light upon bacteria. If plate cultures be prepared and exposed to sunlight, a portion of the plate being protected from its action, as by sticking on a letter cut out of black paper, and the preparation afterwards incubated, it will be found that the colonies develop at the protected portion only, those parts which have been exposed to sunlight remaining sterile. Although this action of sunlight may occasionally be due to chemical changes in the medium, resulting in the production of ozone or other germicidal bodies, the experiments of Marshall Ward and others have conclusively shown that germicidal action may be caused by the direct action of the light, the violet and ultra violet rays being those concerned, and the reel end of the spectrum has no effect. The Rontgen rays seem to have little or no influence "|»m bacteria, but the results obtained are somewhat contradictory. 1 Bacteria being so minute, the actual pressure on a bacterial cell even with these high pressures, is small. If, for example, a bacterium measures 1 /£ by f,,,. a pressure of loot) kgrm. per square centimetre would be but 0-05 grm. (» grain) on the cell. •_>l. Manual of Bacteriology The radium emanations with prolonged exposure and near contact are germicidal to non-sporing organisms.1 Electricity, per se, lias also usually little effect. When the current is passed directly through the cultures electrolysis takes place, and the products formed may destroy the bacteria ; currents of high potential, however, may inhibit growth.2 Living motile bacilli are very sensible to induced currents of electricity, immediately orientating them- selves in the direction of the current, while dead or paralysed bacilli are unaffected.3 Bacterial Products. The chemical changes produced by micro-organisms are chiefly analytic or destructive — the formation of simpler from more complex bodies. This analytic faculty is present to a marked degree in the process known as putrefaction. Putrefaction, is a term applied to the decomposition of organic, especially protein, matter after the death of the animal or plant. It is usually accompanied by the evolution of foul-smelling gases and by the solution of the solid material. A large number of organisms are concerned in this process, particularly a group to which Hauser gave the name of Proteus. The first changes which occur are the formation of proteoses and peptone, then leucin, tyrosin, and glycocol, and basic compounds to which the name of ptomine has been given ; next indole, skatole, and phenol, and volatile fatty acids ; and lastly, mercaptans, sulphuretted hydrogen, marsh gas, am- monia, carbonic acid, and hydrogen. 1 See Green, Proc. Boy. Soc. Lond., vol lxxiii, 1904, p. 375. 2 Lortet, Comp. Bend., t. 119, 1894, p. 403. » Comp. Bend., t. 122, 1890, p. 892. Indole 25 In view of its practical importance in bacteriological analysis and the identification of species, indole may here be referred to at some length. Indole. — Indole (C8H7N) is a product of the putre- factive decomposition of proteins containing a trypto- phane nucleus and is formed during the growth of many organisms, and, since one species may produce it and another allied one may not, the determination of its presence or absence in the culture may be of value in the identification of organisms. The detection of indole is based on the reaction with nitrous acid, with which it gives a fine purplish-red coloration. In order to test for it, the orgnnism is grown in a fluid medium for twenty-four to forty-eight hours or longer, 1 c.c. of a 0"1 per cent, solution of sodium nitrite is added to every 10 c.c. of the culture, and a few drops of pure concentrated sulphuric acid or of hydrochloric acid are allowed to trickle slowly down the side of the test- tube, which is inclined with its mouth away from the operator. As the acid runs down, it is mixed with the fluid; a colour varying from pale pink to pale purple indicates the presence of indole. A control tube, uninoculated, should also be similarly tested to make sure that the reaction is due to the products of the growth of the organism. The culture fluid usually employed is peptone water, preferably 2 per cent., but some samples of " peptone " occasionally fail to give the indole reaction when organisms are grown in media prepared from them ; the right kind of peptone must, therefore, be used. As the dilute solution of sodium nitrite is unstable, a stock 5 per cent, solution may be kept ; 2 c.c. of this solution are diluted to 100 c.c. with distilled water at the time of making the test, and 1 c.c. of this dilution is added to every 10 c.c. of the culture. The addition of the acid liberates free nitrous acid, 26 Manual of Bacteriology which reacts with any indole present, and yields a pink colour. Sometimes when the reaction is apparently absent or feeble, it may be obtained or intensified by placing the tnbe in the blood-heat incubator For half an hour. The sulphuric acid should be pure and tree from oxides of nifcrogerij hence hydrochloric acid is often preferable. A more dedicate method of testing: is to run a little hydrochloric acid down the side of the tube, so that a layer forms at the bottom, the nitrite having been previously added to the culture it required. A pink ring at the juncture of the hydrochloric acid and cul- ture indicates the presence of indole. The pink pig- ment, the product of the reaction, may lie extracted by shaking with a little aniylic alcohol. Other delicate ami more certain reagents for the detection of indole arc para-diniethylaruidobenzaldehyde (15 grm., dis- solved in water 250 c.c, concentrated sulphuric acid 30 c.c. I, which gives a, rose in cherry-red, and /3-naphthaquinone- sodium-niono-sulphonate . 2 per cent, aqueous solution), which gives, when tlx.' mixture is rendered alkaline with caustic potash, a, blue or blue-green colour or precipitate. The coloured compound may be extracted with chloroform, in which it yields a, red solution. Peptone water is by no means a good culture medium, and broth may therefore he employed, hut it should be free from dextrose. Peptone water with the addition of a little rabbit's serum is perhaps the best culture medium for the production of indole. The presence of dextrose, saccharose, glycerin, or lactose in quantity exceeding about 025 per cent, prevents the formation of indole in broth by bacteria. Broth prepared in the ordinary way usually contains a linle dextrose derived from the glycogen in the meat, Indole 27 and this probably explains why the indole reaction is generally much more marked in a peptone water than in a broth culture, although the latter is a better nutrient soil. In order to prepare a soil free from dextrose, T. Smith1 recommends that the acid beef broth used in the preparation of nutrient broth should be inoculated with the colon bacillus and incubated for twenty-four hours, and the peptone beef broth prepared from it. The dextrose is consumed and no indole is formed. Some bncteria not only form indole but also produce nitrites in the culture medium by the reduction of the nitrates present in the peptone, etc., used in making the nutrient medium, in which case the addition of pure sulphuric or hydrochloric acid alone suffices to bring out the pink indole reaction. This forms, therefore, an additional means o£ distinguishing organisms, and is employed especially for the recognition of the cholera spirillum, which, if grown in peptone water, gives the indole reaction (or, as it has been termed, " the cholera red reaction ") on the addition of acid alone. The reaction can be obtained as early as twelve hours alter inoculation, and becomes very marked in twenty-four to forty-eight hours. [f indole is formed only in small quantities, 100 c.c. of the culture may be distilled ; the first 20 c.c. of the distillate will contain the bulk of the indole. This " indole-reaction " is not necessarily always due to indole; the writer has shown2 that the indole-like reaction obtained with cultures of I lie diphtheria and pseudo-diphtheria bacilli is owing to the presence of skatole-carboxylic acid. This substance is distinguished from indole by being non-volatile. To make sure1 of the 1 Jown. «f jasper, Med , vol. ii, I SOT, p. 543. • Traits. Path. Soo. Lond., vol. lii, pt. ii, tool. p. 11:5. 28 Manual of Bacteriology presence of indole, the culture should therefore be made alkaline with caustic soda and distilled. Skatole (methyl indole) seems also to he formed by some organisms. It is volatile like indole, but if a solution con- taining it be boiled with an acid solution of dimethylamido- benzaldehyde (5 per cent, in 10 percent, sulphuric acid) it yields a blue colour, which gives a blue solution in chloroform. Nitrification. — Another important series of changes is that included under the term " nitrification." As mentioned before, protein, albuminoid, and other com- plex nitrogenous matters and urea, all of which are valuable manures for plant life, cease to be so unless bacteria are present. It is necessary, in fact, for the nitrogenous matter to be converted into nitrates, in which form alone is it available for the nutrition of plants. Although so important, extremely small quantities of nitrates are present in the soil ; in fertile soils, for example, under some conditions there may be as little as one part of nitrogen in 1,000,000, and there is often less than ten parts. The bodies yielding nitric acid in the soil iire : (1) free nitrogen; (2) small quantities of nitrates in rain-water; (8) ammonium salts, applied intentionally or carried to the soil by rain or derived from the decay of organic matter; (4) various nitrogenous organic substances arising from the decay of animal and vegetable matters. With regard to the production of nitric acid from nitrogenous organic matters very little was formerly known. In 1877 Schloesing and Miintz by an ingenious experiment showed that nitrification (as the production of nitric acid is termed) of nitrogenous organic matter is brought about by living organisms in the soil. Sewage was passed continuously through a tube Nitrification containing a mixture of ignited quartz 'sand and lime- stone. After three weeks nitrates began to appear in the effluent and increased to such an extent that finally the filtered sewage contained no ammonia. After this had continued for some weeks chloroform vapour was passed at the same time through the tube, with the result that in ten days after the introduction of the chloroform all nitrates disappeared from the effluent. Subsequently the passage of chloroform vapour was discontinued, but nitrification did not resume until the washings from 10 grm. of garden soil were added. Eight days after this addition nitrates again appeared in the effluent (this was confirmed by Warington). Evidently the chloroform vapour acted as an antiseptic and killed the nitrifying organisms, while the addition of soil washings re-inoculated the material. Shortly after this Schloesing and Miintz found that exposure of soil to 100° C. for an hour destroyed the power of inducing nitrification. Soils thus treated were exposed to a current of air, purified by ignition, without nitrification taking place ; the addition of a little unheated mould was, however, sufficient to cause nitrification to recommence. They also tried seeding the sterilised soils with various Hyphomycetes, etc., without result, In 1884 Warington concluded that the factor determining the formation sometimes of nitric acid and sometimes of nitrous acid was a difference in the character of the organisms ; for it is possible to have two similar solutions under identical conditions, and for nitrites to be produced in the one, and nitrates in the other. In 1886 Dr. Munro showed that the process of nitri- fication could take place in solutions practically destitute of organic matter. :;<) Manual of Bacteriology Nitrification in the soil lakes place in three stae-es : I. Amuiunisation. — When complex organic coin- pounds such as albuminoids are applied to the land they are broken up; first they become liquefied, peptone-like bodies being produced; these are then further acted upon and we get alkaloidal substances in small quantity, indole, skatole, leucin, and tyrosin and aniino-aeids, valerianic acid, volatile fatty acids, lactic acid, etc. These changes are brought about by numbers of organisms, among which the varieties of Proteus (formerly known as Bacterium tarino), B. mesenterial*, B. mi/ co ides, B. fluorescens liqu&f miens , and B.putrifieas are the more important. The nitrogenous compounds are then further acted upon and ammonium sails are formed. Accordino- to Bmile Marchal, ammonisation lakes place essentially under the influence of microbes living in the upper layers of the soil. The Bacillus mycoides is one of the most energetic of these, and seems to play a. double role, being atnmonising in the presence both of nitro- genous organic substances mid of nitrates. Urea is ammonised especially by I he Micrococctts uresz. II. Nitrosation. — The ammoniacal sails are next converted into nitrites. The nitrous organisms can probably attack nitrogenous organic substances such as asparagine and milk, hut only feebly, milk being much more rapidly nitrified when the nitrous org'anisms are mixed w ith other species. The organisms bringing about jhis change are short, stumpy, motile bacilli with single polar Hagella which are grouped under the generic name of Pseudumonas. III. Nitratation. — These nitrites are then converted into nitrates. The "nitric" organisms are minute non- motile bacilli known as Nitrobactcr. Nitrification Stages ii and II] are brought about by different species, the nitric organisms having no effect whatever on ammonia, but acting- only after this lias been oxidised into nitrous acid by the nitrous forms. The discovery of Dr. Minim thai organisms will grow in purely inorganic solutions lias been made use of for the isolation of the different species. Solutions such as the following have been used : For the Nitrous Organisms. For tlie Nitric Organisms. Ammonium chloride, Oo -Tin. Potassium nitrite, 0"3 grm. Potassium phosphate, 01 grin. Potassium phosphate. 01] grm. Magnesium sulphate, 0-02 grm. Magnesium sulphate, trOo grm. Calcium chloride, U'01 grin. Calcium carbonate, 5 grm. Calcium carbonate, 5 grm. Distilled water, 1000 C.Q. Distilled water, 1000 c.c. These are seeded with traces of earth, and by carrying on the cultivation for .many generations a large number of organisms are eliminated. This method does not lead to a pure cultivation, for several forms besides the nitrifying organisms persistently maintain themselves in these mineral solutions. So recourse was had to gelatin plate cultivations. Although several organisms were isolated in this manner, none of them possessed the slightest: nitrifying power. Pranhland, and later Wariugton (18(.t(M, succeeded in isolating nitrous organisms by the dilution method. Nitrify- ing solutions were diluted, and traces inoculated into ammoniacal solutions; in some of these nitrification occurred, although no growth could be obtained On gelatin, ami they were found to contain the nitrous organism only. A little, later Winogradsty isolated nitrous organisms, first by modi- fied gelatin plates, and afterwards by the silica jelly method. Warington gives the following directions for the prepara- tion of silica, jelly plates: Sodium carbonate is fused iu the blowpipe, and line white sand is added as long as efferves- cence is produced. The mass is allowed to cool, and is then dissolved in water. The solution is poured into an excess of 32 Manual of Bacteriology very dilute hydrochloric acid (silicic acid and sodium chloride being formed). The solution is dialysed and sterilised. Some of this is placed iu a sterile dish aud is mixed with the following solution and inoculated : Ammonium sulphate . .04 grm. Magnesium sulphate . . . 05 Di-potassium hydrogen phosphate . 01 Calcium chloride .... trace Sodium carbonate .... 0'6-0'9 grm. Water 100 c.c This mixture sets to a jelly in five to fifteen minutes. Winogradsky has also made use of agar for plates, but this medium is not so suitable as the silica jelly. A 2 per cent, aqueous agar is prepared and poured into Petri dishes ; the film is then sown with Proteus, and allowed to grow for seven to ten days. It is then thoroughly washed, collected, melted, and mixed with the salts mentioned above. The object of growing the Profeus upon it as a preliminary is to eliminate the organic matter admixed with the agar. Nitrification in the soil is thus brought about by two groups of organisms. The first oxidises ammonia into nitrous acid, and is isolated by successive cultivation in solutions of ammonium carbonate. The second group oxidises nitrous acid into nitric acid, and may be separated by successive cultivations in a solution of potassium nitrite containing a little sodium bicarbonate. In the soil the nitric and nitrous organisms are equally active. Besides the derivation of nitrogen from nitrogenous compounds, the free atmospheric nitrogen is also " fixed " through the agency of certain micro-organisms and rendered available for plant life. Thus, the Leguminosre are able to obtain their nitrogen directly from the nitrogen of the air. If the roots of a pea, bean, or vetch be examined, numerous Fixation of Nitrogen 33 little nodules will be found upon them ; these contain minute irregular and Y-shaped bodies, which have been termed " bacteroids," and seem to be of the nature of involution forms. On inoculation into suitable cultui'e media1 the bacteroids give rise to a growth of a motile bacillus known as Pseiidomonas radicicola ; this " fixes " the atmospheric nitrogen. The oi'ganisms penetrate the young roots through the root-hairs, mul- tiply and form a filamentous zooglcea, which grows into the tissue of the root and penetrates the cells. Large amounts of nitrogen are taken up by the bacteroids, and are converted into nitrogenous com- pounds which can be assimilated by the plant. Legu- minous plants grown from sterile seeds in a sterile soil dwindle and die, but if inoculated with the organisms derived from another plant of the same species growth becomes vigorous ; if inoculated with those derived from another species growth still takes place, but not nearly to the same extent. The Leguminosse thus store up one of the most important elements of plant food, and hence their value in the rotation of crops. There is apparently no increase of nitrogen compounds in. the soil, the excess found being due to the root residues remaining. A substance termed " nitragin," consisting of a culture of these root organisms, has been prepared as a fertiliser. Nobbe's " nitragin " did not prove a success, apparently because the organisms soon lose their vitality. A better preparation, Cf nitro-bacterine," was devised by Moore of the United. States Department of Agriculture. Besides the leguminous organisms, other bacteria are present in the surface layers of the soil which fix 1 Such as wood-ashes maltose agar. Boil 8 grm. of wood-ashes with 500 o c. of water for one minute ; filter. To 400 c.c. of this extract add 4 grm. maltose and 4 grm. agar. Boil until dissolved : filter, tube, and sterilise. 3 34 Manual of Bacteriology atmospheric niti'ogen. The principal of these are ovoid organisms known as Azotobacter. This group can be cultivated in a mannite medium, e. g. di-potas- sium phosphate 0*2 grm., mannite 20 grm., water 1 litre. This may be used for isolation by converting into an agar medium by the addition of 2 per cent, agar. Prof. Bottomley has succeeded in obtaining a powder preparation of Azotobacter, which retains its vitality for months, and the preparation properly applied to poor soils produces astonishing results. It has been found that partial sterilisation of the soil, e. g. by heat, increases its fertility, whereas it might have been supposed that such a procedure would decrease the fertility by destruction of nitrogen-fixers. Russell and Hutchinson believe that the explanation is that in ordinary soil amoebae and other protozoa devour and keep down the bacteria; by the sterilisation the protozoa are destroyed and the more resistant bacteria arc then free to develop. Besides nitrifying bacteria many de-nitrifying or- organisms occur in the soil. They may (1) reduce nitrates to nitrites; (2) remove oxygen from nitrates and nitrites and form ammonia ; (3) form nitrous and nitric oxides or nitrogen from nitrates and nitrites. Fermentation. — Another important group of changes produced by micro-organisms is that comprised under the comprehensive title of " fermentation/' of which it is difficult to give an accurate definition, for the dis- tinction between it and other chemical changes due to the activity of micro-organisms is conventional rather than scientific. The original conception of the term involved the occurrence of frothing of the fermenting liquid, owing to the escape of gaseous products. Fer- mentation is brought about by the action of ferments, two classes of which are recognised, viz. the living or Fermentations 35 organised ferments, which, in other words, are micro- organisms ; and the unorganised or chemical ferments, bodies such as pepsin, which in minute amount produce changes in a considerable quantity of the substance acted upon, without themselves undergoing alteration. Jt is better to reserve the term " fermentation " for the changes brought about by the organised ferments or living organisms, and to call the unorganised fer- ments enzymes, and the changes which they produce zymolysis. As fermentations are investigated more critically, the tendency is to find that they are brought about by enzymes, extra-cellular or intra-cellular, so that in course of time this distinction may no longer hold good. The following are the chief vaineties of fermenta- tion : The alcoholic fermentation. — This is mainly brought about by the decomposition of sugars of the hexose group (CfiH,206), principally dextrose and hevulose, by yeasts into alcohol and carbonic acid, but some of the bacteria and moulds also produce appreciable quantities of alcohol. Other carbohydrates by the action of enzymes secreted by the organisms may be converted into hexoses, which are then fermented. The general reaction is as follows : C6H1206 = 2C2H60 + 2C03. As a matter of fact small amounts of by-products appear in addition to the alcohol and carbonic acid, viz. glycerin, succinic acid, and higher alcohols. Until 1897 no enzyme had been obtained which would carry out this change; it only occurred when the living yeast-cells were present, but in that year Buchner, by grinding up the living yeast-cells, obtained a juice which decomposed dextrose with the formation of alcohol and 36 Manual of Bacteriology carbonic acid. This " zymase " Buchner claimed to be the alcoholic enzyme of yeast. The lactic acid fermentation. — This is brought about chiefly by bacteria. Hexoses are converted into lactic acid, the reaction being — C6H1206=2(HC3H503), but it is probably not actually so simple as this, for carbonic acid is given off at the same time. A familiar example of this form of fermentation is the souring of milk, in which the lactose is acted upon as follows : C12H22On + H20 = 4C3H603. The butyric acid fermentation. — Butyric acid is formed from carbohydrates by the action of bacteria, mainly the Bacillus butyricus and Clostridium butyricum, the latter an anaerobic organism, some by-products being formed in addition. Milk which has been just boiled usually undergoes the butyric rather than the lactic fermentation, the spores of the butyric organisms sur- viving. Lactic acid is first formed, and this is then converted into butyric acid : 2C3H603 = C4H802 + 2C02 + 2H2. The acetic acid fermentation.— The conversion of alcohol into acetic acid is also due to bacteria, familiar examples of which are the souring of beer and wine. Bacterial enzymes.— Many changes brought about by bacteria and other micro-organisms are due to enzymes, which may be not only intra-cellular but may escape from the cells into the medium in which they are. The most familiar example is the peptonising enzyme produced by bacteria which liquefy gelatin and digest coagulated protein, fibrin, etc. The enzymes differ : an organism Pigment Formation 37 which liquefies gelatin does not necessarily digest blood- serum. The proteolytic enzyme is tryptic in nature and escapes from the cells into the surrounding medium, so that some of the liquefied gelatin free from cells or in which their action is inhibited by an antiseptic, liquefies other gelatin if added to it. Anxiolytic enzymes are also produced, such as amylase (digesting starch), maltase, lactase, inulase, and invertase. Lipases and rennet-like enzymes also occur. " Fermentation " of urea takes place by means of an enzyme secreted by the Micrococcus urese, etc., with the formation of ammonium carbonate. These enzymes do not seem to possess any poisonous action. Formation of pigment.. — Numerous organisms, espe- cially those of air and water, during their growth pro- duce various coloured pigments. They are termed " chromogenic bacteria," examples of which are the Sarcina lutea and Micrococcus cereus, var. flavus, which form citron-yellow pigments; the Bacillus prodigios us and Spirillum ruhrum, red pigments ; the Bacillus violaceus forms a rich violet one ; and the Bacillus pyocyaneit*, a blue. A large number of chromogenic oi'ganisms require oxygen for the production of the pigment, and potato is often the most favourable culture medium. In some cases the medium may become coloui-ed, and the property of fluoi^escence be conferred upon it, as is the case with the Bacillus jluorescens liquefaciens . Usually the pigment is extra-cellular, occasionally, as in B. violaceus, it is intra-cellular. Phoispltorescence, or light-production, is developed by some bacteria., notably by many marine forms, and is well seen in decomposing fish. Some spirilla are also known occasionally to produce phosphorescence. A necrotic action on the tissues is produced by many pathogenic organisms. For example, the tubercle and 38 Manual of Bacteriology glanders bacilli cause necrosis and caseation of the surrounding tissues. Gas production. — This is common to many organisms. The gas may consist of carbonic acid, hydrogen, or marsh gas, and in some cases of foul-smelling sulphur compounds, sulphuretted hydrogen, mercaptans, etc. Sulphuretted hydrogen may be detected by the blackening of lead acetate paper. Methyl mercaptan may be detected by aspirating a current of air through the culture, through a calcium chloride drying-tube, and then through a test-tube or small flask containing isatin dissolved in concentrated sul- phuric acid. The red colour of the isatin solution is changed to olive- or grass-green by the mercaptan. Toxic bacterial products. — Almost without exception the pathogenic action of bacteria is brought about by means of the chemical substances produced in one way or another by their metabolic processes (see also p. 135), The toxic bacterial products may be classified as follows : (1) Decomposition products. — These are substances produced by the decomposition of the medium upon which the bacteria are growing. Thus proteoses appear to be formed by the anthrax bacillus and the pyogenic cocci. The ptomines form another group of these substances. These are a very important group of nitrogenous bodies, analogous to the vegetable alkaloids and mostly solid and crystalline in nature, which are formed by the action of bacteria on protein and albuminoid matter. They often occur naturally in decomposing and putrefying food, meat, fish, etc., and as many of them are virulent poisons they are of considerable practical import. Generally speaking, the poisoning- due to tainted food is due to the absorption of toxic ptomines formed by bacterial action. A number of The Toxins 39 toxic ptomines were isolated by Brieger from cultiva- tions of pathogenic microbes, and great importance was once attached to them. They are referred to in the chapters describing the pathogenic organisms. Brieo-er's work, however, needs revision, for his methods were not such as to exclude alteration by the reagents employed. Stevenson obtained traces of a highly poisonous crystalline ptomine from some sardines that had caused death. Vaughan has isolated a body, tyrotoxicon, apparently identical with diazobenzene, from poisonous cheese and milk. Mytilotoxin (C6H15N03) is the specific poison of toxic mussels. Neurin and muscarin are extremely poisonous and may occur in decomposing flesh. Some of the ptomines produced by putrefaction are very similar to certain vegetable alkaloids and are thus of considerable medico-legal importance. The ptomines are not specific like the true toxins, and toxic ones may be produced by non-pathogenic bacteria. (2) Toxins. — These are the soluble poisons elaborated by the bacteria and excreted by the cells into the surrounding medium. They are regarded by Martin and others as being allied to the proteoses. Eoux and Yersin suggested that the diphtheria poison might be an enzyme, while Brieger and Frankel regard it as albuminous. The toxins are non-basic substances closely related to the proteins and hence have been named tox-albumins, and are considered to be the specific toxic poisons of the pathogenic bacteria. It is difficult or impossible to prepare them in a state of purity and their chemical constitution is therefore un- known, and they are characterised by extreme specifi- city. Such are the poisons of the diphtheria and tetanus bacilli. (3) Endotoxins. — These are toxic substances clabo- 40 Manual of Bacteriology rated by the bacteria which do not to any extent escape from the cells. They are as specific as the toxins and possess analogous properties (see below). (4) Bacterial proteins. — These are toxic constituents of the bacterial cells which do not diffuse from the cells, are not specific, and which probably usually play little part in the production of the disease symptoms. Literature. On Nitrification, see Warington, Journ. Chem. Soc, 1886, et sea.; Franklaud, Cantor Lectures, 1892 ; Nature, 1890, et seq.; Lohnis, Kandbuch der landwirtschaftlichen Balderi- ologie (Borntraeger, Berlin, 1910, full bibliography). On Bacterial Products, see Cellular Toxins, by Vaughan and Novy, 1902 (Bibliog.), Ueber Ptomaine, by Brieger, 1885; Macfadyeu, The Cell as the Unit of Life (Churchill, 1908) ; Wells, Chemical Pathology, 1907. For General Bibliography, see Kolle and Wassermann, Poihogenen MiJcroorganismen. Endotoxins. The majority of pathogenic micro-organisms do not excrete any appreciable amount of toxin ; the toxin remains within the cells. To such an intra-cellular toxin the name of " endo- toxin " has been given. The toxins of the staphylococci and streptococci, the typhoid-colon group, plague, cholera, etc., are endotoxins. Various methods have been employed to pre- pare these endotoxins, such as extraction of the cells by the action of weak alkalies and enzymes, and by autolysis or self -digestion. The late Dr. Allan Macfadyeu conceived that if the intra- cellular toxins (endotoxins) of such organisms as the typhoid bacillus, cholera vibrio, etc., could be obtained free from the bacterial cells, it might be possible to prepare sera (anti- endo- toxic sera) of much more therapeutic potency than the ordi- nary anti-microbic sera. The disintegration of the bacterial cells in the presence of •ntense cold, to prevent chemical change in the bacterial juice Endotoxins 41 42 Manual of Bacteriology obtained, was the method devised by Macfadyeii to attain this end. With the aid of his colleagues, Mr. Kowland and Mr. Barnard, and of his laboratory assistants, Messrs. Burgess and Thompson, apparatus and methods were evolved to effect this. By growing on the surface of agar or other suitable medium in plate bottles (Fig. 15), scraping off the growth and suspend- ing this in salt solution, centrifugalising at high speed, and collecting the bacterial cell-mass on the walls of the centri- fuge vessels, sufficient material is readily obtained to grind or triturate, and thus disintegrate the bacterial cells so as to liberate their contents. This is accomplished by means of a special machine, the essential part of which consists of a metal cone revolving at a high speed in a metal pot, the bottom of which is shaped so as to fit the cone. The pot, with its con- tents, is immersed in a vessel of liquid air or other freezing mixture, and the bacterial mass is ground. After grinding, the ground material is made up with distilled water or with 01 per cent, sodium hydrate, so as to form a 10 per cent, solution (calculated on the original weight of the moist bacterial paste) ; this is centrifugalised, and the fluid is filtered through a sterile Berkefeld filter. The filtrate thus obtained is the endotoxin, and is used to immunise horses and other animals in the same manner as with any other toxin ; it should be used as fresh as possible. The amounts of a typhoid or cholera endotoxin employed for immunising must at first be small, 0'2-0'5 c.c, as it produces considerable disturbance on injection, and the amount is gradually increased. After some weeks' treatment a dose of 20-30 c.c. may be injected. When tests show that the serum has attained the necessary potency, the horse is bled and the serum obtained and bottled. The endotoxins also possess immunising properties to a high degree, and may be used as prophylactic or as curative vaccines ; they markedly raise the opsonic index. Another machine has been devised by Barnard for disinte- grating bacterial and other cells. It is supplied by Messrs. Baker, of High Holborn, and is depicted in Fig. 1, p. 41. Endotoxins 43 The containing vessel consists of a phosphor-bronze body. a, in which five hardened steel balls, b, are placed. The shape of the containing vessel is such that when these balls are at its periphery they accurately fit the inner side of the vessel. The balls are evenly distributed round the vessel by means of a cage, c, and during the time they are running this cage ensures that they are equi-distant and do not collide one with another. At the centre of the metal vessel is a steel cone, d, which is of such a size that it keeps the balls in their proper position in close contact with the periphery of the containing vessel. The vessel is closed by a screw cap, e, through which the steel cone passes, and in which it is free to rotate. Over the whole of this a metal cylinder, f, is placed, and is screwed down, completely sealing the upper opening in the metal vessel. In the top of this metal cylinder a steel bearing, g, is placed, which has freedom of movement in a horizontal direction, but is kept down on the top of the steel cone by the action of a spring. It therefore follows that when this metal cylinder is screwed down the steel cone is pressed on to the balls, and the balls are in their turn forced out to the periphery of the metal pot. The whole appliance is mounted on a cone, h, and a centre, i, which are carried by two uprights attached to the base plate, J ; one end of the shaft is attached to the electric motor. The grinding action is brought about by retarding the revolution of the central cone, d. This has been effected bv mounting on the spindle of the central steel cone, d, a semi- cylindrical mass of iron or lead, k, the weight of which must be such that when the whole apparatus is rotated it is suffi- cient to hold the central cone still. By retarding the cone in this way a drag is placed on the balls, they slide to a certain extent over the inner surface of the pot and exert a grinding action. See Hewlett's Serum Therapy, 1910 ; Hewlett, Proc. Boy. Soc, b., 1909 and 1911; Proc. Boy. Soc. Med., vol. iii, 1909-10 (Pathological Section), p.' 165; Barnard and Hewlett, Proc. Boy. Soc, b., 1911. 44 Manual of Bacteriology CHAPTER II. METHODS OF CULTIVATING AND ISOLATING ORGANISMS. It is necessary for the satisfactory study of micro- organisms in their relation to the various processes of infection and disease, of fermentation, putrefaction, and the like, to separate and isolate the different species occur- ring in a mixture, and, having done so, to cultivate, grow, or propagate each species on suitable soils thixmgh suc- cessive generations. A slight consideration will show that unless we work with pure cultures — that is, cul- tures consisting of a single species — we can never be sure that a particular result is due to a given organism ; in a mixture several or all of the forms present may conduce to the effect nrocluced. With regard to the pathogenic organisms, or disease germs, Koch laid down certain conditions which have been termed "Koch's Postulates" (p. 154), which must be complied with before the relation of an organism to a disease process can be said to be completely demonstrated, one of which is that " the organism must be isolated and cultivated outside the animal body on suitable media for successive generations/' In order to isolate organisms in a state of purity it is absolutely necessary to employ vessels, instruments, and culture media which are sterile, that is, free from any living organisms, and to possess the means of Sterilisation 45 manipulating them in such a way that the entrance of organisms from without is prevented and contamina- tion avoided. Various methods of destroying and of netting rid of organisms are known, such as the use of chemical "germicides," heat, and filtration through porous porcelain. The addition of chemical germicides, such as carbolic acid or corrosive sublimate, is out of Fig. 2. — Hot-air steriliser. the question; for although the vessels and media might be rendered sterile thereby, the growth of the orga- nisms which are being investigated would equally be prevented, so that the two last, viz. heat and filtra- tion, are those which are employed, the former being used for vessels, instruments, and culture media, solid and fluid, the latter for fluid culture media only. Various apparatus are needed for sterilisation and 46 Manual of Bacteriology These will now be the preparation of culture media, described. Hot-air steriliser (Fig. 2).— This is a rectangular box of sheet iron with double walls, having an air- space of nearly an inch between them, and furnished with a door. The bottom should be protected with a loose piece of sheet iron, which can be renewed as it " burns " away. The top is perforated with a couple of holes, through one of which a chemical thermometer, regis- tering to 200° C, is inserted in a cork, while through the other some form of mercurial regulator can be introduced if required, but is not usually needed. In the hot-air steri- liser all thin-glass vessels and cotton-wool are sterilised by heating to a temperature of about 150° C. by means of a Bunsen or a small ring burner under the stei'iliser, which is supported on a suitable iron stand. If the steriliser is placed on a table or other wooden support, a piece of sheet iron, asbestos cardboard or uralite should be laid over the wood to protect it from the heat. An inexpensive substitute for the hot-air steriliser may readily be devised, any iron box or even a biscuit-tin being used for the purpose. Steam steriliser (Fig. 3) — This consists of a cylin- drical or rectangular vessel of tinplate, galvanised iron, or copper, covei'ed on the outside with a layer of felt Fig. 3. — Steam steriliser. Autoclave 47 or asbestos, having a false perforated bottom supported a few inches above the true bottom, and provided with a movable lid. In the steam steriliser or "steamer" the culture media, and thick glass vessels and other apparatus which would crack or be damaged by the hio'h temperature of the hot-air steriliser, are sterilised by steam. The lower chamber of the steamer, below the false bottom, is partly filled with water, which is boiled by means of a Bunsen or ring burner. Above the false bottom the culture media or apparatus are placed, and are sterilised by the steam at 100° C. which fills this space. Here again an inexpen- sive substitute may be de- vised ; the ordinary kitchen saucepan with steamer will do well for many purposes, while a " warren pot "answers admirably. Autoclave (Fig. 4). — This is most useful for many pur- poses, but it is expensive Fig. 4. -Autoclave, and not a necessity, as the steam steriliser can be made to answer almost evei'y purpose for which the autoclave is employed with the expenditure of a little more time and trouble. It consists of a strong boiler of brass or gun-metal with a removable lid, which is attached to the boiler by means of screw-bolts. The lid is provided with a safety valve, a gauge for indicating the pressure and temperature, and a stopcock to relieve the pressure if required. A small quantity of water is placed in the 48 Manual of Bacteriology bottom, and the media or apparatus to be sterilised having- been introduced, the lid is screwed down. It is heated by means of one or more Bunsen burners, which are turned down when the required tempera- ture has been reached. The temperature usually em- ployed is about 115° to 125° C. When sterlising media care should be taken that the vessels are not filled too full, and that the autoclave is allowed to cool down below 100° C. before relieving the pressure by opening the stopcock, or a good deal of the contents may be lost by violent ebullition. Also, while raising the temperature the stopcock should always be left open until steam is being freely generated in order that the air may be expelled. Air-pump. — An exhaust pump is very useful for many purposes, such as evaporating to dryness in vacuo, filtration through porous porcelain filters, etc. Any form will do, but of the more elaborate ones the Fleuss pump (Fig. 5, p. 50) made by the Pulsometer Engineering Company is one of the best. In using it care must be taken that no fluid or moisture gains access to the barrel ; to avoid this it is a good plan to intercept the connecting pipe with a vessel containing strong sulphuric acid (d, Fig. 5), over the surface of which the exhausted air has to pass. A double-necked Woulfe's bottle does well for this, the inlet and outlet tubes extending nearly down to, but not dipping below, the surface of the sulphuric acid. For greasing the vessels, etc., to make air-tight joints, beeswax dissolved in the Fleuss pump oil with the aid of heat to a stiff paste is a good composition, or the resin ointment of the Pharmacopoeia may be used. Bell-jars with ground rims and one or two tubules are useful for evaporation in vacuo. They should Evaporation and Filtration 49 stand on a square of thick ground glass. To make an air-tight joint the surface of the rim of the bell-jar, which must be quite clean, should be well greased and pressed thoroughly home on the ground-glass plate. A thick ridge of grease should then be plastered all round the angle formed by the rim of the bell-jar aud the glass plate. Thick rubber pressure tubing must be used for connections, and all joints should be well greased. For evaporating large quantities of fluid the writer devised a copper stand with shelves, the shelves supporting glass dishes containing alter- nately strong sulphuric acid and the fluid to be evaporated, the whole being placed under a suitable bell-jar. A mercurial gauge is a useful addition to show the amount of exhaust and the occurrence of leakage. The ordinary glass filter pumps used in chemical work and actuated by a stream of water are also useful for many purposes. Porous porcelain filters. — The forms which are generally employed are the Pasteur-Chamberland, the Doulton, and the Berkefeld. These consist of " candles" composed in the first two of unglazed porous porcelain, in the last of a specially prepared diatomaceous earth. The filtration through the Pasteur-Chamberland is much slower than through the Berkefeld. All give a germ- free filtrate, but the last should be employed if the fluid is thick or contains many particles ; a preliminary filtration through paper is an advantage. A useful method of conducting filtration is the following: The filter "candle" (b, Fig. 5, p. 50) is connected by a short length of pressure tubing with a piece of glass tubing passing through a rubber cork in the neck of an ordinary filtering flask c. The - candle » is placed in a jar a, such as a glass measure or urine-jar, which is filled up with the solution to be filtered. The lateral 4 d0 Manual of Bacteriology branch of the Biter flask is then connected with the air-pump. On exhausting, the fluid passes through the filter " candle " over into the filtering flask, in which it is collected. Before use the "candle" should be well scrubbed and some water or per cent, carbolic, run through to clean it, and the whole may be sterilised in the steamer for an hour or two. After use the same process should be repeated to cleanse it. Fig. 5. — Pleuss exhaust pump, arranged for filtration. Flask*, leakers, and test-tubes. — A good supply of these is required of various sizes : Erlenmeyer and ordinary shapes, tall and short forms of beakers, etc. A lew "yeast flasks" are also useful (see Fig. 13, p. 76). Beakers and flasks of "Jena" glass are to be pre- ferred. Enamelled iron ware, jugs, saucepans, mugs, etc., may replace glass for many purposes. The best size of test-tube is 5" x §■" ; a few larger sizes and "boiling tubes" should also be kept. Platinum needles (Fig. 6). — Two or three platinum needles are required. They consist of about two Apparatus and Pipettes 51 inches of platinum wire in a handle of glass rod. One end of a Sflass rod is softened in the Bunsen or blow- pipe flame, and about an eighth of an inch of the platinum wire is embedded in it with a forceps, the wire having1 been first heated to a red heat. The Fig. 6. — Platinum needles. ylass-wire ioint is then well annealed in the flame and allowed to cool slowly. Metal handles may also be used. Two thicknesses of platinum wire are desirable, viz. 0*4 mm. (27-28 B.W.Gr.) for most purposes, but a thicker wire of about 0'7 mm. where stiffness is required, and one or two o in. or more in length are useful. Forceps, needles, etc. — Several forceps are necessary, the ordinary dissecting form in two or three, sizes, one or two pairs of hue pointed, two or three small brass ones, and two or three pairs of the " Cornet" pattern. A few ordinary sewing needles of various sizes mounted in wooden handles serve all purposes. Fig. 7. — Glass pipette. Glass pipettes and capillary tubes. — These are useful for preserving or storing blood or pus, etc., for examination, for sterile water in making film speci- mens, and for many other purposes. A piece of glass tubing is heated in the blowpipe flame until quite soft; it is then taken out uf the flame and the two ends 52 Manual of Bacteriology pulled steadily apart; this forms a capillary tube of greater or lesser length and smaller or larger diameter, and it can be sealed off in convenient lengths. To make a pipette proceed in the same way : seal off the capillary tube two or three inches from the wide tube, then heat this close up to where it was heated before, and draw out again and seal off two or three inches from the bulb. In this way a capillary tube with a bulb at its middle is formed (Fig. 7). "Vaccine tubes," pipettes made of glass tubing drawn out at one end, and Wright's capsules (see Fig. 35, a and (Z, p. 225) are also useful. India-rubber caps.- — A few indiarubber caps for capping test-tube or flask cultures arc required. They retard evaporation and the desiccation of the medium, and prevent the entrance of moulds. For use they should be soaked in 1—500 corrosive sublimate solu- tion ; they should not be Jcept in the solution, as vulcanised rubber absorbs mercuric chloride (Glenny and Walpole). Tinfoil or gutta-percha tissue (sealed down by warming) may also be used to cover the tops of tubes and flasks. Preparation of Sterile Test=tubes, Flasks, etc., for the Reception or Manipulation of Culture Media. To sterilise cotton-wool. — Non-absorbent cotton-wool best or No. 2 quality, should be used for plugging purposes. The wool should be pulled apart so as to assist the penetration of heat ; in the compressed con- dition the interior is difficult to sterilise. The separated wool is placed in the hot-air steriliser and the tempera- ture is slowly raised to 1 15° C. and maintained at this for at least an hour. Above 150° C. cotton- wool becomes brown and brittle. It is a common practice now to use Sterilisation of Glass Ware 53 various coloured wools for the different culture media, especially the carbohydrate ones, so that they are readily distinguishable by the eye. The coloured wools may be purchased, or the ordinary white wool may be dyed with the "Dolly" dyes. Glass vessels. — The vessels (usually test-tubes, flasks, and dishes) are thoroughly washed and rinsed in water, then rinsed with 25 per cent, hydrochloric acid, and afterwards washed well with tap-water and drained. A final rinse with distilled water or alcohol is an advantage, as no deposit then occurs on drying. The cleansed vessels should be dried before sterilising, either in the air or by placing in the hot-air steriliser for half an hour. When dry, the vessels are plugged with a firm plug of the sterilised cotton-wool, and are placed in the hot-air steriliser, the temperature of which is then raised to about 150° C. They should remain at this temperativre for not less than half an hour, after which the steriliser and its contents are allowed to cool slowly. Petri dishes for plate cultures, graduated pipettes, etc., are cleaned as described for tubes and flasks. They may be sterilised and kept in sheet-iron or copper boxes of appropriate size and shape. If tubes, flasks, pipettes, etc., are required in a hurry they may be rapidly sterilised as follows : After wash- ing in water they are rinsed with 5 per cent, carbolic, then with absolute alcohol, and finallv with ether, and are then well flamed over a Bunsen flame, holding in a suitable forceps or holder. The ether evaporates and burns at the mouth, and when dry, a pledget of cotton- wool is held in the forceps and singed in the flame, and, while burning, the tube or flask is plugged with it. When thick glass vessels, such as measures, etc., have to be sterilised, it is not safe to do this in the hot- 54 Manual of Bacteriology air steriliser unless the lieating and cooling are carried out very slowly, as they are very liable to crack. It is preferable, after cleaning and plugging with sterile wool, to steam in the steam steriliser for three to five hours, the heating and cooling being conducted slowly. Culture Media. The ordinary methods of preparing culture media are here given, but " Standard " media, having definite re- actions, are now largely employed (for the method of standardisation, see p. 64). Certain special media will be described as required. In all cases the media are tilled into the cleansed and sterilised vessels, test-tubes, flasks, etc. (p. 53). For ordinary laboratory cultures test-tubes are generally used. Media which are solid at ordinary temperatures, c y. agar, gelatin, and serum, are prepared either as deep, upright tubes (fig. 8, a), for which 8-15 c.c. of the medium are required for a tube, or as sloping tubes (Fig. 8, c), for which 4-5 c.c. are required for a tube. Of fluid media 7-15 c.c. are used for a tube. The prepared media having been introduced into the test-tubes, etc., sterilisation is effected in the steam steriliser (p. 46) by steaming for twenty to thirty minutes on two or three successive days, or in the autoclave (p. 47) by heating to 115°— 120° C. for half an hour on one occasion. Culture media- may also be kept in bulk in flasks ; these need some- what longer sterilisation than tubes. Tubes of some of the culture media can also be purchased ready for use. Certain media can be obtained in powder form (Chop- ping's) from Messrs. Baircl & Tatlock, and in Tabloid form (Thompson's) from Messi*s. Burroughs & Well- come. These are convenient when small quantities are required for occasional use. Broth Media 55 Acid beef-broth. — The basis of the most important culture media, viz. peptone beef-broth, gelatin, and agar-agar, is an infusion of meat prepared usually from beef. In order to prepare this infusion, which may be termed acid beef-broth, proceed as follows : Take 1 lb. of beef ("gravy beef") free from fat, chop fine or mince, add one litre of tap-water, and allow it to simmer in a saucepan for one hour ; cool, remove any solidified fat from the surface, and filter through filter- paper into a clean glass flask. If not required for immediate use, ping the neck of the flask with cotton-wool, and steam in the steam steriliser (or boil) for three- quarters of an hour on two suc- cessive days. It may then be kept until i-equired. Peptone beef- broth. — Take one litre of the acid beef-broth, add tn this 10 grin, of peptone (Witte's) and 5 grm. of common salt (i. p. 1 per cent, peptone and 0'5 per cent, sodium chlor- Fig. 8. — Tubes of culture ide), mix in a flask, and steam in rpLto^c^SlS the steam steriliser until dissol ved. aM™'- When dissolved, remove from the steam steriliser and render slightly alkaline with a 10 per cent, solution of caustic soda (prefer- ably) or of sodium carbonate, glazed litmus-paper being used as an indicator. Having done this, return to the steamer for one hour, then filter through two thicknesses of German filter-paper. It should now be quite clear and bright and may be kept in bulk, after sterilising, or be introduced into test-tubes, etc., and B 56 Manual of Bacteriology sterilised. Beef-broth, if prepared in this manner, may need no clarifying, but if it should filter at all cloudy, cool to 50° C, add the white of an egg beaten up with the shell, and steam for half an hour, filter, and finally sterilise as before. Other preparations of peptone may be used. Instead of meat infusion, meat extracts have been much used of late. The general opinion is, however, that meat- extract media are not such good nutrient soils for many purposes as those made from meat, The following is the composition of " Lemco " broth : Lemco 10-20 grm. Peptone (Witte) .... 10-20 grm. Sod ium chloride ..... 5—10 grm. Water (preferably distilled) . . 1 litre. The constituents are dissolved with the aid of heat, neutra- lised, clarified and filtered. Lemco may also be used to make all the other media for which acid beef-broth is employed. Veal-broth. — For some purposes veal presents advan- tages over beef, e. g. for growing the tubercle. bacillus. When obtained from the butcher's the veal is frequently powdered with flour ; this should be brushed and washed off as completely as possible, as it renders the broth turbid and difficult to clarify. The veal-broth is made in precisely the same way as peptone beef-broth. It is, however, often slightly alkaline, so that less alkali is required for neutralisation. For the cultivation nf the tubercle bacillus about 4 to 6 per cent, of glycerin should be added. Glycerin beef-broth i.s prepared in the same manner, 4 to 6 per cent, of the best glycerin being added to the fluid after filtration. Glncose broth. — For the cultivation of anaerobic organisms the addition of 0-5 to 2 per cent, of grape Culture Media 57 sugar is an advantage. It should be added after filtration. Peptone water. — Add to distilled water 1 to 2 per cent, of Witte's peptone and \ per cent, of common salt, dissolve by heat, make faintly alkaline, steam for one hour and filter. For the cholera vibrio it is an advantage to add 1 per cent, instead of £ per cent, of common salt (Dunham's solution) . Beer-icort. — Procure beer-wort (preferably unhopped) from the brewery. Allow it to stand in a cool place for twelve hours, filter, and then steam for an hour and filter again. Fill into sterile test-tubes and sterilise. Nutrient gelatin. — Take 1 litre of the acid beef-broth in a . large flask and add to it 100 grm. of the best "gold label" gelatin (Coignet's), 10 grm. of peptone, and 5 grm. of common salt. Place in the water-bath or steamer until quite dissolved. Then render faintly alkaline, as for the peptone beef-broth ; cool to 50° 0. and add the white of an egg, stir well, and return to the steamer for one hour. Filter through two thick- nesses of filter-paper in a hot-water funnel (this is best, but it may be done in the steamer at a low temperature, e.g. 35° 0.) Fill into test-tubes and sterilise. After the third steaming the tubes are allowed to solidify, either in the upright or oblique position, according as they are required for stab or surface cultivation. In hot summer weather 15 or even 20 per cent, of gelatin (150 grm. or 200 grm. to the litre) are necessary for the product to remain solid, as nutrient gelatin melts at 24° 0 or '•\ hfh under' longed boiling diminishes and ultimately destroys the gelatinising power of gelatin, s6 the less it is ,l"a'h"1 t,1(' hettcr. It must not be autoclavec! 5° Manual of Bacteriology Glucose gelatin. — Ordinary gelatin with the addition of 1 to 2 per cent, of grape sugar. Beer-wort gelatin. — This is one of the best culture media for yeasts and some of the fungi (e. g. ringworm) Pi ■ocure from the brewery some beer-wort, preferably unhopped, and add to every litre 100 grm. of gelatin. Dissolve, clarify, and filter, as in the case of ordinary gelatin. It is not neutralised. Nutrient, agar-agar. — This is one of our most, valuable culture media, and has the advantage over nutrient gelatin that it remains solid at blood-heat. Agar is a carbohydrate substance of high melting- point and considerable gelatinising power, obtained from Eastern seaweeds. The powdered form is now generally used. Add 15 grm. (i.e. 1| per cent.) of powdered agar to 1 litre of acid beef-broth, together with 10 grm. of peptone and 5 grm. of common salt in a large glass flask, place in the water-bath until dis- solved (half an hour to one hour), and then render alkaline as for peptone beef-broth; allow it to cool to 50° C, and add the white of an egg. Return to the steamer for an hour and a half, then filter through an agar filter-paper (" papier Chardin") in a hot-water funnel or in the steamer. By this treatment a litre of a oar should pass through the filter in two to three hours. If it does not come through clear, add another white of egg and repeat the process. If an autoclave is available, a quicker and better method is, after neutralising and adding the white of an egg, to place in the autoclave with a small beaker inverted over the mouth of the flask, and heat to 131° C. (two atmospheres pressure) for half an hour. Turn the gas out, and allow to cool without opening the stopcock. When cool, open, and filter through the special agar filter-paper in a hot-water funnel; the agar will pass Culture Media 59 through in about ten minutes or a quarter of an hour. Fill into test-tubes and sterilise. Solidify in the upright or oblique position as required. In the case of bar or stick agar, first steep the agar in 1 per cent, acetic acid for a quarter of an hour, then drain and wash it so as to thoroughly remove the acid. The further procedure is the same as detailed above. This yields a very clear, pale product, and is perhaps prefer- able when an autoclave is not available. Glycerin agar. — Add 4 to G per cent, of glycerin to the nutrient agar after filtration and proceed as before. Glucose agar. — One or two per cent, of grape sugar is added to the nutrient agar after filtration. Litmus media. — The addition of neutral litmus to the various culture media is a useful method of demons- trating the production of acid or of alkali by organisms. To prepare the litmus solution take the lump litmus, powder finely, and boil with distilled water so that a saturated solution is obtained. Filter, and preserve in a flask stoppered with cotton-wool, after sterilising by boiling for half an hour on two successive days. For some purposes a special solution of litmus, the Ivubel- Tiemann solution, which can be procured ready for use, is employed. It must not have any antiseptic added to it (as is sometimes done to preserve it for use in the chemical laboratory). .Sufficient of this litmus infusion is added to the nutrient media, after filtration, to tinge them a distinct purplish colour. After steaming the colour has usually disappeared, but returns as the tubes cool. Milk.— Use 'separated milk, but failing this, centri- fugal ise ordinary new milk, or place it in a tall cylinder and allow it to stand overnight in a, cool place, prefer- ab]y in aT1 safe. Then pipette oil' the milk from 60 Manual of Bacteriology the bottom, rejecting the cream. Introduce the separated milk into test-tubes to the depth of about an inch to an inch and a half and steam for one hour on two successive days. The milk is usually tinged with litmus before tubing, forming litmus •milk. Potatoes. — Choose sound potatoes, and scrub them well with water to remove dirt. Cut off the ends, and with a cork-borei', slightly smaller than the test-tubes which are used, bore through the potato so that a cylin- drical piece is removed. Push this out of the borer, and divide it into two por- tions by a very oblique transverse cut, so that two wedge-shaped pieces are obtained, and in this manner prepare as many pieces as there are tubes to be filled. Place them in a basin under the tap, and allow the water to flow over them for about two hours. This prevents the darkening of the potato in the subsequent steaming. The test-tubes for the potato-wedges are prepared as follows : After plugging and sterilising in the ordinary way, introduce a small pledget of sterilised wool into each, push to the bottom, and moisten with a little sterilised distilled water. Drop the tubefo7potatoS potato-wedges into the tubes, plug, and sterilise by steaming for three-quarters of an hour on two successive dcays (Fig. 8, b). The object of the moist wool is to prevent drying, and for the same purpose Roux's tubes (Fig. 9) may be used, the lower bulb being filled with water. Blood-serum. — Clean some glass jars of about 2 to 3 litres capacity, plug with wool, and sterilise in the steamer for an hour on three successive days. Bleed Blood-Serum 61 a horse, with aseptic precautions, and catch the blood in these sterilised jars. Allow the jars to stand in a cool place for twelve hours. Then pipette off the clear serum with a sterile pipette, and fill the sterilised test- tubes to the depth of 2-4 cm. The tubes are then arranged in a sloping- position on the shelves of the serum inspissator, or failing this in a hot-water oven, the temperature of which should be about 50° C. At this temperature they remain for thirty hours ; it is then raised to 65° C, at which temperature the serum coagulates in from four to six hours and the tubes are now ready for use. It is well, however, to place them in the blood-heat incubator for a night, so that any contaminating bacteria may form colonies, and the contaminated tubes may then be rejected. Loeffler's blood-aerum is prepared by adding one part of glucose broth to three parts of the serum before inspissation. The serum inspissator is practically an incubator (see p. 68) with slightly inclined (10-15°) shelves, on which the tubes rest, and thus the serum is coagulated in a sloping position. Fluid serum, etc. — Fluid blood-serum, ascitic and hydrocele fluids, etc., are sometimes useful, and may be used alone or mixed with peptone beef-broth in various proportions. Ascitic oi- hydrocele fluid may be obtained by using sterile trocars, etc., and carrying out the tapping with aseptic precautions, collecting the fluid in sterilised flasks. It is better to collect in several small flasks than in one large one. Fluid blood-serum may be obtained by collecting blood with aseptic precautions in sterilised flasks. When the blood has coagulated and the serum separated, the serum is pipetted off with a sterile pipette into sterile flasks. ,;-> Manual of Bacteriology The flasks of serum, etc., should be kept in a warm place for two or three days to make sure that they are sterile, those in which a growth appears being- rejected. Serum, ascitic fluid, etc., may also be obtained sterile by filtering through a sterilised Berkefeld filter into sterile flasks. Serum, ascitic and hydrocele fluids, etc., may be pre- served in bulk and used as required. The material is collected as aseptically as possible, 5 per cent, of chloroform is added, and the whole is well mixed and kept in a cool place in the dark in a well-stoppered bottle. Subsequently, during the process of sterilisation, the chloroform is volatilised. Serum agar (Kanthack and Stevens). — Ascitic, pleuritic, or hydrocele fluid is collected in clean (not necessarily sterilised) flasks, and allowed to stand over- night in a cool place to allow the sediment or blood to deposit. The clear fluid is then poured off, and to each litre enough of a 10 per cent, caustic potash solu- tion is added to render it very distinctly alkaline — usually about 2 c.c. to every 100 c.c. of the fluid. The alkaline fluid is heated in the autoclave for two to four hours. To this fluid 1*5 to 2 per cent, of agar is added, and the mixture is heated until the agar dissolves. It is then Altered, introduced into test-tubes, sterilised, and solidified in the ordinary way. The addition of 5 per cent, of glycerin and 1 per cent, of glucose is an advantage. Serum agar may also be prepared by adding sterile serum or hydrocele or ascitic fluid, warmed to 45° C, to sterile nutrient agar (2 to 3 per cent, agar) melted and cooled to 45° C. Equal parts of the serum and agar may be mixed, or 1 part of serum to 2 parts of agar. Culture Media Blood aijar. — This may be prepared by smearing the surface of the agar in sloping agar-tubes with blood obtained a'septically from the linger or from a rabbit. Or blood obtained aseptically may be dellbrinated by shaking with glass beads or with a coil of hue wire, and the detibrinated blood, warmed to 45° C, is added to sterile agar liquefied by boiling and cooled to 45° C. Haemoglobin agar may be prepared by hiking detibri- nated blood by the addition of sterile distilled water and adding to the liquid agar as before. Blood agar cannot be sterilised after preparation, and the blood therefore must be sterile. Alkali albumen (Lorrain- Smith). — To 100 c.c. of fresh serum add 1 to 1*5 c.c. of a 10 per cent, caustic soda solution ; mix and introduce into test-tubes in the ordinary way. Place the test-tubes in the slanting- ■ O position in the autoclave at 115° C. for twenty minutes, or in the steamer on three successive days. Egg cultures (Hueppe). — These are very useful for some purposes. A hen's egg is taken and one end sterilised by washing with carbonate of soda solution, rinsing in sterile water, soaking in 1-500 corrosive sublimate solution, and washing in alcohol and in ether. A small hole is then chipped in the shell with a sterile needle and the inoculation made through this. The hole is afterwards closed with a little sterilised wool and collodion. tJschinsky's Fluid. Parts. Sodium chloride . . 5-7 Calcium chloride . . oi Magnesium sulphate . 0-2-0 4 Di-potassium phosphate 2-25 Ammonium lactate . 6-7 Sodium asparaginate . 3-4 Glycerin . . . . :j0_40 Pasteur's Fluid. Parts. Cane sugar . . .10 Tartrate of ammonia . 1 The ash of 1 gnu. of yeast .... Water . . . . 100 b* Manual of Bacteriology Uschinslcy's fluid is a solution of known composition without protein which can be used for investigating the chemical products of bacteria. Pathogenic organisms grow well in it and produce their toxins. Pasteur's fluid is a good culture medium for yeasts, etc.1 Standard Nutrient Media. Slight variations in the composition of the nutrient media have a marked influence upon the characters of the growths of micro-organisms developing upon them. In order to obtain more uniformity for descriptive purposes, etc., a committee of the American Public Health Association drew up a scheme for the prepara- tion of nutrient media of approximately constant composition and reaction. Eyre2 has devoted consider- able attention to this subject, and the following descriptions are based largely upon his papers. (1) Preparation of acid beef-broth. — 1000 c.c. of distilled water are introduced into a large flask, 500 grin, of finely minced fresh lean beef added, and the mixture is heated in a water-bath at 40°— 45° C. for twenty minutes with frequent agitation. It is then boiled for ten minutes, strained, and filtered through paper. To the filtrate sufficient distilled water is added to make up to 1000 c.c. (2) Standardisation.- — This may be most simply de- scribed in the case of acid broth. A 100 c.c. Erlenmeyer flask is rinsed out with boiling distilled water, 25 c.c. of the acid beef-broth are introduced into it, and 0'5 c.c. of phenolphthalein solution is added (0-5 per cent, phenolphthalein in 50 per cent, alcohol). This is kept boiling and decinormal caustic soda 1 Several formulae for synthesised media will be found in the Journal of Experimental Medicine, vol. iii, p. 666. * Brit. Med. Journ., 1900, vol. ii, p. 921 ; 1901, vol. ii, p. 788. Standard Media 65 solution1 is run in from 25 c.c. burette, divided into tenths, until a faint pink tinge appears in the boiling fluid. From the amount of soda solution used the amount of normal or deka-normal soda solution required to neutralise a given volume of the acid beef-broth (e. g. a litre) can be calculated, and this amount is then added. Although neutral to phenolphthalein, the medium is now strongly alkaline to litmus — too alkaline for the optimum growth of most organisms. ^ The reason for this is that the di-sodium hydrogen phosphate (Na3HP04) present in the medium is alkaline to litmus but neutral to phenolphthalein. To reduce (fJin*/' the alkalinity (to litmus) normal hydrochloric acid is then added. The American Committee recommended an acidity of + 1*5 — that is, to every 100 c.c. of the medium neutral to phenolphthalein 1*5 c.c. of the normal hydrochloric acid are added. Eyre advises a reaction of + 1-0 {%. e. 1 c.c. of normal hydrochloric to every 100 c.c), while Chester considers that the acidity should not exceed + 0-5. Whatever the reaction adopted, it should be stated. Similarly, if a medium is used which is alkaline to phenolphthalein, this is expressed by the minus sign; e. g. a reaction of — 1-5 indicates that to every 100 c.c. 1'5 c.c. of normal hydrochloric acid must be added to render it neutral to phenolphthalein, or, what is almost (but not quite) the same thing, that to the neutral medium 1'5 c.c. of normal caustic soda solution have been added to every 100 c.c. Various methods are adopted to obtain the 1 By a "normal" solution is meant the equivalent weight in grammes of a substance dissolved in {%. e. made up to) a litre of water; a" deci- normal" solution contains one tenth of, a deka-normal ten times, this amount. A normal solution of caustic soda contains 40 grin, of pure NaOH (NaOH = 40), of sulphuric acid 49 grm. of pure H„SO, (HoS04 \ ~2~- =49J, Per litre. 66 Manual of Bacteriology- final reaction ; the American Committee recommend first neutralising and then adding sufficient acid (or alkali) ; Eyre, having calculated the acidity, adds only sufficient alkali to reduce the reaction to the required point. Eyre describes the reaction as that represented by the number of c.c.'s of normal alkali or acid per litre, e. g. + 10 on Eyre's scale is equivalent to the American + I/O. In making nutrient broth, agar and gelatin, the salt and peptone and agar or gelatin are added and dissolved, and the titration and neutralisation are carried out as described, on the fluid medium itself, and after neutralisation the whole is heated over a water- bath for half an hour before filtration. The Cultivation and Isolation of Micro = organisms. It should be clearly understood that micro-organisms cannot usually be identified by their microscopical characters alone. We can state from a microscopical examination the form of an organism, that it is a bacillus or a micrococcus, or a sarcina, its size, that it is motile or non-motile, sporing or non-sporing, but we cannot as a rule go beyond this. It is necessary in most cases to ascertain the characters of the growths of organisms on the various culture media before species can be identified, and this is the principal reason for having a varied assortment of nutrient soils. It is likewise necessary for the successful cultivation of pathogenic organisms, i. e. those connected with disease processes and developing in or upon the bodies of man and of animals, to maintain the cultures at a temperatui'e approximating to that of the host. For this purpose some form of incubator is required. This consists of a box or chamber of copper or iron with double walls (Fig. 10), the space between which is filled Incubators 67 with water, the outside being covered with wood or felt, or some other non-conductor. The water between the wall is heated by means of a small burner, the gas supply for which passes through some form of regulator inserted in the water, so that the temperature, indicated by a thermometer inserted through a hole in the top, can be kept constant. The regulator is usually a mercurial one, such as Page's or Reichert's, the principle of its action being that as the temperature rises the mercury expands and at a certain point cuts off the greater part of the gas supply, only sufficient gas then passing to keep the flame of the burner alight. This point can be varied either by a sliding tube, in Page's, or by a screw, in Reichert's, so that the temperature may be set at any desired point. In Hearson's incubator, which is one of the best forms, the regulator consists of a capsule containing a fluid of a certain boiling point, which when ebullition takes place raises a lever and so partially cuts off the gas supply. While the Hearson regulator is a very constant one, it has the disadvantage that it can only be used for a range of temperature of a few degrees unless the capsule be changed. At least one incubator is required, and it is convenient to have two or three. If there be only one the regulator should be set for a temperature of 37° C. ■ if more, another should be kept at about 20° C. The incubator at 37° C. is termed the warm or blood-heat, and that at 20c C. the cool or room temperature one. A warm room or cupboard will serve most of the purposes of the cool incubator. third "wmbatop set for 42° C. is useful for water examination, and a fourth at 25° C. for fermentation work. A substitute for the large and expensive incubator can readily be devised. An ordinary chemical hot- f 63 Manual of Bacteriology water oven may be employed, or simply a smaller tin set in a somewhat larger one, the interspace being filled with water; and, with a little scheming, regulators can be dispensed with by making use of a small gas or Fig. 10. — Hearson's incmbator. lamp flame, varying its size and distance from the bottom until the right temperature has been attained. Gas is a great convenience, but if not available, regulating oil lamps can be obtained to take its place. Electricity has also been adapted for heating incubators. Liquefaction of Gelatin 69 Gelatin will remain solid only at temperatures below 24° O.j and cannot therefore be placed in the blood- heat incubator without becoming for practical purposes a fluid medium. Agar, however — and this is one of its most valuable properties — does not liquefy below a temperature of 97°— 99° 0., though when once liquefied it does not set again until the temperature has fallen to about 45° C. Gelatin is therefore usually reserved for use at low temperatures, while agar, blood-serum, potato, and the fluid media can be used indifferently either at low or at high temperatures. Agar is often a better cultivating medium than gelatin, even at low temperatures, probably because it is so much moister. The growths in fluid media are usually of the nature of a general turbidity and are not particularly charac- teristic, but sometimes an organism produces a film on the surface which another similar organism does not, or the medium remains clear, the growth forming a flocculent deposit, thus affording a distinction. Not only do the characters of the growths of organisms on media differ more or less, but in some instances chemical changes occur in the media which afford valuable information in the differentiation of species. Thus many organisms exert a peptonising effect on gelatin, and render it fluid sooner or later, while others have no such action. Milk is coagulated by some organisms, the coagulation being brought about in one of two ways, either by the production of acids and precipitation of the caseinogen, or by the action of a rennet-like ferment with the formation of a clot of casein. Most organisms which liquefy gelatin coagu- late milk, but the converse is not the case. Agar is carbohydrate, not albuminoid, in nature, and only two or three organisms are known which liquefy it. In fluid media, such as broth and peptone water, chemical tests 70 Manual of Bacteriology can be applied, especially for indole, which is formed by some organisms but not by others. Method of inoculating tubes.— The following is the procedure by which sub-cultures are prepared from an original test-tube or other culture : Tubes of the culture media selected are placed in a test-tube rack. Their mouths are then singed by holding in the Bunsen flame for a few seconds, and with a forceps, also steri- lised by heating in the flame, the wool pings are loosened by a rotatory motion, and then partially withdrawn. The mouth of the original culture-tube is similarly singed and its plug partially Avithdrawn. A platinum needle is selected and carefully straightened The original tube is then taken in the left hand be- tween the thumb and index linger with the palm upwards, and is held obliquely, the mouth of the tube pointing to the right, a tube of sterile medium being held side by side with the original culture in an exactly similar manner. The wire of the platinum needle is then heated to redness by holding nearly vertically in the flame, and the lower part of the handle is also carefully heated. Holding the sterilised needle between the finger and thumb of the right hand, the plug of the original culture is now withdrawn by grasping between the ring and little fingers of the right hand, and is held there while the platinum needle is carefully introduced into the tube Avithout touching the mouth or sides, and a trace of the growth is picked up with it, preferably from the margin. To ensure that the needle is cool, it may first be touched on the medium where there is no growth. The needle is quickly with- drawn without touching the sides of the tube and the plug at once replaced. The plug of the sterile tube is now withdrawn in the same manner, and the inoculated needle introduced. If a typical surface culture is Inoculation of Media 71 desired, a single light streak is made with the needle from the bottom to the top of the medium without penetrating the surface ; if an abundant growth be required for any purpose the whole surface of the medium may be rubbed with the needle; if a stab culture, the needle is plunged steadily into the centre of the medium and withdrawn ; if a fluid one, the growth removed is rubbed up on the side of the tube at the margin of the fluid, and the emulsion washed down by tilting the tube. The inoculation having been completed, the plug is quickly replaced, and the needle is again heated in the name to destroy the remains of the growth upon. it. If the original culture is in a deep stab, or a fluid medium, a looped platinum needle may sometimes be used with advantage. The inocula- tions completed, the mouths of the tubes are singed and the wool plugs pushed in level with the lip. Before replacing the plugs each may, if desired, for greater safety, be taken Avith the forceps, held in the flame for a second or two, and pushed while burning into the tube, and this procedure must always be adopted if the plug be dropped or brush against any- thing. If the tubes have to be kept for any length of time, especially in the blood-heat incubator, each should be capped with a rubber cap, tinfoil, or gutta- percha tissue which has been soaked in 1—500 corrosive sublimate solution . Anaerobic cultures. — Many organisms refuse to grow in the presence of free oxygen, and various expedients have to be adopted to exclude or remove it. The simplest of all is to make the cultivation in a deep stab in glucose agar or gelatin. Narrow test-tubes filled three parts full with the medium are best, and immediately before the inoculation they should be placed upright in a beaker of water, boiled for five minutes, and then 72 Manual of Bacteriology cooled and solidified in cold water. The object of this is to soften the medium so that it does not split, as a dry medium will, when the needle is plunged into it j moreover, the needle track closes up more readily, and the dissolved oxygen is expelled. The tubes being cool, the inoculation is made with a long thin wire, either straight or with a closed loop at the end. It is inoculated and plunged steadily into the centre of the medium, nearly to the bottom, rotated, and then withdrawn, and the wool plug is replaced and singed. The tube is then carefully heated at the upper border of the medium so as to melt this slightly and seal the puncture, and a well-fitting rubber cap is applied while the tube is hot. The heating expels a portion of the air, and, with a well-fitting cap, creates a negative pressure within the tube, so that the residual oxygen is not so readily absorbed, or the tubes may be placed in a Buchner apparatus (see below). The tubes are placed in the incubator at a suitable temperature, and it will be found that the most strictly anaerobic organ - isms can be cultivated in this way. When, however, an organism is required to grow anaerobically on the surface of the medium, or in a fluid medium, some other method must be adopted. The tubes may be placed under the receiver of an air- pump and exhausted as completely as possible. This is not very convenient, for it is difficult without gi-eat care to maintain a vacuum, and special receivei-s must be used when the cultures have to be incubated at blood- heat, while with fluid media ebullitioii causes consider- able difficulty. For fluid cultures Hamilton's method is the simplest of all. The fluid in the tubes is covered with a layer of olive oil 1—2 cm. thick, and the tubes are then sterilised. The layer of oil prevents the access and Anaerobic Cultivation 73 entrance of oxygen. The only disadvantage is that the inoculation, or the withdrawal of culture, must usually be performed with a sterile glass pipette ; if a wire needle be used the material is very liable to be detached in the oil. Another method (Buchner's) is that usually adopted, and consists in absorbing the oxygen by means of alkali and pyrogallic acid, and so cultivating in an atmosphere of nitrogen. This can be carried out in two ways — either in a wide-mouthed bottle with well-fitting glass stopper, sufficiently large to contain the test- tubes, or in a Buchner's tube. For the first the inoculated culture tubes are placed in the bottle, into which a few cubic centimetres of a strong aque- ous solution of p}rrogallic acid have pre- viously been poured. By means of a thistle funnel, an equal volume of 20 per cent, caustic potash1 or soda solution is then added. As quickly as possible the thistle funnel is withdrawn without mixing the solutions, and the stopper, well vaselined, inserted and twisted well Fig.h.— Buchner's home, and some melted paraffin may be tube arranged for poured all round the joint and melted tion.10blC cultlva" in with a hot iron. The solutions in the bottle are now well mixed, and the whole is placed The Buchner's tube (Fig. 11) in a suitable incubator, Thirty-two grm. of pyrogallic acid and 64 grm. of caustic potash dissolved in 100 c.c. of water will absorb 9200 c.c. of oxygen. At the same time some carbon monoxide is evolved (122-5 c.c.) The evolu- tion of CO is a minimum when the potash is in excess and only one fifth of the theoretical absorbable amount of O is absorbed. 74 Manual of Bacteriology is convenient for single test-tube cultures. It consists of a strong glass test-tube, large enough to take an ordinary test-tube, and having a constriction about an inch and a half from the bottom. The constriction supports the test-tube culture, while the mixture of pyrogallic acid and caustic potash fills the portion below the constriction. A well- fitting rubber cork closes the mouth of the tube, and the joint may be paraffined for additional security. If a Buchner's tube is not available, the cotton-wool plug of the culture tube may be pushed into the tube for an inch, some solid pyrogallol is placed on the wool plug, this is just moistened with caustic potash solution and the tube is stoppered with a rubber cork. The displacement of the atmo- sphere by means of hydrogen may be adopted, and is to be preferred for fluid cultures. Hydrogen does not seem to inhibit the growth of any anaerobic organisms, whereas Avhich mio-ht be still more conveniently used has a very decided inhibitory action on some species. The hydrogen is best generated from zinc and sulphuric acid in a Kipp apparatus, or the compressed gas in cylinders, or even coal-gas, may be used. Care must be taken that all joints are tight, and they may be paraffined with advantage. The gas should be passed through a strong solution of caustic potash, and may be passed through some alkaline pyrogallic acid if the most rigorous condition of anaerobiosis is desired, but for Fig. 12.— FriinkeFs tube for anaerobic cultivation, carbon dioxide gas, Anaerobic Cultivation 75 ordinary purposes this is not essential ; it should also pass through two or three fairly firm plugs of cotton- wool to remove organisms; these must be dry, for if moist the passage of the gas may be stopped. For tube cultures F ranker's method may be adopted (Pig. 12). The broth or gelatin is introduced into a large strong test-tube, the mouth of which is plugged with a rubber cork pierced with two holes. Through these holes two pieces of glass tubing pass, one to the bottom of the tube, the other just through the cork. Outside the cork these tubes are bent over at right angles, and each is drawn slightly out so as to contract its lumen at about the middle. The long tube is con- nected with the hydrogen supply, and a current of the gas is passed through and escapes by the shorter tube. After the gas has been passing for twenty minutes to half an hour, and all oxygen has been expelled, the distal, i. e. shorter, tube is sealed off at the contracted portion in the Bunsen or blowpipe flame, and then the proximal or longer one in the same manner. The rubber cork must, of course, fit well, and the joints should be paraffined. If gelatin be the medium, it must be kept fluid in a bath of warm water while the hydrogen is passing. For broth or fluid cultures, which are essential for obtaining toxic products, flasks are used which are fitted with an india-rubber cork pierced with two holes. Through the holes two pieces of glass tubing pass, one to the bottom of the flask, the other just through the cork, as in the Frankel tube described above. The ends of these tubes are plugged with cotton-wool, and the whole— flask, cork, tubes and medium — is sterilised. The medium is inoculated from a recent culture by momentarily removing the cork. Hydrogen is then passed through from a Kipp apparatus, the long tube 76 Manual of Bacteriology being connected with the hydrogen supply. After passing for about half an hour, the tubes are sealed off and the flask is incubated. For convenience of sealing the tubes should be drawn out slightly. As many organisms produce gas during their growth, it may be necessary to provide for its escape, or the flasks may burst owing to the pressure. This can be done by adjusting a mercury valve, and may be carried out in a simple manner by a method devised by the writer. f< Yeast flasks," which can be obtained in various sizes, are made use of, and are filled three parts full with a 2 per cent, grape-sugar bouillon. The neck is corked with a perforated rubber cork (a, Fig. 13), through which a glass tube, B, passes to the bottom of the flask, projecting two inches above the rubber cork and here plugged with cotton-wool. The lateral tube of the yeast flask is also plugged with cotton- wool, care being taken that the plugs are loose enough to allow air to pass freely. The whole is sterilised and inoculated. The glass tube, b, which passes through the rubber cork, is then connected with a Kipp or other hydrogen-generating apparatus by means of a rubber tube, and a current of hydrogen is Fig. 13. — Yeast flask arranged for anaerobic cultivation. Plate Cultivations 77 passed through the flask. The hydrogen bubbles through the bouillon and escapes by the lateral tube. AfteAhe gas lias been passing for half an hour a small tube containing mercury, c, is applied to the end of the lateral branch, so that the open end just dips below the surface of the mercury, and the tube b, which passes through the rubber cork, is sealed off in the blowpipe flame, care being taken that all the air has been expelled from the flask by a free current of hydrogen. The flask, with the capsule of mercury applied to the end of the lateral branch, can then be placed in the incubator. The mercury thus forms a valve through which air cannot enter, while gases formed by the growth of the organism have free exit. For large flasks, the lateral tube may be just bent down and a little capsule of mercury hung on. The addition of £ to 1 per cent, of sodium formate to the culture media much simplifies anaerobic cultiva- tion ; the tetanus bacillus, for example, can be grown in formate broth in a stoppered bottle without any elaborate precaution for excluding the last traces of air. The sodium formate should be added immediately before the last sterilisation, not previously, or decom- position may occur. Sodium sulphindigotate (0*3 per cent.) may be similarly used. With such a bi'oth, Dean's bottle may be used for anaerobic cultivation. This consists of a bottle around the neck of which a gutter for mercury is formed. A loose glass cap fits over the mouth of the bottle, and its edge dips into the mercury in the gutter, thus sealing the bottle. Plate cultivations. — The method of plate culture' is one of the most important in bacteriology. It is used for three purposes : (1) for obtaining pure cultivations, i. e. cultures containing a single species, from a mixture s 78 Manual of Bacteriology of organisms; (2) for the enumeration of organisms- and (3) for ascertaining the characters of the colonies of organisms as an aid in the identification of species Before the introduction of plate cultivations pure cultures of organisms could only be obtained by chance or by the dilution method, which was also by no mean' certain. The dilution method consisted in estimating approximately the number of organisms in a given volume of fluid by means of an instrument on the same principle as the hasmatocytometer. The fluid is then diluted by the addition of some sterile fluid so that a given volume of the dilution contains a single organism only, assuming the organisms to be evenly distributed throughout the fluid. By transferring this volume to tubes of sterile media pure cultivations can in some cases be obtained, a single organism having been sown in a tube. It is obvious, however, that this method is at best an uncertain one, but the plate-culture method to a large extent obviates this uncertainty. It depends upon the following principles: Gelatin and agar media, when melted, remain fluid down to 25° and 45° C. re- spectively, temperatures which will not affect the vitality even of delicate organisms. By inoculating the fluid gelatin or agar, thoroughly mixing, and then pouring on to a level sterilised surface, so that the medium solidifies in a thin film ("plating"), the organisms, wherever they may be situated, are fixed and are unable to wander, and, being in a good nutrient soil, grow and multiply and ultimately form visible growths or colonies. Many of these colonies will have arisen from a single organism ; the growth, therefore, is "pure/; i.e. consists of a single species, and pure cultures can be obtained by inoculating tubes of sterile media from them. Plate Cultivations 79 When suitable, sterile nutrient gelatin is usually employed for the preparation of plate cultivations, as if is more easily manipulated than agar. Three tubes of sterile nutrient gelatin are melted at a low tempera- ture in a beaker of water (gelatin melts at 24 C. ; the temperature should not exceed about 45° C.) The tubes may be termed respectively 1, 2, and 3. Tube No. 1 is inoculated, by means of a platinum needle, with a trace of the growth from which pure cultivations are desired. The trace of growth is thoroughly mixed up and distributed throughout the melted gelatin. If this mixture be " plated," so many organisms may be present in the film that the colonies which develop will not be separate, but will form a confluent growth. To obviate this difficulty a second and a third dilution are prepared. The second dilution is made by inocu- Fig. 14. -Petri dish for plate lating the tube of melted cultivation, gelatin No. 2 with one platinum loopful from tube No. 1, and thoroughly mix- ing up ; and to be quite sure that the resulting colonies will be isolated from one another, a third dilu- tion is prepared in the same manner by inoculating the tube of melted gelatin No. 3 with two to four platinum loopfuls from tube No. 2. The organisms having been distributed throughout the gelatin by rolling and gentle shaking, the wool plug is in each case withdrawn from the mouth of the tube, the mouth of the tube is sterilised in the Bunsen burner to prevent contamina- tion, then cooled for a few seconds, and finally the melted gelatin is pointed on to a level sterile glass surface. For- merly plates of glass were used (hence the name) ; but now 80 Manual of Bacteriology shallow glass dishes with lids, about three or four inches m diameter, known as Petri dishes (Fig. 14), are almost always employed. They are previously sterilised m the hot-air steriliser in suitable iron or copper boxes holding a dozen or so ; the melted gelatin having been poured in, the dish is tilted to diffuse the gelatin over the bottom of the dish, placed on a level surface for the gelatin to set, and then stored in the cool incubator. The plates are examined daily, with a hand lens if necessary, or with a low power of the microscope, the dish being turned bottom upwards on the stage of the microscope for this purpose. When the colonies have developed, inoculations can be made from them by means of a platinum needle on to tubes of sterile media. The colonies, having arisen from single organisms, are pure, and the resulting sub-cultures are therefore also pure (it sometimes happens that the colonies are mixed owing to two or more organisms being close together). Different species of organisms usually form colonies having different appearances, so that the colonies are an aid in diagnosis and enable the various species to be picked out from a mixture. The colonies in gelatin are as a rule much more distinctive than those in agar. Whereas the plate cultivation prepared from tube No. 1 is generally too crowded, plates 2 or 3, or both, can be made use of, and it is apparent that, to make certain of isolating all the organisms from a mixture, several sets of plates should be prepared. Flat bottles (Fig. 15) may likewise be used for plate culturing, and are also very useful for growing organisms in bulk for the exami- nation of the constituents and actions of the bacterial cells. In addition to the isolation of species from mixtures and for diagnosis, plate cultures are also used to enumerate organisms. Assuming that every colony arises from a single organism, which is approximately Plate Cultivations 81 the case, the number of colonies represents the number of organisms originally introduced into the gelatin, and if a known weight or volume of the material inoculated be used, the number of organisms in it can be calcu- lated. For example, in the bacteriological examination of water a measured volume of the water is added to melted gelatin by means of a sterilised pipette, and by counting the resulting colonies the number of organisms originally present in 1 c.c. of the water can be estimated. Agar plate cultures may be prepared in a similar way. The agar must, however, be brought to a tem- perature of nearly boiling before it melts ; it is then allowed to cool to nearly 45 C. and the tubes are inoculated in the same manner as for a gelatin plate culture described above. Unless the manipula- tions be carried out expeditiously the agar will solidify, or the agar film in the Petri dish be lumpy. Agar plates should usually be inverted during incubation, or the growth may become confluent owing to the condensa- tion water carrying the organisms all over the film. Fm. 15.-" Plate " The plate-culture method can be modi- bottles, fied to suit particular circumstances : for example, the melted gelatin or agar, uninoculated, may be poured into the dishes and allowed to solidify, and the film then inoculated by streaking or painting with the material, or by pouring a few drops of broth con- taming the organisms upon it. This is practically the only way in which blood-serum can be used, the sterile blood-serum being placed in the Petri dish, solidified in the inapissator in the same manner as for blood- serum tubes, and the coagulated film inoculated. 6 82 Manual of Bacteriology lor many purposes plates are unnecessary, the same result being obtained by rubbing over the surface of two or three tubes of sloping agar or gelatin succes- sively the once charged needle, straight or looped. In the second or third tubes isolated colonies generally develop. J The plate-culture method often fails if the organism to be isolated forms but a small minority of the total organisms present in the mixture ; the only alternative then is to multiply the number of plates, which, how- ever, may entail great labour in their examination. Single-cdl cultures.— With large cells, such as yeasts, it is not difficult to obtain growths from single cells by making miniature plate cultures on ruled cover-glasses, examining microscopically, and ascertaining the places m the film where single cells are located (see Chapter XVI). But with the minute bacterial cells this method is inapplicable. By the use of Burri's Indian ink method,1 however, single-cell cultures of bacteria can be obtained. Fluid Indian ink is diluted with 6-10 volumes of dis- tilled water and the mixture is sterilised in the auto- clave. Several loopfuls of this are deposited in series on a sterile slide. The first drop is inoculated with the culture which is being investigated, the second drop is inoculated from the first, the third from the second, and so on. A fine mapping-pen, sterilised in the flame, is then dipped into the third, fourth, or fifth drops, and the trace of Indian ink mixture so picked up is depo- sited on a gelatin or agar plate. The droplet is covered with a sterilised cover-glass and is examined with a -i- in. or ^ in. objective, with a high eyepiece. An oi-ga- nism shows up white on a black background. Many drops are deposited on the plate and examined, and 1 Das Taschverfahren (G. Fischer, 1909). Gunther Wagner's ink (Hanover) is recommended and is supplied by Griibler. Roll Cultures 83 those in which only a single organism can be found are noted and the plate is then incubated so that colonies may form, from which sub-cultures may be prepared. Esmarch's roll cultures. — Another modification of the plate-culture method is known as Bsmarch's roll culture. For this purpose large test-tubes ("boiling tubes"), at least an inch in diameter and six inches long, are sterilised and plugged with cotton- wool. The sterile melted gelatin, about 10 c.c, is poured in and inoculated, the wool plug replaced, and the tube held in the horizontal position and rotated under a stream of cold water, or in warm weather on a block of ice, until the gelatin has set. In this way the gelatin forms a thin film over the inside of the tube, but a little practice is required to get it evenly distributed. The colonies then develop in the film of gelatin, which is quite analogous to a film in a Petri dish. Anaerobic plate cultivations are sometimes required. The plate culture after preparation as described above, using a deep Petri dish, is inverted, and some alkaline pyrogallol is placed in the lid; this absorbs the oxygen within the dish. The preparation must be kept under observation for the next hour or so, and more alkaline pyrogallol is added from time to time to compensate for the rise of fluid within the dish until absorption of the oxygen from the contained air is complete. In Botkin's method a bell-jar standing in a glass dish is made use of. The Petri dishes are placed on a support within the bell-jar, and mercury or oil is poured into the glass dish. By means of a piece of bent glass tubing a stream of hydrogen is passed into the bell-jar under its rim so as to displace the air, which bubbles out through the oil or mercury. When the an- has been entirely displaced the glass tube is removed, the bell-jar weighted, and the whole placed 84 Manual of Bacteriology id the incubator. Bulloch's apparatus is somewhat similar to this. Wide-mouthed jars with well-ground glass lids, which are luted down, are very convenient, the oxygen being absorbed with alkaline pyrogallol placed at the bottom, and the Petri dishes stacked on a glass capsule or other support to raise them above the fluid. The Esmarch roll cultures can be adapted for anaerobic plate cultures. The wool plug is replaced by a rubber cork with two holes, through which inlet and outlet glass tubes pass, as in Frankel's anaerobic tubes (Fig. 15). The roll culture having been prepared, and the film having set, hydrogen is passed in and the tubes are sealed off, or, better still, the hydrogen is allowed to bubble through the inoculated melted gelatin, the test-tube meanwhile being kept in a little wa rm water to prevent the gelatin from solidifying, the tubes are sealed oil', and the roll culture is then prepared. For the detection of fermentation and gas production, stab cultures in glucose agar or shake cultures in gela- tin may be employed. For the latter a tube of gelatin1 is melted at a low ,„ _ , , temperature, inoculated with the org-a- Fig. 16.— Durham's . r . ' . ..,.„ . ° fermentation tube, uism, and allowed to solidity m the upright position ; the organism is thereby distributed throughout the medium. Fermentation with gas production is indicated by the presence of gas bub- bles, or even by the disruption of the medium. Durham's fermentation tubes are very convenient for showing 1 Lemco gelatin frequently gives no gas ; a meat-broth gelatin should therefore be used for gelatin shake cultures. Fermentation Tubes 85 fermentation. These are test-tubes containing- suitable fluid media (10 c.c. each) into which small glass tubes closed at the upper end are placed ; the latter become filled during the sterilisation. The tubes are inoculated and incubated, and if fermentation occurs the little tube becomes filled with gas (Fig. 16). Binhorn's sacchari- meter may also be used (Fig. 17). The tube is filled with the medium, sterilised, inoculated, and incubated. Fig. 17. — Einhorn's saccharimeter. Any gas produced collects in the closed limb of the tube. When the amount of gas ceases to increase, a little strong caustic potash solution may be added; this absorbs the C00, the residue probably being hydrogen, and thus the H : C02 ratio may be deter- mined. The most suitable media for fermentation are peptone broth, the acid beef-broth for which has been treated with the colon bacillus (see p. 27), 1-2 per cent, peptone water, or a medium which has been Manual of Bacteriology largely used by Houston, Gordon, and others, consist- ing of a 1 per cent, solution of « Lemco " in distilled water with the addition of peptone 1 per cent., sodium bicarbonate Ol per cent.; to either medium is added 1-2 per cent, of glucose, lactose, saccharose, starch, mulin, mannitol, dulcitol, etc., and the mixture is tinged with litmus. The fermentation tube has been much used of late for the examination of fasces in abnormal intestinal con- ditions. For this purpose 1 grm. of fasces is thoroughly emulsified in 10 c.c. of physiological salt solution and 1 c.c. of the suspension is introduced into the fer- mentation tube, the long arm of which is 95 mm. long. The media employed are 1 per cent, dextrose, lactose, and saccharose broths made with " Lemco " (as above) or with sugar-free meat broth (see p. 27). With such tubes normal stools yield the following amounts of gas ■} Dextrose. Lactose. Saccharose. 2675 . . 29-9 . . 19-5 mm. 1 See Herter and Kendall, Studies from the Rockefeller Institute (Reprints), x, 1910. Preparation of Tissues 87 CHAPTER III. THE PREPARATION OP TISSUES AND ORGANISMS FOR STAINING AND MOUNTING. STAINING AND STAINING METHODS. A selection of the numerous methods devised for the pi-eparation and staining- of tissues, bacteria, etc., is here given. Special methods occasionally employed will be described when required. Preparation of Tissues. As the demonstration of the bacteria in the tissues is of the primary importance, the elaborate methods which have been described for fixing the tissue elements are not essential in bactei'iology, unless of course the relation of the bacteria to the tissue elements is being studied. The tissues should always be obtained as fresh as possible, because within a few hours of death they are invaded by numerous bacteria, derived from the air and from the intestine, which may mask the original bacterial infection and lead to serious mistakes if this source of error be not carefully borne in mind. In all cases the tissue should be cut into pieces of convenient size, not more than about 1 cm. in thickness, and organs if kept en masse should be sliced. Having been thus prepared, the material may be treated by one of the following methods ; 88 Manual of Bacteriology (a) Place directly i„ methylated spirit1 for a week or a fortnight. (6) Place in methylated spirit 1 part, water 2 parts for twenty-four to forty-eight hours, transfer to methy- lated spmt and water, equal parts, then to methylated sprit, and finally to absolute alcohol for like periods (c) Place m rectified spirit (86 per cent, alcohol) containing 1 per cent of corrosive sublimate for twelve to forty-eight hours, and pass through increasing strengths of alcohol as in (6). (d) Place for six to twenty hours in a saturated aqueous solution of corrosive sublimate. This is prepared by saturating boiling distilled water with the corrosive sublimate, cooling, and filtering Keep m the dark. When removed from the corrosive sub- limate solution the tissues must be washed in a stream of running water for an hour, or, better, placed for a day m 70 per cent, alcohol deeply coloured with iodine, to remove the excess of corrosive sublimate and prevent precipitation. The tissues are then passed through increasing strengths of alcohol, as in (6). (e) Formalin, a 40 per cent, aqueous solution of formic aldehyde, is an excellent fixing agent. A solu- tion of 1 part of formalin and 9 parts of water, or better, physiological salt solution, may be used/the pieces of tissue remaining in this for twelve to twenty- four hours. They are then washed in running water for an hour or two and passed through increasing strengths of alcohol, as in (b). 1 Methylated spirit free from mineral naphtha should he used and can be obtained in quantities of five bulk gallons, "for scientific purposes only," by special order from the Inland Revenue Authorities, Somerset House, W.C. If it cannot be procured, absolute alcohol must be employed. Duty-free absolute alcohol can also be obtained under somewhat similar conditions and is much cheaper than the ordinary. Section Cutting 89 All tissues after fixing and hardening should he preserved in dilute alcohol — water 1 part, absolute alcohol or methylated spirit 2 parts. The methods (c), (d) , and (e) are to be recom- mended, especially the two last, as the tissue elements are well fixed thereby. In all cases the fixing fluid should be used in considerable excess. Fixing fluids containing potassium bichromate (as in Miiller's fluid) and chromic acid seem to prevent the bacteria from staining with any certainty, and should be avoided. Section Cutting. In order satisfactorily to demonstrate bacteria in tissues, and their relation to the tissue elements, it is usually necessary to prepare sections. For this pm*- pose either the freezing or the paraffin method should be employed. (a) Freezing method. — The tissue, in suitable pieces, must first be soaked in water to remove the alcohol. A convenient way of doing this is to place the material in a wide-mouthed bottle, into the mouth of which an ordinary glass funnel is introduced, and the bottle with the funnel is placed under a stream of running water; the funnel, while allowing the water to flow out, retains the pieces of tissue in the bottle. With running water the alcohol will be completely removed in from one to two hours; in still water, which should be changed two or three times, this result may not be attained for several hours, during which time there is an ever-increasing risk of bacterial contamination from without. It is essential to remove all the alcohol, or the I issue will not freeze. When the alcohol has been removed, which is known by the tissue sinking in the water (lung is an exception 90 Manual of Bacteriology —it always floats unless solid from any cause), the pieces are transferred to a strong- mucilage of gum acacia : Gum acacia 5 grm Cane sugar 0.5 *rm Water 100 c.c. Fig. 18. — Swift's ether-freezing microtome. Add a piece of thymol or a little carbolic acid to preveut decomposition. Hamilton saturates the solution with boric acid. In this gum solution the pieces remain for twelvre to forty-eight hours, according to their size and the time at the disposal of the investigator, and are then cut on one of the numerous ether-freezing microtomes now to be obtained, such as Swift's (Fig. 18) or Cathcart's. A microtome in which the freezing is effected by Paraffin Sections 91 carbonic acid is now largely employed and acts well. Liquid carbonic acid, contained in a cylinder, sprays by its own pressure on to the under surface of the plate on which the block of tissue rests ; the tissue quickly freezes and is then cut. This form of microtome works satisfactorily in the hottest weather. The material must not be frozen so hard that the sections roll up and fall off the knife ; the sugar in the above solution should prevent this. The sections are trans- ferred successively to two or three lots of distilled water, preferably slightly warmed, to remove the gum, and can then be stained or preserved in equal parts of methylated spirit and water until wanted. Bacteria seem to retain their staining properties better in the tissue in bulk than in sections. Although the bacteria may stain well in sections for some time after preparation, it frequently happens that in a month or two they refuse to stain. Such is certainly the case with anthrax tissues, but not with tubercle or leprosy, the bacilli in sections of the latter seeming to retain their staining properties unaffected for a considerable time. (b) Paraffin method. — Nothing can surpass the paraffin method for the thinness and beauty of the sections obtainable by it, and for some friable tissues, such as actinomycosis, it is almost essential. The tissue, in suitable pieces for cutting, is transferred from the diluted spirit preservative solution to pure methy- lated spirit for two or three hours, and then to absolute alcohol — which may have to be changed once unless a fairly large volume is employed — for from four to twenty-four hours. It is then removed from the alcohol, lightly dried between the folds of a dry cloth or piece of blotting-paper to remove the superfluous alcohol, and placed in an excess of xylol, in which it 92 Manual of Bacteriology remains for from four fr, * £ ■> cWP(q T)- °Ur .t0 ^nty-four hours until cleared. This 1S recognised by the material assuming a more or less semi-transparent condition, and the pro cess may be much accelerated by warming the xylol to from 37° to 50° 0. in the blood-heat incubator' or paraffin oven the bottle containing the xylol being well stoppered. When cleared it is ready "to go into he bath of melted paraffin. A paraffin of a fairly high melting-point is perhaps the best, viz. 45° to 55° 0, and ia placed in glass capsules in an oven which can be kept uniformly heated to the required tempera- ture. An ordinary chemical hot-water oven answers the purpose quite well, and is heated by a special form of small Bunsen burner with mica chimney, the tem- perature being regulated by some form of mercurial regulator, winch is set a degree or two above the melting-point of the paraffin employed. The tissue is taken out of the xylol, blotted to remove the excess and placed in the melted paraffin for six to twenty hours'. It is then embedded by pouring a little of the melted paraffin into a watch-glass, or into a small box formed of folded paper or lead-foil, or by bringing together two L-shaped pieces of brass on a glass plate so that a rectangular cavity is produced. The pieces of tissue are then taken out with a small warmed forceps or needle, adjusted to the position they are required to occupy,' and more melted paraffin is poured in, so as to cover them. When a film of solid paraffin has formed, the whole is immersed in cold water so as to cool it rapidly. A new paraffin is frequently crystalline in structure, and acts much better after it has been kept melted for some weeks, or is much improved by heating nearly to its boiling-point for five or six days (P. T. Beale). The xylol for clearing may be used several times and the Paraffin Sections 93 paraffin repeatedly, the remains of old tissues being removed. The time which the tissues require to re- main in the alcohol, xylol, and paraffin depends upon their size ; very small pieces may be treated in a few hours, large ones may require two or three days. Other clearing agents, such as chloroform, turpen- tine and cedar oil, may be used instead of xylol. The paraffin method is usually straightforward, but small pieces of tissue must not be left too long either in* Fig. 19. — Cambridge rocking microtome. absolute alcohol or in the paraffin bath, for they are liable to become too hard to cut. Thyroid tissue and skin are also rather troublesome ; they become very hard unless the whole process is carried out as rapidly as possible. If the pieces of tissue be large, the cap- sule of melted paraffin containing the tissue may be placed under the receiver of an air-pump, which is then exhausted. This causes the paraffin to penetrate better, and the process may be repeated two or three times during the period of infiltration. A special form of paraffin oven has been devised by Cheatle for infil- 94 Manual of Bacteriology fcrat'ing under diminished pressure, and is made by Hearson, of Regent Street. In order to prepare sections from material embedded in paraffin some form of microtome must be employed. An ether-freezing microtome can be employed with some manipulation, the paraffin block being placed in a little melted paraffin on the freezing plate so that it is cemented there, and sections are cut with the razor or plane iron, as though it had been frozen (it is not. to be frozen). It is better, however, to use some special form of microtome, the Cambridge "Rocker" (Fig. 19), or a modification of it, or the " Minot," being perhaps the best. The block of paraffin con- taining the tissue is trimmed with a knife to remove the excess, and is cemented to the carrier of the micro- tome with a little melted paraffin, or by melting the paraffin on it with a hot iron (end of a file, etc.) or a match. The union may be made more secure by melting the paraffin around the base of the block with a hot iron. Having fixed the paraffin block to the carrier, sections may then be cut of any degree of thinness. In order to do this it is essential for the knife or razor to have a keen edge and one of the right nature, for a knife may be perfectly sharp and yet the sections as they are cut roll up in such a manner that it is difficult to flatten them. Though this may be due to a wrong consistence of the paraffin, owing to cold weather or some other factor, in the majority of instances it is the ' edge of the knife which is at fault. Provided the knife be sharp, stropping on the palm of the hand will usually remedy this difficulty. The paraffin being of the right consistence, and the knife in good order, the sections as they are cut should be flat and should adhere together at adjacent margins so that a ribbon of greater or shorter length is formed. Paraffin Sections 95 Satisfactory sections having been obtained, they are transferred with a needle or camel's-hair brush to a tin pan containing a little water, or spirit and water, warmed to about 40° C. The sections float and the paraffin soften*, so that they spread out perfectly flat (the water must not be hot enough to melt the paraffin). A clean slide is then introduced underneath the section, raised so that the section is lifted up on it, and by fixing the section with a needle and tilting the slide the section is deposited in the required position on the slide and allowed to dry. If preferred, the section may be transferred to a slide flooded with water, which is warmed over the Bunsen. The slides can be manipu- lated in an hour or two if dried at 37° C, but it is best to allow them to dry in the incubator all night. It will be found after this treatment that the sections adhere sufficiently firmly to the slides for all the ordinary methods of staining to be carried out without detaching them, which would be fatal. The sections must be fairly thin, however; if they are at all thick they do not adhere nearly so well. Should the sections have to be subjected to pro- longed treatment during staining, etc., they may be cemented to the slides by the following method. Equal parts of egg-white and glycerin are mixed and filtered, and to every 100 c.c. of the mixture 1 grm. of sodium salicylate is added. The slide is smeared thinly with this, the section is transferred to it in the manner just described, and the slides are dried. Supposing that, while cutting, the sections, in spite of all precautions, curl up instead of lying flat, it is still often possible to obtain a few that can be mounted. They may sometimes be unrolled by cautious manipu- lation with a couple of needles after having been softened by warming, or a needle or knife-blade may 96 Manual of Bacteriology be held close to the edge of the microtome knife during enttmg so that the sections, instead of curlW shd! up against this guide. S' m labelled pdl boxes and cut all at once or from time to time as required, or the ribbons of sections may be preserved in I box m a cool place until wanted. The slides also, with the ectxons attached can be kept until it is convenien to stain if preserved free from dust in a slide box. Cover-glass and Film Specimens. The satisfactory preparation of cover-glass and film specimens is one of the most important in bacteriology for they are used for the examination of cultivations of bacteria, and of blood or other fluids or secretions organs, etc., for the presence of micro-organisms. It is necessary in the first place to have clean cover- glasses of the right kind ; they must be thin, otherwise the higher powers cannot be employed to examine the preparations, and those described as "No. 1" should be purchased, "f-in. squares" being a convenient size. These serve both for cover-glass specimens and for covering sections • it is well also to have a few of the same thickness but larger, viz. fin. or 1-in. squares, for large sections. In order to clean them they should be gently boiled in a porcelain dish with 10 per cent, carbonate of soda solution for a few minutes, well washed, and then treated with strong sulphuric acid warmed carefully in a porcelain dish, for a few minutes. The acid having been poured off, they are well rinsed in several changes of water, and should be kept in a stoppered glass capsule in absolute alcohol. Slides may be used instead of cover-glasses, and should bs cleaned Film Preparations 97 and manipulated in the same manner as described for cover-glasses. A clean cover-glass (or slide) is taken, dried with a clean soft linen or silk rag or handkerchief, or with Japanese paper, or it may be momentarily introduced into the Bunsen flame and the spirit burnt off, and placed flat on a convenient support on the work-table —a white glazed tile is excellent— with the corner pro- jecting so that it can be conveniently picked up with the forceps.1 A droplet {%. e. small drop) of water is then placed on it, in the middle, by means of a looped platinum needle, or with a small glass pipette (Fig.^ 7). Theoretically, sterilised physiological salt solution2 should be used, a few cubic centimetres being boiled in a sterilised and plugged test-tube for two or three minutes and cooled; but ordinary tap-water may usually be employed. A thin film of organisms has now to be formed on the cover-glass, and the following is the method of procedure with a culture on a solid medium such as agar or gelatin. The culture tube and platinum needle are held and manipulated in precisely the same manner as that, described for the inoculation of tubes (p. 70), a trace of the growth being removed from the tube and the wool-plug immediately replaced. A mere trace of the growth from a culture should be taken, just sufficient to soil the tip of the platinum needle, or the preparation will be too crowded, and this is rubbed up well with the droplet of water on the cover-glass, so as to form an emulsion, which is then spread over the surface. As a general rule the 1 The writer has devised a useful support for staining. It consists of a square of plate glass, painted half white and half black at the back, and having a narrow strip of thick glass cemented across it on which the covers rest. It is made by Messrs. Baird & Tatlock. 2 0"75-0'95 per cent, of sodium chloride dissolved in distilled water. 7 98 Manual of Bacteriology material should be well emulsified, but in some instances tins is inadvisable, as a particular formation or characteristic grouping- may be disturbed thereby m winch case, after a slight admixture with the water' the emulsion is gently spread. The thin film on the cover-glass is allowed to dry, or may be dried by gentle warming- over the Bunsen flame, preferably holding the preparation in the fingers and moving backwards and forwards over the flame. Having dried the film, it has next to be fixed, which is accomplished by holding the cover-glass, film side up, in the forceps and passing fairly rapidly three times through the Biinsen flame (slides 6-9 times). The object of this "fixing" is to thoroughly dry the film and coagulate albuminous material, whereby the film adheres better to the glass, and is not so likely to be detached in the subsequent processes of staining and washing, etc. Fixing may also tend to diminish the staining capacity of the extraneous matter mixed with the organisms. The preparations are now ready for staining. When the culture is in a fluid medium, such as broth, the tube is manipulated in the same way, the deposit at the bottom having been shaken up if neces- sary, and a loopful or two of the fluid removed with a looped platinum needle, transferred to the cover-glass, spread out, dried, and fixed as before, but as the medium is fluid there is usually no need to acid a droplet of water. If a specimen of blood, pus, or sputum is required, the procedure is much the same. A little of the material is taken up with a looped platinum needle and spread in a thin film over the cover-glass or slide, which is then dried and fixed. If necessary, a droplet of water or physiological salt solution may be used to dilute the material so as to obtain a thinner film. If a Film Preparations 99 specimen is to be made from an organ, a particle of the pulp is picked up and an emulsion made as before, or a small piece of the organ may be held in sterile forceps and the cut surface gently smeared over the cover-glass or slide, which is then dried and fixed; these are termed " smear preparations." To obtain the best results it is preferable before staining to submit films of blood1 or pus or smear pre- parations to the action of some chemical fixing agent, unless the film is stained with Irishman's solution, which both fixes and stains. The simplest method of doing this is to immerse the films, after cur-drying, in a mixture of equal parts of absolute alcohol and ether for five to fifteen minutes. In hot countries a saturated aqueous solution of corrosive sublimate (five to fifteen minutes) is perhaps as good as anything. Another method, combining both fixing and staining, is to immerse the films as soon as they are prepared and without drying in the following solution : Absolute alcohol saturated with eosin . 25 c.c. Pure ether , 25 c.c. Alcoholic solution of corrosive sublimate (2 grin, in 10 c.c.) . . . .5 drops For four cover-glasses 5 to 10 c.c. of this solution are required, and they should remain in it three to four minutes (it may be prolonged for hours without harm) ; they are then removed with a forceps and well rinsed in water, stained for not more than a minute in a saturated aqueous solution of methylene blue, washed quickly, dehydrated in absolute alcohol, cleared in xylol, and mounted in xylol balsam. The solution may be used for fixing blood, pus, sputum, etc., if the eosin 1 For the method of preparing blood-films see the section on " Malaria," Chapter XVIII. 100 Manual of Bacteriology be omitted, and the preparations may then be stained or otherwise treated in any desired manner.1 Scott2 recommends the following as giving the most perfect results with blood films, etc. : (1) Hold the freshly prepared and still wet film in the mouth of a wide-mouthed bottle half filled with the ordinary formalin solution, film side downwards, for five seconds. (2) Drop, while still wet, film downwards, into abso- lute alcohol. Leave for fifteen minutes, or, for con- venience, for any time up to forty-eight hours. The preparations may then be stained with methy- lene blue or hematoxylin, and eosin, or with Irishman's stain. (See also under "Malaria" and " Trypano- sornes," Chapter XVIII.) Impression specimens. — These are employed to examine and preserve permanently the colonies or growths of organisms so that their characteristic forma- tion may be observed. With plate cultivations this is very simple. A clean cover-glass is sterilised in the flame and, having cooled, is cautiously lowered on to a selected colony with a sterile needle, avoiding all lateral movement. It is gently pressed on to the colony and then carefully raised by means of a couple of needles ; the colony should adhere to the glass, and may be dried and fixed. The colonies in gelatin tube cultures may also be used if the gelatin is removed from the tube. This can be done by dipping the tube for a few seconds into hot water ; the gelatin round the walls of the tube will be melted, and the gelatin mass can then be tilted out of the tube on to a glass dish or tile. 1 Gulland, Brit. Med. Journ., 1897, vol. i, p. 65. a Journ. Path, and Bad., vol. vii, No. 1, p. 131. Stains and Staining Methods Stains and Staining Methods. Micro-organisms being so minute and transparent, it is usual to stain or dye them, so that they can be more readily examined. In some instances organisms may have a peculiar staining reaction, which may serve as an aid to their identification. But when an organism is being investigated, examination m the fresh and livino- condition must never be omitted, for at is only thus°that its motility and life-history can be studied. Only general methods are detailed here ; special ones will be given when they are required. (1) Loffler's alkaline methylene blue. Saturated alcoholic solution of methylene blue . 30 c.c. Solution of caustic potash, 0 01 per cent. . 100 c.c. A very useful staining solution. Cultures should be quite fresh, or the organisms do not stain well. When the organisms are mixed with extraneous material, as in smears, or there is much debris, this is one of the best staining solu- tions to employ. Methylene blue preparations are, however, not very permanent, and in hot countries rapidly fade. Thionine blue is then preferable. Cover-glass specimens are stained for three to ten minutes, and sections half to twenty-four hours. (2) Carbol-methylene blue (TCiihne). Methylene blue I'5 grm- Absolute alcohol ...... 10 c.c. Five per cent, aqueous solution of carbolic acid 100 c.c. A more intense staining solution than the former, and very useful for sections, which are stained for from half to six hours. (3) Anilin gentian violet. Saturated alcoholic solution of gentian violet . 30 c.c. Anilin water 100 c.c. 102 Manual of Bacteriology withh90?ilinf7tr11iS,Prepared b^baki^ 3 c.c. of anibn with 90 c.c. of dxstxUed water, allowing the mixture to stand fox a few minutes, and filtering. Instead of anilin water 1 violet) aqUe°US Cai'b0liC ^ USed ^YholS^Ln This solution is a useful general stain for films, which are stained for two or three minutes, and is employed in Gram's method of staining. It does not keep well. (4) Carbol-fuchsin (Ziehl-Neelsen solution). Fuchsin 1 art Absolute alcohol . n A ^ , -p. 10 parts *ive per cent, aqueous solution of carbolic acid 100 parts The fuchsin is dissolved in the absolute alcohol and then mixed with the carbolic acid solution. It must always be filtered before use. An intense staining solution. For film specimens it is best diluted with five to ten parts of water; stain for two to five minutes. (•"3) Carbol-thionine blue (Nicolle). Saturated solution of thionine blue in alcohol (90 per cent.) 10 c c One per cent, aqueous solution of carbolic acid 100 c.c. Sections can be strained in from a few minutes to half an hour. This solution may be used for a modified Gram's method (see p. 109). Can be substituted for methylene blue for all purposes, and is more permanent than the latter. (6) Chenzinski's solution (after Klein). Saturated aqueous solution of methylene blue (Griibler's) . . . .." 50 c.c. Eosine (soluble in alcohol) Absolute alcohol .... Distilled water .... Film specimens, after fixing, are placed in absolute alcohol for half a minute, dried, and placed, film downwards, in a 0"5 grin. 70 c.c. 130 c.c. Staining Solutions 103 watch-glass of the stain, which is then warmed until it steams^ The specimen is then taken out and well washed an tan water rinsed in distilled water, dried, and mounted. Actions are immersed in absolute alcohol for seven, minutes, stained in the cold for six to twelve hour well washed in distilled water, and passed through absolute alcohol and xylol, and mounted. (7) Bosin (alcohol-soluble and water-soluble). A somewhat diffuse stain. Is used for counter-staining the tissues in Gram's method, and for staining red blood- corpuscles and acidophils granules in leucocytes. A 1 to 1 per cent, aqueous or alcoholic solution may be used, "and the staining should not, as a rule, be prolonged for more than about half a minute. (8) Bismarck brown (Vesuvin). A saturated aqueous solution should be prepared and diluted somewhat for use. A good counter-stain for the tissues in Grain's method. Stain for two to five minutes. (9) Orange-robin. Prepare saturated aqueous solutions of orange G. and rubin S. Mix equal volumes and dilute with water until of a light port-wine colour. Stain tissues for five to fifteen minutes. A good contrast stain for tuberculosis and actinomycosis. (10) Picro-carmine. This is best bought ready prepared. Sections are stained in the solution for half to one hour, washed, then placed in a watch-glass of spirit, to which three or four drops of hydro- chloric acid have been added, for two or three minutes, then well washed in water. The section can now be counter- stained with Loffler's blue or by Gram's method. (11) Hematoxylin. Ehrlich's formula is one of the best and simplest to use, 104 Manual of Bacteriology and can be obtained ready for use. It must be « ripe." It freiat£x:l:and not a bacteriai sto- - (1) Distilled water, one to two minutes (2) Stain with the hematoxylin solution for five to thirty minutes In some cases the solution is preferably diluted somewhat with distilled water. """tea (3) Einse in distilled water. J4) Einse in distilled water containing a trace of acetic (5) Treat with distilled water containing a trace of ammonia The sections remain in this until they assume a deep blue colour. (Tap-water, five to ten minutes, may also be used.) J (6) They can then be dehydrated, cleared and mounted or counter-stained with eosin, orange-rubin, or Van-Gieson, and then mounted. Hematoxylin makes a good contrast stain for the tubercle and the leprosy bacillus and for Actinomyces. Mayer's hemalum (see section on the 'Amoeba coli ') and Delafield's hematoxylin are also good hematoxylin stains. (12) Ehrlich-Biondi triple stain. This is best bought ready for use. It is a good histological stain for tissues and leucocytes. Actinomycosis stains well by it. Stain for ten to sixty minutes, then treat with methylated spirit until the section becomes greenish. Pass through absolute alcohol, clear, and mount. (13) Leishman's stain. Like the Jenner, Wright, and other similar ones, a modifi- cation of the Eomanowsky stain, a double compound of eosin and methylene blue. The solution will keep for some time, but is best freshly prepared. Griibler's powder or Burroughs Wellcome's soloid may be used, and is dissolved in pure (Merck's or Kahlbaum's) methyl alcohol. Failure fre- quently proceeds from the use of a so-called pure methvl Gram's Method 105 alcohol, which is not really so. (For method of using, see << Malaria," Chapter XVIII.) (14) Giemsa stain. An eosin-aznr mixture dissolved in glycerin and methyl alcohol. Useful for blood-films, film preparations, etc., and has been much used to demonstrate the spirochaetes m syphilitic material. (For method of using, see " Syphilis.") ' Safranin and acid fuchsin are also used as counter-stains. Malachite green, neutral red, and rosein may be used for intra-vitam staining of protozoa, etc. Eosin, orange-rubin, hsematoxylin, and picro-carmine keep well in solution; the remainder may or may not, and are best used fairly fresh. All stains should be filtered before use, and may be conveniently kept in bottles having a funnel fitted with a filter-paper, so that they are always ready. Or smaller bottles may be used, fitted with pipettes, and several arranged in a stand. The best stains are Griibler's, which can be obtained from many agents in this country. Messrs. Burroughs, Wellcome & Co. supply most of the anilin dyes and some other reagents, iodine, etc., in "soloids," which are very convenient and good. Gram's method. — This is a most useful method, espe- cially for sections, specimens of blood, or smear or impression preparations, as the tissue or ground substance can be counter-stained so that the organisms show up in marked contrast. Ordinai'y cover-glass specimens do not usually require this method, unless debris or ground substance is present and the best result is desired. Unfortunately Gram's method is not applicable for all oi'ganisms, as many do not retain their colour by the process. This disadvantage, however, is counter-balanced by the fact that it forms a valuable means of distinguishing organisms, and is always one of the points to be noted in bacteriological diagnosis. Most of the moulds, yeast, streptothrix and 106 Manual of Bacteriology sarcina forms, and cocci stain by it, though there are exceptions; the spirilla and protozoa do not stain by it but as regards the bacilli no rule can be laid down (see p. J 08). Cover-glass specimens are stained for five to ten minutes, and sections for ten minutes to half an hour, m anilm-or carbol-geutian violet solution. Drain off the superfluous stain, and then immerse, without washing, in the following solution for one half to two minutes : Iodiue. • .... 1 part Potassium iodide .... 2 parts Distilled water . . 300 parts The purple colour of the gentian violet changes to a dirty yellowish-brown, and sections become much like a used tea-leaf. The specimens must not be passed on to the next solution until they have assumed the brown colour. Cover-glass specimens are best imnwrsed in the solution in a watch-glass, film side up, or they and slide specimens may be flooded with the iodine solution two or three times. The specimens are removed from the iodine solution, drained, and then immersed in alcohol, preferably methylated spirit. In this the purple colour of the gentian violet returns and is dissolved out, so that they ultimately become colourless ; this is aided by moving them gently about, and for sections two or more baths of alcohol may be an advantage, a fresh one being substituted when the first has become deeply coloured. Film specimens decolorise much more readily than sections, and they should be removed from the alcohol when no more colour dissolves out, or the stain may be entirely removed; usually twenty to forty seconds in the alcohol suffices, thick preparations taking Gram's Method 107 longer than thin ones. It is also important with cover- o-lass specimens to remember on which side ot the glass the film is, for it may be very difficult to ascertain this when the specimen has been decolorised. After decolorising, film specimens are washed m water, dried and mounted, or, after washing, the ground substance may be counter-stained, if required, with eosin for a few seconds, or Bismarck brown for two or three minutes, washed again in water, dried, and mounted. Sections after decolorising are passed through absolute alcohol and xylol before mounting, or, if Required to be counter-stained, are immersed in eosin for fifteen to thirty seconds, or Bismarck brown for three to five minutes, and then passed through methylated spirit, absolute alcohol, and xylol. Sections frequently are somewhat difficult to de- colorise with alcohol alone, in which case it is well to treat them with a slightly acid alcohol (3 per cent, of hydrochloric acid) for a few seconds, and then return to the alcohol (Giinther's method). The iodine in Gram's method seems to act as a mor- dant, precipitating the stain in a relatively insoluble form in certain species of bacteria. The staining of organisms by Gram is relative ; some forms do not stain at all, are Gram-negative — i. e. the colour is removed by the alcohol with the greatest facility ; others stain intensely, are Gram-pasitive, but even these may become decolorised by prolonged treatment with alcohol. The best procedure is to have three watch-glasses of methylated spirit, and to transfer the cover-glass specimen from one to another as the stain is dissolved out; in the last bath, immediately the final trace of colour is so;en to be dissolved out, the prepara- tion should be removed, washed in water, dried, and mounted. In order to ascertain whether an organism 108 Manual of Bacteriology is or is not stained by Gram's method, it is sometimes useful to mix with it in making the preparation some undoubted Gram-staining organism— e. g. if a bacillus, v^U4^- with tne Micrococcus pyogenes- if a coccus, with B. anthracis or B. suUilix. The admixed organism then : ^(/TC ^serves as an index. Q~^\ft^^^e following organisms are Gram-positive: B. antfyacis , )r,)[~C\M^//> B- diphtheria, B. tettstjii, B. Welchii, B. bdtitlinus, B. U0r*- culosis, B. smegmatis, B. lepnv, B. mvrisepticus, Actinomyces, j^K ■ B. subtUis, B. mesentericus, B. megaterium, B. mycoides, the \ ,p pyogenic cocci, the streptococci, including the pneumococcus, most cocci, yeasts, moulds, and streptothrices. The following organisms are Gram-negative : B. typhosus, B. enteritidis, B. dysenteries, B. coli, B. pestis, B. influenzae, B. mallei, B. pseudo-tuberculosis, B. pgocyaneus ,J3^J>d.fi,mci t i« maligni, B. Chauvsei (usually), B. prodigiosus, B.protevs, the septicemic bacilli, such as chicken cholera, the spirilla and vibrios, spirochaetes and protozoa, M. gonorrhoea, M. menin- gitidis, M. melitensis, aud M. catarrhdis. Gram's method of staining depends upon the formation of an iodiue-pararosanilin-protein compound which is not readily dissociable in the case of the Gram-positive organisms. Para- rosanilin dyes, such as gentian violet, methyl violet and victoria blue, are alone suitable for the method. In Claudius's modification of Gram's method,1 stain- ing is done in a 1 per cent, aqueous solution of methyl violet (films for one minute, sections for two minutes). The preparations are washed, treated with a half- saturated aqueous solution of picric acid for one to two minutes, washed again, and dried with filter-paper. Decolorisation is then carried out in the case of covers with chloroform, in that of sections with clove oil. After decolorising, the preparations are treated with 1 Ann. de I'Inst. Pasteur, xi, 1897, p. 332. Weigert's Method 109 , nllfP(l Bv this method the ordinary xylol and mounted. By . d . a]g0 the bacmi of Gram-positive organisms aie stained, aiso ConnteY. procJ*e -'ions wKethe, o^r~, should be manipulated on the suae. 11 y Wlth the anilin gentian violet and treat d w^h Weigert's iodine solution (iodine 4-5 per cent, potas- sium iodide 6 per cent.) as m the sample , & am method The iodine is then removed with filtei-papei and the sections are flooded with anilin oil two or three times This removes the colour and dehydrates. the anilin oil is removed by flooding two or three times with xylol. Thionine blue may be used for Gram's method, the carbol solution being employed (No. 5, p. 102). Sections are stained for two or three minutes, then treated with an iodine solution somewhat stronger than Gram s (200 parts of water instead of 800 parts). The sections, after remaining in this for one to two minutes, are decolorised in alcohol containing 1 per cent, of acetone (methylated spirit does very well), and subsequently treated as in Gram's method. The Staining of Film Specimens. To stain cover-glass, smear, and impression prepara- tions, after fixing, the film is flooded with a drop or two of the solution, or the preparation, if a cover- glass, may be floated, film side down, on the solution con- tained in a watch-glass; if it should sink it makes little difference. Various baths or pots can be obtained for staining slides. Warming intensifies the staining properties of all staining solutions, and may be neces- 110 Manual of Bacteriology e sary if deep staining is required or if the temperature ot the laboratory be low (see also p. 114) When stained sufficiently, the preparation is rinsed in a beaker or tumbler of water- or in a fine stream of water, preferably distilled, to remove the superfluous colour, after which it is dried and mounted on a glass slide, film side down, in a drop of solution of Canada balsam in xylol. The preparation may be dried either by gentle warming over the Bunsen flame after the film has been blotted with filter-paper, or the film may be allowed to dry spontaneously in the air, in which case it should always be set up on edge to drain, pre- ferably on a ledge of filter-paper, which is folded into a sort of compressed ^ (z) shape. The preparation, must be completely dried before being mounted in balsam. To prevent the stain flowing all over a slide, two lines may be drawn across the slide with a grease pencil, one on either side of the area to be stained. If there be much debris or other material which, when stained, would interfere with a clear view of the organisms, various expedients may be adopted. One is to stain for a short time with a solution which docs not give a very dense colour, the best for this purpose being Loffler's methylene blue, or Gram's method may be made use of if the organism stains by it, and will give the best result of any. Another plan is to treat the specimen with acetic acid before staining ; it may be just dipped in glacial acetic acid and immediately washed in distilled water, or immersed in 20 per cent, acetic acid for five to ten minutes, washed in distilled water, and then stained. A third is, after staining and washing, to rinse the preparation in dilute alcohol (methylated spirit 1 part, water 1 or 2 parts), and immediately wash again in water to stop the further action of the alcohol. If the film be thick, two or Frozen Sections 111 three rinses in the dilute alcohol may be necessary. This process gives excellent results with the sarcime Ut Je Lining agent should be aniHn gentian vio e or dilute carbol-fuchsin and not Loffler s blue, unless it is allowed to act for fifteen to twenty minutes, lie treatment with acetic acid before staining may be combined with decolorisation with alcohol after Preparations can always be examined m water with the Mn objective, after washing and before perma- nently mounting, in order to see whether they are satisfactory. A drop of water is placed on the slide the specimen is mounted in this, film side down, and the unper surface of the cover-glass is dried with filter-paper. If satisfactory, the preparation can be slipped off, dried, and mounted in balsam; or if not stained sufficiently, or if stained too deeply, it can be stained again, or further decolorised, as the case may be. If the film is on a slide, a drop of water is put on and covered with a cover-glass. Treatment of Sections for Staining and Mounting. (a) Frozen sections.— -If preserved in spirit they should be rinsed in distilled water before staining, unless the staining solution is an alcoholic one, in which case this is unnecessary. After staining they are well rinsed in water or methylated spirit to remove the excess of stain, and then dehydrated and cleared before being mounted. For dehydrating, if they have been washed iu water, they should be well rinsed in methylated spirit1 to remove the excess of water, and then transferred to absolute alcohol for a few seconds 1 Absolute alcohol may of coarse be employed instead of the first bath of methylated (or rectified) spirit, but methylated answers just as well and is less expensive (but see note, p. 88). 112 Manual of Bacteriology to two minutes, the time varying witli the size and thickness of the section. In many eases— for instance, when the anilin dyes have been used for staining-— the sections must be passed as rapidly as possible, con- sistent with thorough dehydration, through the absolute alcohol to avoid removing too much of the colour. If it is important to avoid any decolorisation, anilin oil may be used for dehydration, as in Weigert's method (pp. 109 and 115). For clearing, xylol or cedar oil is the best agent, for neither dissolves the anilin dyes; they will only clear, however, out of absolute alcohol: hence the preliminary rinsing of water-washed sections with methylated spirit to prevent dilution of the subse- quent bath of absolute alcohol. Oil of cloves can also be employed, but has the disadvantage that it dissolves the anilin dyes, and the colour of stained sections treated with it is apt to be less permanent ; it has the advantage, however, of clearing out of methylated spirit, absolute alcohol being unnecessary. The alcohol and clearing agents are conveniently placed in watch- glasses or small shallow glass capsules. The section is known to be cleared when it appears quite transparent and almost invisible when the watch-glass or capsule containing it is held over a dark surface. If it appears cloudy and opaque it is not properly cleared, which results from insufficient clearing or dehydrating. If the section does not clear in a minute or two it is evidently not sufficiently dehydrated, and should be returned to a fresh bath of absolute alcohol for a short time, and then transferred again to the clearing agent. Care should be taken that watch-glasses, etc., used for the absolute alcohol and clearing agent are perfectly dry. The clearing agent, especially clove oil, can be used many times before becoming useless. For transferring the sections from one solution to Paraffin Sections 113 another an ordinary needle, fixed in a light wooden handle, suffices, or, better still, a piece of glass drawn out at one end, the section being carefully lifted by one corner to prevent crumpling ; but for the final process of mounting it is necessary to use a section lifter or cigarette-paper. The section, spread out with care, is raised by means of the section lifter or cigarette- paper introduced under it, and transferred to the slide, anv crinkles are removed by spreading with a needle, the superfluous clearing agent is drained off, a drop of xylol balsam put on, and it is then covered with a clean cover- o-lass. If clove oil has been used as the clearing agent, the section, after draining, should be blotted with two or three thicknesses of filter-paper to remove as much oil as possible before putting on the balsam. In blotting firm pi'essure should be used, and the section will then adhere to the glass slide and not to the blotting-paper. With delicate sections all the processes of staining, dehydrating, clearing, for clearing, etc. etc., may be carried out on the slide. (b) Paraffin sections. — The sections having been safely fixed on the slide (p. 95), it is necessary, in order to stain and mount, to remove the paraffin by solution in xylol. The slides with attached sections are treated as follows : Immerse in (1) xylol for one or two minutes ; (2) absolute alcohol one to two minutes to remove the xylol ; (3) methylated spirit ; (4) distilled water. They are now ready for staining, and are to be flooded with the staining solution or immersed in it, and after staining they are treated in the same manner, but in the reverse order, i. e. (1) distilled water ; (2) 8 114 Manual of Bacteriology methylated spirit; (3) absolute alcohol; (4) xylol. On being removed from the xylol the slides are drained for a few seconds, a drop of xylol balsam is then put on, and the section covered with a clean cover-glass. Glass pots (Fig. 20) filled with the alcohol, xylol, etc., are convenient for the treatment of paraffin sections, the slide with .the section upon it being- immersed in the fluid. Section Staining-. When Gram's method is applicable it gives by far the best results, and should always be employed. If, however, the organisms are decolorised in Gram's process some other method must be adopted. One of the best is to stain for from ten minutes to six or eight hours in Loffler's methylene blue. Fresh easily staining organisms will be sufficiently stained in ten or fifteen minutes, but when the organism is difficult to stain, as glanders, six to eight hours may not be too long a time. Warming intensifies the staining properties of all staining solutions ; for frozen sections the watch- glass of stain may be warmed on a sand-bath or asbestos cardboard, or in the blood-heat incubator. Sections on the slide may be flooded with the stain and warmed on a piece of asbestos cardboard placed over a Bunsen flame, or a penny may be heated in the Bunsen and the preparation laid on it, the coin being re-heated as often as required. The stain may be prevented from flooding the slide by confining it between grease-pencil lines as described for films (p. 110). After staining, the sections are well rinsed in distilled water and then slightly decolorised by rinsing for half a minute or so in a watch-glass of 1 per cent, acetic acid in distilled water. Section Staining 115 They are then again washed and passed as rapidly as possible through alcohol, cleared in xylol, and mounted. Carbol-methylene blue or carbol-thionine blue may be used instead of the Loffler's solution, the staining taking from a few minutes to half an hour. If a contrast stain be desired the sections may be treated for a few seconds with the eosin solution after the dilute acetic, but a better method is to use the Chenzinski solution, which is strongly recommended by Klein (see p. 102). If staining be prolonged evapora- tion must be prevented. In the case of a section mounted on the slide and flooded with stain, the slide should be placed on a piece of wet blotting-paper on a tile and covered with the lid of a Petri dish. The micro-organisms in sections stained with Loffler's blue are very liable to become decolorised unless the dehydration is expeditiously performed. To avoid this Unna's method may be adopted. After staining and decolorising with acidulated water as described, the sections are placed on the slide (if not already mounted thereon), gently warmed, and so dried; they are then treated with xylol and mounted in balsain. The tissue elements, however, are apt to suffer. A better method is, after decolorising with the dilute acid, to dehydrate with anilin oil instead of alcohol, the section being treated with fresh anilin two or three times, then with a mixture of equal parts of anilin and xylol, and finally with two or three baths of xylol. Capsule Staining1. Many organisms, especially in the tissues or body fluids, are invested with a capsule of gelatinous matter, probably derived from the membrane of the bacterial 116 Manual of Bacteriology cell, and differing in composition in different species. The capsule may be as thick as the bacterial cell itself, and appears, in the unstained state or after staining by the ordinary methods, as a clear halo or zone surround- ing the organism. Organisms in films of albuminous matter often appear to be surrounded by a clear halo, which must not be mistaken for a capsule. As organisms frequently lose their capsules on ordinary culture media, Moore recommends cultivating in fluid serum to obtain the re-development of the capsule. In order to stain the capsule one of the following methods may be adopted : 1. Stain the preparations by just dipping in the following solution : Carbol-fuchsiu . . . 1 part Distilled water . . . .1 part Rinse in water and then stain for fifteen seconds in a very weak aqueous solution of gentian violet (0-l per cent.). Rinse in water, dry, and mount. 2. McConlceys method. — The following solution is pre- pared : Methyl green . . . . 1*5 grm. Dahlia 05 grm. Distilled water . . . 100 c.c. When dissolved, 10 c.c. of a saturated alcoholic solution of fuchsin are added, and the whole is made up to 200 c.c. with distilled water. The stain should not be used for a fortnight, and should be kept in a dark place. Specimens are stained for five minutes or longer, then thoroughly washed in a stream of water, dried and mounted. 3. Friedldnder's method (for tissues). — Mix, Concentrated alcoholic solution of gentian violet ... 50 parts Distilled water . . .100 parts Acetic acid .... 10 parts Spore Staining 117 Stain the sections in this solution in the warm incubator for twenty-four hours. Rinse well in 1 per cent, acetic acid, pass through alcohol and xylol, and mount in balsam. Spore Staining. When spore-bearing bacteria are stained by the ordinary methods the spores are just tinted, or remain uncoloured with the outlines more or less stained. This seems to be due to the fact that the spores are surrounded with a slightly permeable membrane which inhibits the entrance of the staining agent. By staining by some method which causes the penetration of the stain, and then cautiously decolorising, it is possible to remove the colour from everything except the spores, the impermeable membrane of which in the same way prevents the full action of the decolorising agent. (a) Simple method.— A film is prepared in the ordinary way. If a cover-glass, it is floated on a watch-glass, or, if a slide, it is flooded with carbol-fuchsin, and the stain is warmed for twenty minutes. After being washed in water the prepara- tion is rinsed for a second or Wo in 1 per cent, sulphuric acid and again washed at once in water. If there is still a good deal of the red colour remaining, the film may be once more rinsed in the acid, but if nearly colourless it should be mounted in water and examined with the -J -hi. objective. If the spores alone are well stained the preparation may be counter- stained with Loffler's methylene blue for two to five minutes, washed, dried, and mounted. If, however, the bacilli as well as the spores retain the red colour, the preparation must be further decolorised in the acid, while if everything has been decolorised, it may be re-stained with warm carbol- fuchsin. The spores sometimes stain better if the preparation be fixed by passing through the flame twelve times instead of 118 Manual of Bacteriology three, as is usual. To obtain good preparations and ones showing the spores in situ, the specimens should be made as soon as spores have definitely developed in the cultures. Spore staining often requires a good deal of patience, and in many instances it is difficult to obtain a satisfactory pre- paration by this simple method, in which case that of Moeller should be made use of, and rarely fails. (b) Moeller 0 method.— Prepare the cover-glass or slide specimen in the ordinary way. Treat with absolute alcohol for two minutes, and then with chloroform for two minutes. Wash in water and treat with a 5 pereent. solution of chromic acid for two minutes, wash, and then stain with warm carbol- fuchsin for ten minutes. Wash, decolorise carefully in 1 per cent, sulphuric acid, again wash, and counter-stain with Loffler's methyle .e blue for one minute ; wash, dry, and mount. Some organisms, such as the B. mesentericus, stain better if treated with the chromic acid for five to ten minutes. Flagella Staining. Many organisms possess delicate protoplasmic pro- cesses— flag-ella — in greater or less number ; but these are not visible when the organism is examined in the living condition (except by the use of dark-ground illumination), nor when the ordinary staining methods are employed. In order to demonstrate them it is necessary to make use of some special method, in which a mordant is essential. One of the earliest devised was that of Loffler, which Avith care gave fair results. It is not, however, nearly so satisfactoiy as some more recent ones, so is omitted. For all methods of flagella staining the cover-glasses or slides must be absolutely clean, the cultures recent, and the growth sufficiently diluted to obtain the organisms in an isolated condition. (a) Stephens s method. — This is a modification of the well- Flag-ella Staining 119 known Van Ermengen method,1 and has been communicated to the writer by Dr. J. W. W. Stephens. To clean slides.— Rub the slides with a clean cloth and place on a piece of clean wire gauze and heat with a smokeless flame for some minutes (by this means grease is completely removed). Eemove the slides when cool, not before. To make the emulsion.— All methods are unsatisfactory. Eub a little of the culture in a small drop of tap-water in a watch-glass. Then transfer a drop with the smallest possible platinum loop to a minute drop of water on the slide. Mix and spread with the platinum wire as quickly as possible. The film thus made should dry immediately if a small drop only of water has been vised. Age of the culture— A twenty-four hours' culture does quite well (a younger one is perhaps better, but flagella can be shown for a week or fortnight or more) . I. The mordant : Osmic acid, 2 per cent. . . 1 part. Tannic acid, 20 per cent, watery solution 3 or 4 parts II. Silver solution : Silver nitrate . . 1 per cent. III. Gallic acid, 2 per cent, solution . 1 part Ammonia fort. . ... 1 part To be mixed before vising and to be used immediately. To stain. — Place the mordant on the film for one or two minutes or less (time unimportant). 1. Wash in tap-water thoroughly. 2. Shake off as much water as possible. 3. Place a few drops of silver nitrate on the slide for a few seconds or longer. 4. Shake off: all excess. 5. Allow one drop of the ammonia-gallic solution to fall on the middle of the slide from a small pipette. A wave spreads away from the centre to each end of the slide. As 1 Centr. f. Balct., xv, 1894, p. 969. 120 Manual of Bacteriology soon as the film is seen standing out clearly and black in the centre (in a few seconds), wash off in tap-water. 6. Add again a drop or two of the silver solution and allow it to act for half a minute or thereabouts. 7. Wash in tap-water, blot, and dry over the flame. 8. It is best not to mount in balsam or in cedar-wood oil, as the preparations rapidly fade in these. If done with any care, the film should now appear black and distinct to the naked eye with no precipitate, and the flagella will be found to be stained distinctly and intensely with hardly any ground substance, or at least insufficient to interfere with a clear view of them. (b) Pitfield's method.— Two solutions are freshly prepared : A. Saturated aqueous solution of alum . 10 c.c. Saturated alcoholic solution of gentian violet ...... 1 c.c. b. Tannic acid ...... 1 grin. Distilled water 10 c.c. The solutions should be made with cold water, filtered, and preserved in separate bottles. For use equal quantities are mixed together. The specimens are flooded with the mixture and held over the flame until it nearly boils ; they are then laid aside, with the hot stain on them, for one minute, and are finally washed in water. After washing, the preparations are flooded with anilin gentian violet for one second, washed in water, dried and mounted. (c) McCrories method1 {modified by Morton-). — Prepare the following solutions : a. Tannic acid .... 1 grm. Potash alum ..... 1 grm. Distilled water .... 40 c.c. b. " Night " blue 0'5 grm. Absolute alcohol .... 20 c.c. Mix and filter. 1 Brit. Med. Joum., 1897, vol. i, p. 971. 2 Trans. Jenner hist. Prev. Med., vol. ii, p. 242. Preservation of Specimens 121 The prepared slides are stained with this solution (winch shouhl l avs be filtered before use) for two minutes the Son being changed two or three times, washed ^eiitiy in running water, and then counter- stained m anihn gentian violet for one to two minutes, washed, dried, and mounted. Preservation of Cultures. Gelatin and agar cultures may be satisfactorily preserved bv submitting them to the action of formaldehyde vapour for some hours by soaking the wool plug of the culture tube m formalin and plugging the tube with it. The tube may then be sealed with gutta-percha tissue, sealing-wax, or paraffin wax, or best of all in the blowpipe flame. Plate cultivations may also be exposed to the vapour and the lid of the dish afterwards cemented on, or the cultures may be made in the flat bottles ("Soyka's bottles") devised for the purpose, and after development treated like tube cultures. Preservation of Pathological Specimens. These may be preserved in the ordinary way in spirit, but a much better method, by which the natural colour of the specimen is retained, is the following. The specimens are first washed in water, and then placed in the following solu- tion for twenty-four to forty-eight hours : Formalin 6 parts Sodium chloride .... 1 part Sodium sulphate .... 2 parts Magnesium sulphate ... 2 parts Tap-water 100 parts After being taken from the formalin solution the specimens are placed in methylated spirit for ten minutes, and then in a fresh bath of methylated; in this the colour to a large extent returns, and they should be carefully watched and not allowed to remain in it for more than an hour. They a,re then mounted in the following mixture : 122 Manual of Bacteriology Glycerine 400 c.c. Potassium acetate .... 200 o-rni Water 2000 c.c. ' A trace of formalin should be added to this. The writer has preserved meat infected with B. prodigiosus very satisfactorily by the following method. Slices were cut off and placed in the formalin solution given above for a few hours. They were then well drained and placed in suitable glass capsules. Ordinary nutrient gelatin was melted and sufficient poured in to cover the specimens, and when it had set a little formalin was poured on and allowed to remain for a few days. It was then poured off and the glass top cemented down. For further information on preparation of tissues, section cutting, staining methods, etc., see The MicrotomisV s Vade- Mecum.BoRes-'Lee ; Practical Histology, Schafer ; Methods of Morbid Histology and Clinical Pathology, Walker Hall and Herxheimer ; and Lehrbuch der Mikroskopischen Technik, Rawitz. Investigation of Microbial Diseases CHAPTER IV. METHODS OF INVESTIGATING MICROBIAL DISEASES THE IN- OCULATION AND DISSECTION OP ANIMALS HANGING DROP CULTIVATION— INTERLAMELLAll FILMS THE MICROSCOPE. The systematic study of a condition dependent on the activity of micro-organisms is in many instances no light matter. When only one or two forms are present and these are readily cultivated it may be comparatively easy, but when there are many the investigation may become exceedingly complicated. The first step to be taken is to ascertain by careful microscopical examina- tion the general characters of any organisms that may be present in the material, and their distribution both in the fresh condition and in stained perparations, and if possible at different stages of the disease. ^ In disease conditions, for example, the blood and secretions may be examined both before and after death, but in the latter it must be remembered that soon after the fatal event adventitious organisms rapidly make their appearance, gaining access from the air and from the intestinal tract. If organisms be detected an attempt should be made to determine whether there is any predominant form and if this is constantly present at different stages. If organisms are found, it simplifies matters ; but if not, it cannot therefore be said that they are absent, for they may be few in number, and 124 Manual of Bacteriology consequently be missed in a microscopical examination ; or they may be confined to a particular locality or tissue, or are present only at one stage of the infection. In addition to the microscopical examination, cultures must be made on various media, those media being chosen which will probably be suitable for the growth of the organism present in the particular condition ; for example, in the examination of animal diseases, media rich in protein, such as blood-serum, peptone-agar, and gelatin, will be the most serviceable. In the examina- tion of plant diseases, vegetable infusions prepared from the plant itself or from other sources, and enriched by the addition of vegetable proteins, and carbo- hydrates, should be chosen. In fermentations, beer- Avort, grape or fruit juice, and saccharine solutions should be made use of; while for the nitrifying orga- nisms, solutions containing nitrates and nitrites, salts of ammonia, urea, and asparagin will have to be employed. In addition, it will in most cases be advisable, and in all safer, in order to isolate the various species, to make plate cultivations, either in Petri dishes (p. 79), or by streaking several sloped tubes of agar, etc. (p. 81). Having obtained pure cultivations it will be necessary to determine the species of organism,1 if it has been previously isolated and described, or to give a careful description of it, if it be a new one, for the use of subsequent investigators. In the identification or description of an organism all the following features must be carefully noted : 1 The descriptions of a large number of species of bacteria have been collected and tabulated in convenient form by Chester in A Manual of Determinative Bacteriology (Macmillan & Co., 1901). The terms he suggests for describing bacterial growths, etc., might well be adopted by bacteriologists. A committee of the Society of American Bacteriologists has drawn up an elaborate chart for the description of species of organisms. Identification of Organisms 125 1 The morphology of the organism under various con- ditions, its size, form, and motility, the presence of flagella, and their number, arrangement, and character. •2 The presence or absence of spore formation, its nature, the conditions under which it occurs, and any peculiarities m the germination of the spores, and their size and location m the cell. 3. The peculiarities of staining, and the staining reaction with Gram's and the Ziehl-Neelsen methods. 4. The characters of the colonies in gelatin, agar, and other media, both surface and deep. 5. The characters of the growth on a variety of culture media at different temperatures— for example, for a pathogenic organism, on blood-serum, agar, and gelatin (surface and stab cultures), in broth and on potato ; liquefaction or not of the gelatin ; the growth in milk, with or without curdling, and the reaction therein; and the fermentation reactions on carbohydrates, glucosides, alcohols, etc. ; the nature of the gas, if any, formed therefrom, and the H : C02 ratio. 6. The behaviour towards oxygen— is it aerobic or anaerobic ? 7. The range of growth at different temperatures. 8. The reducing power by growing in litmus broth wliicli becomes decolorised, or by the formation of nitrites in a solution containing nitrates. 9. The production of indole with or without nitrites. 10. The production of pigment and the conditions under which it occurs. 11. The pathogenic action on various animals if it be a disease germ, or the changes which it produces if it be an organism connected with other conditions. 12. The chemical changes which it induces. 13 The thermal death-point and the action of germicides and antiseptics upon it (see Chapter XXII). For descriptive purposes, "standard" culture media should always be employed, and the acidity or alkalinity of the medium stated (p. 64). 126 Manual of Bacteriology It must never be forgotten that under cultivation the properties of organisms may be considerably modified, and due allowance must be made for this. For example, pathogenic organisms may lose their virulence more or less completely, pigment production be lost, and fermentive action modified. An instance of tlie latter is given by Percy Frankland ; a bacillus isolated by him possessed the power of fermenting calcium glycerate, but after cultivation on ordinary gelatin it completely failed to do so (see also p. 6). To obviate these difficulties the organisms should be cultivated under as nearly natural conditions as possible and sub-cultivation avoided so far as can be. No general rule can be given as to the duration of life of cultures on artificial media. Most organisms will retain their vitality for at least three or four weeks without being transferred to a fresh soil, some for many months ; a few must be sub-cultured every week, or they will die out ; while there are still a small number, such as leprosy and relapsing fever, which so far have rarely or never been cultivated. On the whole, organisms retain their vitality best on gelatin. For an organism to retain its virulence it is, as a I'ule, necessary to pass it through a susceptible animal at longer or shorter intervals, and to enhance the virulence recourse must be had to a succession of passages through susceptible and then less susceptible animals. In this way the virulence of organisms has been increased to a point far greater than is ever met with naturally, as in the case of the Streptococcus pyogenes. If an organism retains its virulence, even slightly, it is generally possible, by employing large doses, to enhance this by passage through a susceptible animal. Another method may also be adopted, namely, to inject along with it some other pathogenic form, Animal Inoculation 127 such as the Streptococcus pyogenes ; the combination will kill the animal, and the slightly virulent organism can be recovered and will be found to have increased in virulence. A third method is to inject the organism into a susceptible animal together with a lethal dose of toxin obtained from a virulent form of the same species, or with some substance, such as lactic acid, which lowers the vitality of the tissues. The slightly virulent organism will then be able to grow under the more favourable conditions, and a form which has become completely non-virulent can be made to regain its lost virulence. Collodion sacks are now frequently used to study the action upon animals of the dialysable products produced by micro-organisms which do not form any appreciable amount of toxin in vitro, for cultivating species which are difficult to grow by ordinary methods, for studying the phenomena of infection when the micro-organisms are protected from the phagocytes, and for other purposes. A glass rod or small test-tube, according to the size desired, is dipped into a beaker containing the ordinary {not flexible) collodion, is then withdrawn and allowed to dry, and the process is repeated two or three times. In order to detach the collodion from the glass, the whole is dipped for a few seconds alternately into strong spirit and into watei*, the collodion loosens, and may be easily peeled off the glass. The sack may be sterilised by placing in a test-tube and heating to 150° C. in the hot-air steriliser. For the inoculation of animals various methods may be adopted. In some cases, after clipping the hair, the organism may be introduced by rubbing into the skin after scarification, or, a small incision having been made through the skin, a small quantity of a culture may be introduced on a platinum needle ; or a broth 128 Manual of Bacteriology culture or an emulsion, made with sterilised water or broth, may be injected with a sterilised syringe subcutaneously, intra-peritoneally, or into the muscular or other tissues or organs as required, since the seat of -inoculation may have to be varied for the different species to produce their pathogenic effect. For injection purposes a syringe like an antitoxin syringe, i. e. with asbestos or metal piston and glass barrel that can be boiled, may be used. Several sizes are kept in stock, 1 c.c, 2 c.c.j and 5 c.c. at least being required. An all-glass syringe is a still better form, but is expensive. For accurate dosage, the piston-rod should be graduated and have a nut travelling on a screw up and down it. Before use the syringe with the needle should be boiled for ten minutes to sterilise it ; after use it may be well rinsed and again boiled. The needles should be wiped dry and a wire inserted, or they may be kept in a bottle of xylol. Guinea-pigs and rabbits are usually inoculated in the thigh or abdomen ; mice in the dorsal region or at the root of the tail (dorsally), the hair being clipped, and the skin disinfected, but this is not generally necessary. Numerous mechanical holders have been devised for animals, but are not as a rule required. Rabbits may be inoculated intra-venously by one of the large veins in the ear. The ear is shaved, and the skin is well washed with a little alcohol with vigorous rubbing ; the base of the ear is lightly pinched so as to obstruct the venous but not the arterial circulation, and render the vein prominent, and the injection is made with a small syringe fitted with a fine needle, the needle being passed into the vein towards the base of the ear. After the withdrawal of the needle the wound is compressed for a little and may be dressed with some antiseptic wool and collodion. Post-Mortem Examinations 129 The phenomena occurring after inoculation must be noted. Usually these are not very obvious in the rodents, but loss of appetite, sluggishness, staring coat, convulsions, etc., may be observed. The weight of the animal is a good index of what is happening. If the infection is serious, the weight rapidly falls ; if the animal is to recover, its weight soon begins to increase after the preliminary fall. The temperature in the rectum may also be taken, but is not so valuable, as in the guinea-pig variations occur from mere handling or other slight causes. The temperature of the guinea-pig averages 38-6°, but varies between 36° and 39° C. (Eyre). The examination of the dead animal should be carried out with as little delay as possible. For dissection, the body should be pinned out on the back on a board, which may stand in a shallow enamelled iron pan, by pins or nails through the feet, and the abdomen well soaked with antiseptic solution, not so much to sterilise the skin as to prevent the hair from getting into the incision ; to obtain complete sterilisation of the skin, it is preferable to clip or shave the hair and then sear with a red-hot iron. Knives, forceps, scissors, etc., should be well boiled in an enamelled-iron mug or pie- dish, the water being kept boiling during the progress of the dissection and the instruments rinsed from time to time in it. A little sodium carbonate may with advantage be added to the water. A small enamelled- iron fish-kettle with perforated strainer forms an excellent steriliser for instruments, or a surgical instru- ment steriliser may be used. An incision is made and the skm well reflected and pinned out • the knife and forceps should then be re-sterilised, or fresh sterile in- struments taken, for the deeper incision and opening the body cavities ; these again must be re-sterilised, or a third set of instruments employed for incising the organs. 9 130 Manual of Bacteriology During' the progress of the dissection the condition of the tissues at the seat of the inoculation should be noted, and likewise the conditions of the serous membranes and the various organs. hi many diseases the organism is met with most abundantly in the spleen, in others in the blood, and in some at the seat of inoculation. When a systematic examination is made, film specimens and cultures on two or three media, aerobic and anaerobic, should be prepared from the seat of inoculation, the spleen, liver, lungs, and heai't-blood, and in some cases from the serous membranes, muscles, or central nervous system in addition, the carcase being in the intervals covered with a bell-jar which has been rinsed in, or with filter-paper moistened with, antiseptic solution. An assistant is often useful or even necessary. The greatest care must be taken to avoid dropping or splashing or otherwise disseminating infective material, any stains being immediately swabbed up with antiseptic solution ; and the operator must exercise every precaution to prevent the infection of himself and others. It is convenient to have some efficient antiseptic solution near at hand ; it may be kept in a large bottle on a wall bracket and drawn off as required by a syphon tube provided with a tap or spring clip. The most generally used antiseptics are 5 -pev cent, carbolic, and 1-500 corrosive sublimate, but 2 per cent, cyllin or kerol or 3 per cent, lysol is cheaper and more efficient. The access of flies to the carcase must also be guarded against, as they might carry infection. When finished with, the carcase should be efficiently disinfected and disposed of without delay, preferably by burning it, together with the board on which it has been pinned out. If the carcase be left, especially in warm weather, for even a few hours before the examination is carried Bleeding 131 out, the tissues are liable to become invaded and infected by organisms from the respiratory and digestive tracts. In the ■post-mortem room, infection of the tissues is very common ; out of fifty cases, Symes1 found only seventeen to be sterile. Ford states that even in normal animals, killed and immediately examined, bacteria are present in 70 per cent, of the internal organs.2 When the blood of an animal is required several expedients may be adopted. If the animal is not needed for further experiment, it may be decapitated and the blood collected in a porcelain dish ; but if a sample only is wanted, and the animal has to be further treated, as in antitoxin work, it may be bled from the carotid, the vessel being afterwards ligatured. In the rabbit a small artery passes superficially across the inner aspect of the thigh • this permits of the withdrawal of a small quantity of blood without the necessity of an operation such as is required to expose the carotid. The simplest method of all is to introduce the fine point of a piece of glass tubing, drawn out and bent to a convenient angle, or the needle of a syringe, into one of the ear veins and aspirate the blood into 'it Or the vein may be punctured and the blood allowed to drip into a small tube. Blood may be obtained from a patient for the agglutination reaction, for microscopical examination, or for culture experiments, by pricking the finger or the lobe of the ear with a sterile needle, preferably a flat one of the « Hagedorn » type, or with half a steel pen (nib) or a glass point ; for disinfection, the skin may be rubbed with a little alcohol or ether. The blood may be collected in vaccine tubes, small bulbous tubes (Fig. 7, p. 51), or Wright's tubes (Fig. 35, p. 225). 1 Lancet, 1899, vol. i, p. 365. 2 Journ. of Hygiene, vol. i, No. 2, 1901, p. 277. 132 Manual of Bacteriology Organisms, in natural infections in man, are usually present only in small numbers in the blood, and for demonstrating them by culture methods it is necessaiy to withdraw 2—5 c.c. from a superficial or deep vein by means of a sterile syringe under aseptic conditions, and to inseminate broth tubes or agar plates each with ^ c.c. of the blood. Although the modern methods of isolation and cultivation have rendered immense service to bacteriology, they have also had the effect of diminishing the attention paid to the exact morphology and biology of organisms. At the present time there is a tendency to investigate bacteria en masse rather than to study them as individual living forms, and the following remarks by the late Marshall Ward1 may be aptly quoted in this connection : " We must remember that De Bary and Brefeld had aimed at obtaining a single spore isolated under the microscope, and tracing its behaviour from germination continuously to the production of spores again ; and when we learn how serious were the errors into which the earlier investigators of the mould fungi and yeasts fell, owing to their failure to trace the development continuously from spore to spore, and the triumphs obtained afterwards by the methods of pure cultures, it is not difficult to see how inconclusive and dangerous all inferences as to the morphology of such minute organisms as bacteria must be unless the plant has been so observed. As a matter of fact, the introduction and gradual specialisation of Koch's method of rapid isolation of colonies encouraged the very dangers they were primarily intended to avoid. It was soon discovered that pure cultures could be obtained so readily that the characteristic differences of the colonies in the mass could presumably be made use of for diagnostic purposes, and a school of bacteriologists arose who no longer thought it necessary to patiently follow the 1 Nature, vol. lvi, 1897, p. 455 et seq. Irrigation Method 133 behaviour of the single spore or bacillus under the microscope, but regarded it as sufficient to describe the form, colour, markings, and physiological changes of the bacterial colonies themselves on and in different media, and were content to remove specimens occasionally, dry and stain them, and describe their forms and sizes as they appeared under these conditions. To the botanist, and from the point of view of scientific morphology, this mode of procedure may be compared to what would happen if we were to frame our notions of species of oak or beech according to their behaviour in pure forests, or of a grass or clover according to the appearance of the fields and prairies composed more or less entirely of it, or — and this is a more apt comparison, because we can obtain colonies as pure as those of the bacteriologist — of a mould fungus according to the shape, size, and colour, etc., of the patches which grow on bread, jam, gelatine, and so forth." Examination in the Fresh State. One essential procedure in the investigation of an organism is its examination in the fresh and living con- dition. This may be done by placing a droplet of sterile water, broth, or salt solution on the slide, inoculating with a trace of the organism or growth, and covering with a cover-glass and examining microscopically. The action of stains and reagents on the organisms may be observed by the irrigation method. A drop of the stain or reagent (c, Fig. 21) is placed on the slide, A, just in contact with one margin of the cover-glass, B, and is drawn through the preparation by means of a small piece of filter-paper, v, placed on the other side, a torn margin touching the film of fluid at one edge of the cover-glass. The filter-paper absorbs the fluid from under the cover-glass, leaving the cells and other particles behind, and at the same time the reagent on the opposite side 134 Manual of Bacteriology flows under the cover-glass to take the place of the absorbed fluid. Afterwards the excess of the reagent or stain may be washed away by running in water under the cover-glass in a like manner. Care must be taken that no fluid gets on to the upper surface of the cover-glass, which must always be kept dry. The advantage of this method is that it may be applied while the specimen is being examined under the microscope, and the action of the reagent on a particular cell or granule can, with a little care, be watched. If the cells be large and it is desirable to avoid pressure of the cover-glass, a fine hair or bristle may be so placed Pig. 21. — Method of irrigation. on the slide that when the cover-glass is lowered one edge, l'ests on it. If the specimen has to be kept for any length of time, the film of fluid will before long evaporate and the preparation become dry. To prevent this a ring of oil or vaseline may be painted round the margin of the cover-glass so as to seal it to the slide. A simple method for keeping organisms under examination for a lengthened period of time, and of watching their growth and development, is by the use of hanging-drop preparations. To prepare a hanging drop, a ring of vaseline is painted round the margin of the hollow of a hollow-ground slide (or other cell, see below). A cover-glass is sterilised by flaming in the Bunsen, care being taken not to heat sufficiently to Hanging-Drop Preparations melt it, A droplet of some sterile fluid medium — water, broth, wort, sugar solution, etc.— is then placed in the centre of the cover-glass with a sterile platinum loop. This droplet is then inoculated with the organism which is to be observed, care being taken not to add too many organisms — a few isolated organisms and small groups in each field is what should be aimed at. The vaselined cell is now taken and turned over, so that the ring of vaseline is downwards, and is then placed on the cover-glass, in such a way that the droplet is situated in the middle of the hollow, but not touching the slide at any point. The cover-glass adheres to the slide by means of the vaseline, and on quickly inverting the whole, so that the fluid has no time to run, it will be found that the droplet is hanging Fig. 22. — Hanging-drop preparation. from the under sirrface of the cover-glass in a cell which is hermetically sealed by the vaseline, and evaporation is thus rendered impossible (Fig. 22). Such a preparation, in fact, can be kept for a week or ten clays in a warm incubator without drying up. Great care must be exercised in examining a hanging- drop specimen microscopically, especially with the immersion lenses, for the slightest pressure breaks the unsupported cover-glass. It often saves time first to centre the drop with the low power before examining with the immersion lens; an ink or pencil dot at the margin of the drop aids focussing. The light must be di minished by closing the diaphragm, lowering the condenser, etc. (p. 140), and artificial light is generally preferable to daylight. The central parts of the drop only should be examined, not the margin. 136 Manual of Bacteriology Instead of hollow slides, various devices may be employed to form the cell. Metal, glass, or vulcanite rings, or rings cut out of thin sheet lead, tin-foil, card- board, or two or three thicknesses of paper or filter- paper may be cemented on to slides with vaseline, Hollis's glue, gold size, or Canada balsam, or even a thick ring of vaseline may be used. The only certain method for ascertaining whether an organism is motile or not — often an important clue to its identification — is by the use of hanging-drops. Actively motile organisms may frequently be met with in a resting stage, although still alive, and various factors may bring about this condition, such as old age, exhaustion of nutriment, excessive heat or cold, electric shocks, and the like. The absence of movement of an organism in a specimen prepared from an ordinary culture, particularly if more than a day old, does not necessarily prove that it is non-motile. A hanging-drop should be prepared with a nutrient medium (the best, perhaps, is glucose broth) and placed under conditions of temperature, etc., favourable to the growth of the organism, and examined after an interval of an hour or so, or better still at intervals of half an hour for three or four hours. In this time the old cells will revivify, and new ones will have been produced, and if the organism be a motile one, more or less active move- ment of some of the cells is almost sure to be observed. It is necessaiy to beware of two fallacies in connection with motility — not to mistake for it the so-called Brownian movement, which is a vibratory one back- wards and forwards about one point, and common to all fine particles suspended in a fluid; and not to be misled by a flotation of the cells due to currents set up in the fluid from some cause or other — all the particles then tending to move in the same direction. Interlamellar Films 137 Another purpose for which the hanging-drop cultiva- tion may be employed is that of obtaining a permanent record of the various phases through which an organism may pass during its development. If a number of these cultivations be made, sa,y twenty, in an exactly similar manner, and afterwards kept under identical conditions, and if at the end of every half-hour one of the preparations be taken, its cover-glass carefully re- moved, and the droplet dried and stained, a permanent record of the life-history of the organism is obtained extending over ten hours. Various more elaborate forms of cells for hanging- drop pi'eparations can be obtained, some being provided with inlet and exit tubes for the passage of various gases. For anaerobic preparations cells are made having a groove at the bottom into which a mixture of pyrogallic acid and potash is introduced. The observation of hanging-drop cultivations at blood-heat can be carried out on some form of warm stage. Interlamellar films.1 — Another method of investi- gating the life-history of organisms, especially moulds and protozoa, is by means of interlamellar films. A glass slide 1£ by 3 in. is sterilised in the Bunsen flame, and while hot three small drops of sealing-wax are placed on it, so arranged that they form the apices of an equilateral triangle, the side of which measures about one inch, and a drop of sterile nutrient medium is deposited between them. A cover-glass of about li in. in diameter is then sterilised in the Bunsen flame, a droplet of a suitable nutrient medium is placed upon it and inoculated with the organism to be observed, and the prepared cover-glass is picked up with sterilised forceps, inverted, and lowered on to the slide. The 1 Delepine, Lancet, 1891, vol. i, June 13th. 138 Manual of Bacteriology nutrient medium is thus contained between the slide and the cover-glass, and by using a hot wire, and so softening the sealing-wax, it can be spread out to form as thin a layer as desired. The preparation is kept in a moist chamber to prevent evaporation, and can be studied when required. The Microscope. A bacteriological microscope should be of the monocular form, and have a rack-and-pinion coarse adjustment and an efficient fine adjustment. The stage, preferably of vulcanite, should be large and roomy and quite plain, with two or more holes at its margin to receive spring clips for fixing the slide. For the ordinary examination of specimens a mechanical stage is not needed; in fact it hampers that freedom of mani- pulation which is so useful for the rapid examination of a specimen. For some purposes a mechanical stage is very useful, and for a critical survey of the whole of a specimen, e. g. a blood-film, it is essential. A detach- able form is to be preferred (Fig. 23), so that, if required, the stage may be free for the examination of plate cultivations, etc. A sub-stage condenser is essential for all work in which high powers are employed, and also enhances the value of low powers. It consists of a system of lenses below the stage, by means of which the light is concentrated on the object. It should have a rack-and- pinion, or a sow, adjustment for focussing, and be pro- vided with some form of diaphragm for modifying the light, preferably an " iris." The condenser must be centred — that is, adjusted so that its optical axis corre- sponds with the optical axis of the objective ; and for this purpose it is usually provided with two lateral Illumination 139 screws working at right angles to each other, by means of which its position relative to the optical axis can be altered. In order to centre, a diaphragm with small aperture is used, and the hole in the diaphragm is focussed with a low power ; then, by means of the lateral screws, this hole is brought into the centre of the field. Below the sub-stage condenser a mirror with concave and plane surfaces should be fitted, the plane surface being used with the condensei', as a general rule. The Fig. 23. — Swift's detachable mechanical stag-e. concave mirror may be used for illumination with low- power objectives, the condenser being detached or swung out of position. The necessity for careful illumination must be insisted upon • in fact, to obtain the best results the light should be readjusted for every specimen by mirror, diaphragm and condenser, %. e. " critical " illumination should be aimed at. A good specimen may be utterly spoilt, visually, by faulty illumination j while an indifferent one may be made to look passable by proper illumination. In the examina- tion of micro-organisms in the fresh or living and unstained condition, it is necessary, as a rule, to 140 Manual of Bacteriology diminish the light by means of a small diaphragm, or by racking- down the condenser, or by both; while for stained or opaque objects the full aperture of the diaphragm, or thereabouts, may generally be employed. It must be remembered, however, that the resolving power of a lens (see below) is diminished by closing the diaphragm and by throwing the condenser out of focus; the illumination then becomes " non-critical." For fine work, if the illumination is too intense, this should be diminished by diminishing the source of light or by interposing a coloured screen, such as Gifford's, which consists of a cell containing a solution of malachite green in which is inserted a piece of green signal glass. Coloui-ed glass may also be interposed. The microscopist should accustom himself to examine speci- mens both by daylight and by artificial light ; hanging- drop specimens are usually best seen with the latter. For artificial light, probably nothing surpasses a paraffin lamp with flat wick, the edge of the flame being always used, while to obtain the best results the mirror should be removed, and the flame used direct by elevating and tilting the microscope somewhat. For the finest woi'k, daylight illumination is inadmissible. An admirable form of electric lamp is the "Barnard," made by Messrs. Swift & Son, the source of illumina- tion being a Nernst lamp. For ordinary routine work, an incandescent carbon or metal filament electric lamp, a Nernst lamp, or an argand or in- candescent gas burner may be used. Various devices have been introduced for the employment of mono- chromatic illumination, e. g. the quartz mercury vapour lamp by Barnard. With the filament, Nernst, or incandescent gas, lamps, the image of the filament or mantle is troublesome when the condenser is in focus; this may be obviated to Eyepieces and Objectives 141 some extent by the use of frosted bulbs or by inter- posing- a screen of line ground glass, by the use of Gordon's glass rod illuminator, or by interposing a spherical flask filled with water or dilute copper sulphate solution. Incandescent bulbs may be frosted by dipping in a 15 per cent, solution of caustic soda and allowing to dry. Two eyepieces are sufficient, and the lower-power ones are to be preferred, such as the b and c of the Ens'lish, or the 2 and 3 of the Continental, makers. Although increased magnification can be obtained by the use of a high-power eyepiece, it is at the expense of definition, the image losing its sharpness, because the eyepiece magnifies the image formed by the objective, and any imperfections in the latter are made more apparent, so that the use of very high eyepieces is not to be recommended, except with the finest lenses; moreover, as will be pointed out later, it is useless to increase the amplification beyond a certain point. With regard to the length of the tube of the micro- scope, this differs in the English and Continental systems. The standard English tube-length is 8'75 in., the Continental is 6*3 in., and is usually adopted, but the longer tube gives greater amplification. The tube of the microscope is generally provided with an inner, or draw-tube, by means of which its length can be nearly doubled ; this gives increased amplifica- tion, but at the expense of definition, at least with the higher powers which are corrected or adjusted for a definite tube-length. The lenses or objectives must next be considered. For powers higher than the -Un., or thereabouts, it is advisable, for many reasons, to employ the immer- sion system of objectives. With these lenses a drop either of water, in the water-immersion system, or of 142 Manual of Bacteriology cellar oil, in the oil-immersion one, is placed on the cover-glass, and. the objective is racked down so that its front lens touches and is immersed in either the water or oil, as the case may be. It is a good plan then to raise the objective very slightly by means of the coarse adjustment, still, however, keeping it in contact with the drop of water or oil. The observer then, looking down the microscope, very cautiously and gradually racks down again with the coarse adjustment until the object comes into view, and finishes the focussing with the fine adjustment. The fine adjust- ment should only be used after the object has been brought into view by means of the coarse adjustment. After the examination has been concluded for the day, the lens should be cai'efully wij:>ed with a soft rag, or preferably with a piece of soft Japanese paper, to remove the water or oil. If the oil should happen to dry on the lens, it may be removed by wiping with a soft rag or Japanese paper moistened with xylol, quickly drying with another rag or paper. Instead of cedar- oil, a liquid paraffin has also been used. The TV in. (2 mm.) oil-immersion lens is the one usually selected. It combines sufficient magnification for most purposes, with adequate working distance for convenience in using. If expense is not an object, the Zeiss * in. (3 mm.) apochromatic oil-immersion lens is a very fine one for general use. By means of the compensating oculars sufficient magnification can be obtained, Avhile the working distance is greater, the field is largei', and the penetrative power is greater than with the in. lens. The immersion system of objectives has many advantages : the loss of light is less, the distance between the cover-glass and the front of the objective — the working distance, as it is Immersion Lenses 143 termed — is greater, and more can be seen with an immersion lens than with a dry lens of equal magnifying power. This can he best illustrated by means of two simple diagrams. In Fig. 24 let c d represent the surface of a fluid, either water or oil, and let a b be drawn perpendicular to this surface, and cutting it at y. Let r y represent a ray of light proceeding from a rarer medium, such as air, into a denser one, water or oil. As is well known, this ray when it enters either the water or the oil does not continue in the same direction, but is " refracted " or bent nearer the perpendicular r vr o £ Fig. 24.— Diagram to illustrate the refraction of light. a b, the bending being more marked with oil than with water. Thus we may suppose that the direction of the ray in water would be represented by the line y w, and in oil by the dotted line y o. Conversely, a ray of light proceeding from a denser medium into a rarer is bent away from the perpendicular, and the rays w y in water, and o y in oil, would, on emerging into air, proceed m the direction y r. In Fig. 25 (which for convenience is drawn somewhat out of proportion) let s represent an ordinary glass micro-slide, x a, layer of Canada balsam in which the object is mounted and covered with the cover-glass o, while l is the objective 144 Manual of Bacteriology with its front lens. Let the object be illuminated by the ray of light J y; this on entering the glass of the slide and the Canada balsam will be refracted or bent nearer the perpen- dicular and will proceed in the direction y t. Canada balsam, and also cedar oil, produce about the same amount of " refraction," or bending of a ray of light, as crown glass, and hence these three substances — crown glass, Canada balsam, cedar oil — are said to have the same " refractive index," and, consequently, the glass of the slide, the Canada balsam, v V or Fig. 25. — Diagram to illustrate the course of rays of light through an objective. and the cover-glass act as one homogeneous medium, and the hue y t is a straight one. In the first place, let us suppose that the objective l is a dry one, having a layer of air between its front lens and the cover-glass ; then the ray of light, on emerging from the cover-glass into the air, is now bent away from the perpendicular and pursues a direction practi- cally parallel to its former one, represented by the Hue t w, and misses the lens altogether — the lens is unable to take it up. If, however, we suppose that our objective is an oil- immersion one, and that a drop of cedar oil takes the place of the layer of air between the cover-glass and the front lens in the foregoing example, then the glass slide, Canada balsam, Oil-Immersion Lenses 145 cover-glass, cedar oil, an 1 the front lens of the objective form practically one medium ; tlioy all have the same refractive index and produce the same amount of refraction or bending of a ray of light. Therefore the direction of the ray forms a straight line in all these, and the ray passes into the objective as is represented by the broken line t — v. More important still, however, is that which happens to rays which fall on the slide at a very oblique angle. In the same figure (Fig. 25) let e / represent such a ray ; on entering the slide it will be refracted, and its passage through the slide, balsam, and cover-glass may be represented by / 7c. As before, let us suppose that in the first place our objective is a dry one, and that we have a layer of air between the cover- glass and its front lens. In this case, if the angle which / 7c makes with the perpendicular is greater than about 39° or 40°, the ray, instead of' emerging from the cover-glass into the layer of air, is totally reflected by the cover-glass and pursues a course roughly represented by Jc p, so that it never enters the objective. If, however, we employ an oil- immersion objective, with oil instead of air between the cover- glass and its front lens, then, as before, the slide, balsam, cover-glass, oil, and front lens of the objective form practi- cally one homogeneous whole, and the ray e / 7c, instead of being totally reflected, continues its course in a straight line, and is taken up by the objective, as is represented by the dotted line 7c v. Hence we see that the same rays which are unable to enter a dry objective are admitted by an oil- lmmersion one, and that an oil-immersion lens can 'take up rays which fall on the slide at a very oblique angle. In order that these oblique rays may be present, ready to be taken up by the oil-immersion objective, it is necessary to employ a sub-stage condenser. It is only by means of a sub- stage condenser that a "wide-angled cone of rays," as it is termed, is obtained. Hence to make full use of an oil- immersion objective-to "get most out of it "-it is abso- lutely essential to employ a sub-stage condenser, and for the finest work a special " oil-immersion condenser " is employed. 10 146 Manual of Bacteriology It will also be obvious that although a water-immersion objective admits more rays than a dry oue, it does not admit so many as an oil- immersion. It must be pointed out, however, that Canada balsam, or some medium having the same or a higher refractive index, must be used for mounting to obtain the full advantage of the oil-immersion system. The oil-immersion can of course be used for examining objects mounted in water, etc., cedar oil being still used between the cover-glass and the lens. It is to be noted that a dry objective cannot be used as an immersion one, nor an immersion objective dry, as the construction differs in the two cases. Of late " dark ground illumination " has been much employed, particularly for the examination of living objects. In this special condensers are used, the central rays passing through which are " stopped out," so that the object is illuminated only by very oblique rays and appears white on a dark background. A stop has also to be introduced into the objective, and slides and cover-glasses of special thickness together with brilliant illumination are necessary The lenses in the objective are formed by cementing together different kinds of glass in order to correct for " spherical " and for " chromatic " aberration. The rays passing through the margin and the centre of a simple lens are not focussed at the same point, and a distorted image is the result ; this is known as " spherical aberra- tion," while the violet and red ends of the spectrum, being of different ref rangibility, and a simple lens acting like a prism, coloured fringes are observed ; this is termed " chromatic aberration." The apochromatic system of objectives and eyepieces has these defects very perfectly corrected by the use of special glass and fluorite, correction being partly effected in the objective, and this is completed by combination with the special eyepieces. The latter, termed " compensating oculars," Resolving: Power 147 are therefore essential for perfect correction with apochromatic objectives, but can also be used with ordinary lenses. For photographic purposes apochro- matic lenses are far superior to achromatic ones. Apochromatic objectives are, however, expensive, and though advantac/eous are not really necessary for ordinary bacteriological work. In consequence of certain optical principles, the " diffraction " theory, for details of which the reader must refer elsewhere,1 it is useless to increase the magnifying power of objectives beyond a certain point ; for, although the object viewed appears larger, no more details of structure can be made out. The use of the immersion system increases the " resolving power," or the amount of detail which can be seen. Thus, if a number of fine equidistant parallel lines be ruled on a glass plate, it is impossible to see with a dry lens, using white light, more than about 90,000 lines to the inch as isolated lines. If more are ruled they will not appear, and practically nothing is visible. With a water-immersion objective it is possible to see about 120,000 lines to the inch, and with an oil-immersion as many as 146,000 lines to the inch, as separate lines— a clear gain in resolving power in the latter case of about one half over a dry lens 2 As it is necessary, in order to see such fine structures as hues ruled 50,000 or more to the inch must be, to have considerable amplification in addition to resolvino- power, not much is gained, in ordinary work at any rate, by adopting the immersion system for the lower power objectives, such as the |~in. By the physical theory of microscopical visibility, it can | See Carpenter on the Microscope, edited by Dallinger. (Churchill ) 148 Manual of Bacteriology be shown that objects having a diameter of less than about 0-16 fx cauuot be seen with the best optical appliances. If, then, a micro-organism is less in size than this it could not be seen microscopically, and this fact may explain why it is that in certain undoubted infective diseases no micro-organism has yet been isolated. Of the existence of such " ultra- microscopic" organisms we have proof. The finest porcelain filters, such as the Chamberland B, do not allow visible particles to pass through, yet in several instances, if the infective material be filtered through such a filter, the filtrate is still infective. This is the case with the blood serum in yellow fever, Cape horse sickness,1 dog distemper, hog cholera and swine fever, in bird and cattle plagues, and with the juice of bird molluscum. The organism of cattle pleuro-pneumonia is just on the limit of visibility. The rabic and vaccine viruses also seem capable of passing through a Berkfeld V. These experiments do not necessarily prove that the organism in all stages is invisible.2 Siedentopf and Zsigmondy have devised a method whereby ultra-microscopical particles may be rendered visible, but inasmuch as they appear merely as luminous points, it is questionable whether the method will be of great service in bacteriology. There is no real necessity in bacteriological work for the immersion objective to be provided with a " correction collar." The " correction collar " is an additional screw in the objective by means of which the distance between some of its constituent lenses can be altered to " correct " for varying thicknesses of cover- glass, etc., and though necessary with the higher power dry lenses, it is theoretically unnecessary with the immersion system. Nevertheless, as slight variations do occur in the various media, glass, oil, etc., and they may not form a truly homogeneous whole, for the finest work the correction collar is still desirable. So much 1 Journ. Comp. Path, and Therap., vol. xiii, 1900, p. 1. 2 See Roux, Bull, de I'Inst. Past., vol. i, 1903, pp. 1 and 49 Remlinger, ibid., vol. iv, 1906, pp. 337 and 385. Measurement of Organisms 149 for the high-power objectives. As regards the lower powers, which, of course, are dry, a f-in. and a in. are generally selected. The f-in. is a more serviceable lens than the 1-in. which is often recom- mended. A very useful accessory is a " double " or " triple nosepiece." This consists of a light metal framework, which is attached to the lower end of the tube of the microscope, on to which two or three objectives can be screwed. The framework can be rotated, thus bringing each objective in succession into the optical axis of the instrument, and the necessity for unscrewing and screwing on each time an objec- tive is changed is obviated. A microscope such as described, with sub-stage condenser, two eyepieces, a f-in. and a -J-in. dry and a TV-in. oil-immersion objectives, triple nosepiece, etc., complete in case, can be obtained for about £15, and it is well to add another sovereign or two for superior finish. Both British and Continental firms supply microscopes arranged as indicated, and in this department the English makers hold their own. The measurement of micro-organisms is carried out by means of a stage micrometer, alone, or in combination with an eyepiece micrometer. The former consists of a scale of tenths and hundredths of a millimetre or hundredths and thousandths of an inch ruled in fine lines on a glass plate, by means of which the measure- ments can be made by focussing the scale under the microscope. The stage micrometer is placed in position on the stage and the scale is focussed with the par- ticular ocular, objective, and tube length which are to be used. A drawing of the scale is made with a camera lucida ; the micrometer is then removed and the object placed in position and a second drawing is made of the object on the scale already drawn/ A 150 Manual of Bacteriology simpler and less expensive arrangement is to make nse of a disc of glass ruled with equidistant fine lines, which can be placed in the eyepiece by unscrewing the top lens and dropping it on the diaphragm below. The value of the divisions in the eyepiece scale is first ascer- tained by means of the stage micrometer. The stage micrometer is then removed and the object to be measured put in its place, and its dimensions are deter- mined by means of the eyepiece scale. With the eyepiece micrometer, the value of the divisions is first ascertained by means of the stage micrometer, which is then replaced by the object. If the objective or the eyepiece be changed the value of the divisions of the eyepiece scale in both cases will be altered, and must again be determined by means of the stage micrometer. The unit for microscopical measurement is the micron (some- times erroneously termed a micro-millimetre), which measures one thousandth of a millimetre, or t^-J^ of an inch, nearly, and is designated by the sign fx. If a micrometer is not available, rough measurements may be carried out by comparison with, a red blood- corpuscle. The majority of the red corpuscles of normal human blood measure 7-5 p. in diameter. Infection 151 CHAPTER V. INFECTION VEGETABLE AND ANIMAL PARASITES THE IN- FECTIVE PROCESS ANTI-BODIES ANTI-SERA AND ANTITOXINS IMMUNITY. Infection. By the term infection is meant the invasion of the living- tissues by living micro-organisms which grow and multiply at the expense of the host. A disease produced by the growth and multiplication of micro- organisms is termed an infective disease, and is trans- missible in most instances by inoculation. If the micro-organisms are from time to time discharged from the body of the host, either with the excreta, secretions, desquamated particles, or in some other way, the disease becomes infectious or contagious, according to the ease with which another individual becomes injected, and the material which conveys the infection is often termed the contagion. Thus, in scarlatina and smallpox the contagion is very readily conveyed from person to person even for a distance through the air, and these are infectious diseases. Ringworm and syphdis, as a rule, require more or less close contact for infection to take place, and these are, therefore, contagious diseases ; while malaria is neither infectious nor contagions, since persons in the neighbourhood never directly contract the disease, though it can be conveyed by inoculation, and it is therefore infective 152 Manual of Bacteriology only. But the distinction between infectious and con- tagious is mainly one of degree, and these terms have now to a large extent been discarded. Excluding individual susceptibility, the relative infectivity of a disease probably depends on three factors : (1) the contagion is freely given off aerially and is not destroyed thereby ; (2) the contagion gains access by the respira- tory tract; and (3) the relative virulence of the con- tagion ; in some instances the smallest amount of the contagion is sufficient to infect. If the contagion can gain access only through a wound or the digestive tract, the chances of infection may be largely reduced. In certain instances infection is conveyed by an inter- mediary, e. g. the mosquito in malaria, and in such cases infectivity will obviously depend on the presence and abundance of the intermediary. Infection is mani- festly a part of the whole subject of parasitism, which includes the animal and vegetable parasites which develop in the animal body. If, however, the subject of parasitism is considered more closely, it will be seen that there is a vast difference between, say, a condition caused by the echinococcus or by the round worm, in which the effects are largely mechanical and in which relatively little poison is produced by the parasite, and the disease diphtheria caused by the diphtheria bacillus, in which the diphtheria bacilli have little or no action mechanically, but elaborate virulent chemical poisons which cause a general intoxication. Some parasites also may produce a general infection, e. g. anthrax, others only a local infection, e.g. ringworm. Parasites may therefore be divided into infective and non-infective, though there is a series of connecting links between these, and the two groups cannot be sharply separated. The infective parasites are: (1) vegetable micro-organisms, chiefly bacteria,, a few yeasts Phenomena of Disease 153 and some moulds; (2) many protozoa; and (3) a few metazoa, generally worms. The non-infective 'parasites are the animal parasites generally, particularly many worms. The production of the phenomena of disease by pathogenic organisms has been ascribed to (1) the using up of the oxygen which should go to the tissues; (2) the using up of the proteins of the body and of the food ; (3) the effects of plugging of the vessels by the microbes; and (4) the effects of substances or " toxins," having a poisonous action, formed by the microbes. Of these, the first three are quite subsidiary, embolism and thrombosis being perhaps the most important, and the toxins are the chief factors which induce the patho- genic effects. These toxins are substances of a very complex composition, probably allied to the proteins; in some instances they seem to be of the nature of enzymes or ferments, and they are direct products of the bacterial cells. The toxins of most pathogenic organisms, e.g. typhoid, cholera, plague, etc., are more or less integral parts of the bacterial cells ; they are "endotoxins," and are not excreted to any extent into the surrounding medium, but may gain access to it by autolysis of some of the organisms. A few organisms, notably Bacillus diphtheria and Bacillus tetani, produce extra-cellular toxins which are found in the culture liquid. The toxins are classified by Sidney Martin,1 as follows (see also p. 38) : (1) Poisons produced by the digestive or the destructive action of bacteria on proteins in the culture medium. Examples of these are the poisons of the Bacillus anthracis and of the pus-producing staphylo- COCCI • (2) Poisons which are the result of the digestive or 1 Manual of General Pathology, p. 7(i. 1 154 Manual of Bacteriology destructive action of bacteria on proteins, but formed as an excretion (the toxin) of the bacterium. The Bacillus diphtherias is the best example of this. A similar combination of poisons is found in snake-venom. (3) Poisons which are excretions only, such as those pi-oduced by the tetanus bacillus. (4) Poisons which are typically intra-cellular, but which may also be excretory. The poisons produced by the typhoid bacillus, the Bacillus coli, the Bacillus enter itidis of Graertner, and the cholera vibrio belong to this group. The Infective Process. With regard to the pathogenic micro-organisms, or disease germs, Koch laid down the following conditions, which have been termed " Kuril's postulates," which must be complied with before the relation of an organism to a disease process can be said completely to be demonstrated : (1) The organism in question must be present in the tissues, fluids, or organs of the animal affected with, or dead from, the disease. (2) The organism must be isolated and cultivated outside the body on suitable media for successive generations. (3) The isolated and cultivated organism, on inocu- lation into a suitable animal, should reproduce the disease. (4) In the inoculated animal the same organism must be found. To these may be added : (5) Chemical products with a similar physiological action should be obtainable from the artificial cultures of the micro-organism, and from the tissues of man or animals dead of the disease. Modes of Infection 155 (6) Specific serum and other reactions, agglutinative, bacteriolytic, complement fixation, etc., generally obtain- able, under certain conditions, if the blood of the infected person or animal be allowed to act on the specific organism producing the infection. It is true that one or more of these conditions may not be fulfilled in all cases, but on general evidence the disease is classed as infective. Thus the Treponema pallidum of syphilis cannot be cultivated, and the organism of rabies is quite unknown. The modes of infection, or entrance of the infective agent into the body, are varied. The infective agent may enter by (1) the gastro-intestinal tract, e. g. typhoid, cholera, and glanders ; (2) the respiratory tract, e.g. pneumonia and influenza, and occasionally typhoid, plague, etc. ; (3) by inoculution, not necessarily only of the skin, but also of the mucous membranes, e. g. the septic diseases, glanders, tetanus, etc. The extreme infectivity of some diseases — e. g. variola, scarlatina, influenza, etc. — may be due to the fact that infection takes place by the respiratory tract. In certain in- stances the infection is conveyed in some special way, e. g. by mosquitoes in malaria and in yellow fever. Nor is infection necessarily confined to one mode of entrance ; m plague, for example, infection by the skin is com- monest, but it is not infrequent by the respiratory, and may occur by the digestive, tract. The infecting agent may remain localised, giving rise to a local infection, or it may be widespread through the body, a septi- caemia^ or general infection. The absorption of chemical products from a local site of infection may produee general symptoms; this is intoxication, as ■ Septicaemia" and "a septicaemia" have different meanings The former is applied to a general infection with the so-called septic organisms, the latter to a general infection with any organism 156 Manual of Bacteriology occurs in cholera, in which the microbe is limited to the bowel, in the early stage of diphtheria, in which the diphtheria bacillus is limited to the membrane, and in a local abscess. Fever is usually one of the results both of intoxication and of general infection. Infection, if recovery ensues, is usually followed by remarkable alterations in the blood and tissues. One of these is the production of immunity or insuscepti- bility to the same infecting agent; this will be con- sidered later (p. 204). Agglutinins, substances which cause clumping of the infecting organism, are also generally produced (p. 193). Anti-bodies.1 Another remarkable property, and one of consider- able importance in immunity, conferred by the injection into an animal of complex substances, such as bacterial toxins, bacteria, blood-corpuscles, cells and cellular proteins, ferments, etc., is the development of anti-bodies. Thus an animal injected with sub-lethal doses of abacterial toxin, e. g. diphtheria toxin, acquires a tolerance towards the toxin, becomes immunised, and a substance is developed in the blood that an- tagonises the toxin which was injected ; this substance is known as antitoxin. If bacteria be injected, the fresh blood in vitro has a solvent action on the bacteria (bacteriolysis) ; if blood-corpuscles be injected, the fresh blood has a solvent action on the same kind of blood-corpuscles (haemolysis) ; if cells be injected, the blood has a solvent action on the cells (cytolysis), and so on. If ferments be injected, anti-ferments are formed and will prevent the specific action of the 1 All the subjects dealt with in the subsequent portion of this chapter are discussed in detail by Emery, Immunity and Specific Therapy, 1909. Anti-Bodies and Antitoxins 157 ferment. With doubtful exceptions,1 it is only com- plex bodies of protein nature, or allied to the proteins, which give rise to the production of anti-bodies on inoculation ; alkaloids, carbohydrates, mineral poisons, etc., do not give rise to anti-bodies, though some in- susceptibility to them may be produced (see also (jAk^ p. 216). Any substance which gives rise to an ^ anti-body may be termed an " antigen," These anti-bodies, etc., may first be considered, after which ., immunity will be discussed. Anti-bodies are probably formed for the most part in the spleen, lymph-glands and bone-marrow by leucocytes, or by endothelial cells, or by both. Antitoxins. — The anti-bodies produced by the inocn- lation of an animal with bacterial toxins or toxic proteins (e. g. ricin, abrin, ;md snake-venom) are known as antitoxins, and are of considerable practical impor- tance. An animal injected with increasing amounts of the toxin acquires a high degree of immunity, and its blood-serum injected into a second animal confers on the latter a similar immunity against the toxin with Avhich the first animal was injected, but not against other toxins ; the serum is specific. The anti-serum formed by the injection of toxin is antitoxic and not anti-microbic, and the diphtheria bacillus will grow and multiply iri*diphtheria antitoxin. Since, however, such an organism as the diphtheria or the tetanus bacillus produces its pathogenic effects through the toxin which it forms, the an^oxin will counteract the effects of the micro-organism as well as of_its toxin. The neutralisa- tion of the micro-organism,Tiowever, may not be quite complete, a certain amount of local reaction or necrosis ensuing. 1 Ford has described the formation of an anti-body by the injection of a poisonous glucoside derived from fungi. 158 Manual of Bacteriology Antitoxins are prepared by injecting animals — pre- ferably horses, but goats, rabbits, etc., may also be employed — with bacterial toxins or with cultures. With those organisms which produce powerful toxins, such as diphtheria and tetanus, it is customary to grow the organism in a fluid medium so that an active and virulent toxin is obtained. The culture is then filtered through a Berkefeld or Pasteur- Chamber- land filter and the toxic filtrate inoculated sub- cutaneously into an animal, generally a horse, commencing with sub-lethal doses. The dose of toxin can be gradually increased, and concurrently with the increase in insusceptibility the blood-serum acquires antitoxic properties. The treat- ment is tedious, and the activity of the antitoxic serum is largely dependent upon the amount and activity of the toxin injected. The requisite degree of strength having been attained, the horse is bled with aseptic precautions, the blood is allowed to coagulate, and the serum is bottled for use. Antitoxin may be obtained in a concentrated form by " salting out " the globulin constituents of an antitoxic serum (p. 175), and_a=driecL product may be prepared by ej^iporatiiig the serum to dryness in vacuo at 40° C. (10 c.c. serum = 1 grm. dry residue). The mode of production of the antitoxin by the injection of the toxin has been the subject of various theories. By some it has been supposed that the anti- toxin is modified toxin, the modification being brought about by the vital activities of the cells. But the amount of antitoxin produced does not necessaj^y_bear, ^ryTelatioiLtg the quantity of toxin injected. Wood- head records instances in which the amount of antitoxin formed amounted to 40,000 times the equivalent amount of toxin injected, and substances which_Jncreaie_the Side-Chain Theory 159 secretive properties of glandular ce-lls, such as pjjo- carpiue, enormously increase the output, so to speak, of antitoxin. In view of these facts Ehrlich has elaborated his "side-chain theory/' a theory which, whether it be the real explanation or no, has received a considerable amount of experimental support, and has had far- reaching- effects in stimulating- research. Ehrlich believes that the chemical activities which are the manifestations of the vital activities of the living- cell are due to a very large nucleus or chemical molecule Fig. 26. — Diagram to represent the cell with its various com- bining groups or side-chains. (After Ehrlich.) Pig. 27.— First stage in anti- toxin formation. (Black = toxin molecule. (After Ehr- lich.) having- a ring structure, analo^-nns to the benzene ring, and having attached "tolTa" number bt atomic groups or « side-chains." A " side-chain " is an atomic group, a carbon atom of which is linked to one of the carbon atoms in a ring. These atomic groups or side-chains are unstable in nature, and enter freely into combina- tion with other suitable groups should these be pre- sented to them, and thus the physiological activities of the cell, assimilation, nutrition, etc., are carried out (Fig. 26). Now Ehrlich supposes that, antitoxin is merely an excess of cevt^j^c-h^ w]licli are 160 Manual of Bacteriology normally present and subserve some of the ordinary functions of the cell and which have become free in ii . 1 ■ 11 the blood. The antitoxins being specific, by this assumption the difficulty is obviated of supposing that special chemical groups or molecules exist preformed ready to combine with a number of different toxins on the remote chance that some one of these may at some time or other come within the particular sphere of action of one of those groups. Moreover, small amounts of anti-bodies, such as antitoxin, bacteriolysin, agglutinin, etc., are met with in normal untreated Fio. 28.— Second stage in anti- Fig. 29.-Third stage m anti- toxin formation. (After Ehr- toxin formation. Side-chains lich.) beginning to be produced in excess. (After Ehrlicli.) animals and in man. While some have supposed that the small amount of diphtheria antitoxin (equivalent to half a unit or so) present in human blood-serum is due to an infection with the diphtheria bacillus (not neces- sarily an attack of diphtheria), it seems more_rational to suppose that this antitoxin is due to a natural libera- tion of_such side-chains fromjhg protoplasm and that ^rtificlaTl^tntoxin prod^tjonT? merely a ver£j£reat stimulation of this natural process. The toxin molecule, according to Ehrlicli, possesses at least tWfixative atomic groups or side-chains. One Side-chain Theory 161 of these, the " haptophore J£rjm_rj^. conditions the union of the toxin molecule with cell-protoplasm ; the other, the " toxophore group," conditions its toxic action. Similarly, in order that the cell may suffer the full effect of the action of the toxin, it also must possess two receptive groups or side-chains having a maximum affinity for the haptophore and toxophore groups of the toxin ; these may be termed the " receptor " and "toxo- phile " groups respectively (see Fig. 31). The relationship of each fixative group of the corresponding groups — viz. that of the toxin and that of the side-chain of the cell — must be most intimate, and analogous to the relations to each other of a male and a female screw (Pasteur) or of a lock and its key (E. Fisher). The ^genesis of antitoxin-on the " side-chain theory" takes place in the following manner: Toxin being introduced, the haptophore groups of the toxin mole- cules imite with J^e__p^-ticlJar receptor side-chain^ of ^ the protoplasm for which they have an affinity (Fig. 27). By this combination the physiological activities of the cell are mterfered with, a~~dele7t~ is created, the cell is damaged (it is only necessary to consider the case of one cell, or, more strictly of one molecular group of the cell -protoplasm). -But through its recuperative powers the cell soon recovers by the formation of new r^^^JT' chains to takethTplace of those which have hee~7^T out of action. Onjnjecting more toxin, the toxin cojriW^vj^^ receptors and a defectis again created (Fig. 28). Once more the cell re^onds, and a fr|sh^eries__of receptors is developed (Fig. 29) But by this continual stTmulatioiiTa^tt were, the cell cJ*f&2^^ receptors ine*ce«s~ tt^^l2l^^dg^eateci and^iWelf- th6Se re^ptors are ^r^eTlnluch numbers that 162 Manual of Bacteriology they can no longer remain attached to the cell but beoome^frftft in the plasma (Fig. 30). These receytor side-chains, detached from the cell and floating free in ■ the blood-stream, constitute the antitoxin. This excessive production of side-chains after stimulation by repeated injections of toxin is not a phenomenon confined to antitoxin formation, but is a general physiological law , enunciated by Weigert ; as a result of repeated stimu- lation, over-production or hyper-compensation is the rule. ^ Ehrlich has termed the diverse free receptors which UdlUu** » n occur in the body fluids in - r 1 *v\fl various circumstances 'jhap- The existence of both hap- tophore and toxophore groups in the toxin molecule is sug- gested by the following experi- ments. If tetanus toxin be injected into the blood-stream Fig. 30.— Foiirth stage in anti- of an animal it rapidly dis- toxin formation. Side-chain, appears within a few seconds i. e. antitoxin, free in the \ . , .„ blood. (After Ehrlich.) of the m]ection, and even it the animal be at once bled, the blood withdrawn being replaced by fresh blood, tetanus ensues, but not till after the lapse of an incubation period of some hours. The tetanus toxin, therefore, immediately becomes fixed or anchored to the tissues of the central nervous system. Evidently the toxin molecule enters at once into combination with the nerve-tissues by means of its haptophore group ; this after a time brings the cells withm the sphere of influence of the toxophore group, and after a certain incubation period toxic symptoms ensue. The affinity of tetanus toxin for nerve tissues may be 3hown in another way. If tetanus toxin be emulsified Side-chain Theory 163 with fresh guinea-pig brain, the emulsion will be found to be innocuous on injection, owing to a combination between the two having taken place. The cerebral cortex of a highly susceptible animal (e. g. mouse) has a marked neutralising power, of a less susceptible animal (e. g. rabbit, fowl) a feebler, and of an insuscep- tible animal (e. g. frog, tortoise) no neutralising power.1 Moreover, both diphtheria and tetanus toxins may be converted into non-toxic modifications (" tolcolZs7TwTih5ir F?black7 S?ffimanC STCh6m^ t0 reP«»ent the union of toxin mwwixm. . that is to say, according to Ehrlich ' The cornbinaL , *' *' ™* specific and of the ^^^^^^^^^ See Noon, Journ. of Hyg To ^J(Sf*Wean antitoxin an<* toxin. Bordet, Ann. de Vlnst. Past xvii 1903 P" ^ BeSredka an<* 164 Manual of Bacteriology the antitoxin (Fig. 32), and therefore the toxophore groups cannot exert their influence because the toxin is now _j — ' " - — > ^ unable to unite with the protoplasm, its liajjtojfljore or binding groups being already occupied. In a poisonous toxin, such as diphtheria or tetanus toxin, the toxophore group is more readily destroyed than the haptophore group, and by heating a toxin for some time to 60°-70° C. its toxicity is destroyed, but it still retains an affinity for antitoxnn_ If some antitoxin be mixed with such heated toxin it will be found that the capacity of the former for neutralising active toxin is much dimi- nished— in other words, al- though the toxophore groups of the heated toxin have been destroyed, the binding or haptophore groups still remain. Toxin which has Y Fig. 32.— Neutralisation of toxin by antitoxin in the blood. (After Ehrlich.) b^en_ke^t_Joji_sojiu3 time_ decreases in toxicity, but com- retains the power of bining with antitoxin, again showing that haptophore or 'binding groups are present (such derivatives of toxin possessing haptophore groups are termed "toxoids"). Wassernmnn and Bruck have obtained presumptive evidence of the existence of the second stage in anti- toxin formation, viz. the increased production of receptors by the cells. Using tetanus toxin which had been kept for some time and had lost its toxicity, but which still combined with antitoxin— that is, toxoids with hapto- phore groups were still present-they found that on in- iecting it into animals no antitoxin was formed as a result of the injection. They then performed some experiments based on the following line of reasoning : If the old non- Side-chain Theory 165 poisonous tetanus toxin containing these toxoids be first injected into an animal, and after a short interval, some fresh, actively poisonous tetanus toxin, more of the active toxin ought to be required to kill this animal than a normal one, because, owing to the previous toxoid injection, part of the cell receptors susceptible to tetanus toxin are already occupied. Provided Ehrlichias theory be correct, so that this binding of the toxoid really occurs, the conditions should be entirely different, when, instead of injecting the toxin shortly after the toxoid, a longer time elapsed — one to three days — before the injection of the active tetanus toxin. For in that case Weigert's law should come into play and the receptors should have increased in number — i. e. the organism would now possess more sensitive groups than before. This should be manifest by the fact that, in contrast to the first experiment, the fatal dose of active tetanus toxin ought now to be smaller than previously; m other words, a smaller dose should now tetanise the animal in a shorter time. The experiments yielded results which were exactly in accordance with these theoretical considerations. A guinea-pig was injected with some of the non-poisonous toxoid, and then, one hour later, with the active tetanus toxin. It was found that much more toxin was required to kill this animal than a normal guinea-pig of equal size. If, on the contrary, an interval of one to three days were allowed to elapse, it was then found that a close of tetanus toxin which would not even tetanise a normal guinea-pig was sufficient to kill this one. The fact that no antitoxin is formed— i. e. no receptors !,r" fchrust off-by the single injection of the non- poisonous toxin or toxoid Wassermann ascribes to the ack of stimulus which he suggests resides in the fcoxophore groups. 166 Manual of Bacteriology The slow combination of the haptophore and receptor groups has been proved by Wassermann in another way. The researches of Meyer and Ransom have shown that tetanus toxin is absorbed by the nerve-trunks, not by the blood and lymph-channels, while tetanus antitoxin is absorbed by the latter — the blood and lymph-channels. Adrenalin is a substance which strongly contracts the capillaries, and thus tends to block absorption in a particular area. The following experiment was devised: Tetanus toxin and antitoxin were mixed in such propor- tions that the mixture was innocuous to animals, i. e. it was just neutral. If this mixture be injected into the hind paw of a guinea-pig no tetanus develops. When, however, some adrenalin is injected into the hind paw of a similai'-sized guinea-pig, and a few minutes are allowed to elapse so that the capillaries may contract, and then the mixture of toxin and antitoxin is injected, typical tetanus ensues. The explanation of this is that the channel of absorption for the tetanus antitoxin, the vessels, is blocked by the adrenalin, while that for the toxin, the nerve path, remains open. The toxin and antitoxin had not yet combined, or such combination as had occurred is a loose one and becomes dissociated, and, therefore, the toxin travelled along the nerves to the central nervous system, with the production of tetanus. The experiment, however, succeeds only within a certain period, not exceeding an hour after mixture of the toxin and antitoxin, because after this the toxin- antitoxin combination becomes a stable one. If a longer time — say three or four hours — is allowed to elapse, it will be found that, even in the adrenalin animal, no tetanus is produced, because by this time the combination, previously a loose one, is so firm thai the substances can no longer be dissociated. This union can be hastened by employing more tetanus Antitoxin Treatment 167 antitoxin, for with an excess of antitoxin, even after only half an hour, it is impossible by means of adrenalin to free the tetanus toxin. This experiment, therefore, shows that the combination of tetanus toxin with anti- toxin takes place slowly and is at first a loose one, and that the union becomes firmer and firmer with time. It also suggests the possibility of hastening the combina- tion by increasing the amount of antitoxin — a point of considerable practical value in serum therapy. The above considerations are of importance in the antitoxin treatment of disease. Antitoxin, in the strict sense, is not anti-inicrobic, and therefore antiseptic treatment of the throat in diphtheria, and of the wound in tetanus, should be pursued. The fact that the toxophore group of the toxin does not come into action as a rule for many hours at least (an exception is snake-venom) is a fortunate coincidence, for the antitoxin may, therefore, act before tissue damage has occurred. Antitoxin cannot repair tissue damage if this has been produced by the toxin, but it can, and does, prevent the occurrence of further damage by neutralising any fresh amounts of toxin that may be absorbed. Hence the necessity for early treatment. Toxin already anchored to the tissues by its baptophore group may for some time be dissociated from them if a multiple of the simple neutralising dose of antitoxin be injected, and the quantity necessary to accomplish this rises rapidly as the interval between the intro- duction of the toxin and of the antitoxin increases ; hence the necessity for the use of antitoxin in large excess. Probably the union between tissue and toxiu at first is a loose one, and a large amount of antitoxin by mass action transfers the affinity of the toxin from the tissue to itself. An essential condition in antitoxic treatment is the adminis- tration of a sufficient amount of anti-serum, and this does not depend on the actual volume of serum injected. The anti-serum may be regarded as a solution containing a variable amount of the antitoxic or anti-microbic constituent, and for 168 Manual of Bacteriology therapeutic use its strength must be ascertained, and is for convenience described in arbitrary units. The dose of antitoxin is dependent upon the gravity of the disease, and not on the age of the patient, for evidently Just as much toxin may be formed in a cbild^asjnjui ad nit The antitoxins are ^"strictly specific; diphtheria antitoxin, for example, has not the slightest influence in tetanus. To obtain an immediate reaction to antitoxin it should be ^dmiuistej^mtra-venously. A subcutaneous injection may not be completely absorbed in less than thirty-six hours. In cases of mixed infection, e. g. where diphtheria bacilli are associated with streptococci or staphylococci, the diph- theria antitoxin will have no influence on the streptococcic or staphylococcic infection. The complications and accidents of antitoxin treatment are few and usually unimportant. Abscess and other local troubles at the seat of inoculation should not occur if proper antiseptic precautions be taken. Urticaria or other rashes and joint pains are by far the most troublesome com- plications. These are due to the injection of foreign serum, and not to the antitoxin, for the serum of an untreated horse produces a like effect. Repeated injections of serum at short intervals may be continued for a long period without inducing more disturbance than that caused by one or two or a few injections, but if twelve days or more elapse between • two injections a condition of " supersensitation," due to anaphylaxis, ensues (see p. 176). This consists in the rapid appearance of rashes, joint pains, pyrexia, etc., or even of grave symptoms, faintness, vomiting, dyspnoea, convulsions, collapse, etc. Anti-sera may be used as prophylactics, but the immunity produced by them does not last more than three weeks. Various hypotheses1 have been advanced to explain » See Craw, Proc. Roy. Soc. Land., B. vol. lxxvi, 1905, p. 179 ; Journ. of H„a , vol. vii, 1907, p. 501 ; and ibid., vol. ix, 1909, p. 46 ; Arrhcnus, fmmuno-c?lem^r1/j1907;MadsenJ B;it. Med. Journ ^1904, vol. n, p. 567 ; Bordet, Ann. de I'Inst. Pasteur, xvii, p. 161 3 MoKendrick, Proc. Boy. Soc. Land., b, vol. lxxxiii, 1911, p. 493. Toxin —Antitoxin Reaction 169 the manner in which toxin is neutralised by antitoxin. Roux and Buclmer suggested that the antitoxin in sonic way renders the cells and tissues insusceptible to the toxin and Buclmer performed experiments showing that while mice are more susceptible than guinea-pigs to tetanus toxin, a tetanus toxin-antitoxin mixture which is just neutral for mice is distinctly toxic for guinea-pigs. To explain this Ehrlich suggested that there may be present in a toxin solution, several toxic substances, some of which exert a toxic action on the guinea-pig* but not on the mouse. Madsen and Dreyer showed that a mixture of diphtheria toxin and antitoxin which is inno- cuous to guinea-pigs on subcutaneous inoculation is lethal to rabbits on intra-venous injection, and in order to explain this Ehrlich made a similar assumption. Mor- genroth, however, has shown that the difference in the latter case depends on the mode of injection. The reaction between the toxin and antitoxin takes time to complete : there is an interval probably of some hours at 20° C. before equilibrium is reached (see also p. 171). Wh en a recently prepared mixture of toxin and antitoxin is ^injected subcutaneously, absorption is slow an rl in the meanwhile the toxin and antitoxin combine, but when the mixture is injected into the veins, the toxin is fixed by the tissuesbefore it has had time to combTnTVith the antitoxin, and poisonin^ensues. If the mixture be kepfc for some hours beforejnjection, intravenous in- jection is then innocuous. Ehrlich concluded That diphtheria toxin is neutralised by diphtheria antitoxin much in the same way as a strong base is neutralised by a strong acid, and that the course of neutralisation suggests the presence in the toxin of several toxic and atoxic substances (toxoids and fcoxones), all of which combine with, though they have different affinities for, the antitoxin 170 Manual of Bacteriology Arrhenius and Madsen, however, believe that the toxin-antitoxin reaction is analogous to the action of an acid on an alcohol, and that the chemical laws of mass action apply equally to the two. The chief reaction is considered to be between two substances only, toxin and antitoxin, that it is reversible, and that when the system lias reached equilibrium, a fraction of toxin and also of antitoxin remain free, this fraction of toxin producing- the " toxone effect" (see p. 172). If equiva- lent quantities of acetic acid and alcohol are mixed, the reaction is never complete ; the acid and alcohol never entirely disappear, because the water formed reacts with tiit' ethyl acetate, re-converting- it into acid and alcohol. Such a reaction is termed reversible, and this particular case could be thus represented : CH^.COOH + C3H - .OH;CH3.COO02H5 + H20. Bordet has suggested that the fixation of toxin by antitoxin is an adsorption phenomenon, similar to the fixation of a dye by a tissue. These hypotheses may now be examined more in detail. Bhrlich's experiments 1 on diphtheria toxin seemed to show that the neutralisation of toxin by antitoxin follows the laws of simple chemical combina- tions such as the neutralisation of a strong base (NaOH) by a strong acid (HOI). If so, it would be expected that antitoxin would neutralise proportionate amounts of toxin ; but this is not so, and Ehrlich was forced to the conclusion that toxin is a complex mixture of proto-, deutero-, and trito-toxin, and toxone, with different toxicities and different avidities for antitoxin. More- over when toxin is kept it decreases in toxicity, thong b still retaining much of its avidity for antitoxin. Ehrlich 1 See Trans. Jenner Inst. Prev. Med., vol. ii, p. 1 ; Croonian Lect., Boy. Soc. Land., 1900 ; and p. 293. Toxin— Antitoxin Reaction 171 assumed, therefore, that the toxin becomes transformed into substances termed toxoids, which are non-toxic but retain their affinity for antitoxin. This he explained as due to destruction of the unstable toxophore groups, with the retention of the more stable haptophore groups. That the neutralisation of toxin by antitoxin is due to a chemical combination between the tAvo seems to be proved by the work of Martin and Cherry. Brodie,' and Martin and Cherry,2 making use of a Chamberland filter, the pores of which had been rendered very fine by saturating with gelatin, found that toxin would pass through such a filter but that antitoxin would not, presumably because the molecule of the latter is larger. By mixing diphtheria toxin and antitoxin in such propor- tion that the latter was in sufficient quantity to neutralise the toxin, and subjecting the mixture to filtration through a gelatin filter, the filtrate was found to be non-toxic. Now since toxin can pass through such a filter, the inference is that the toxin has combined with the antitoxin. Using snake-venom and its anti- serum or anti-venin, another method was employed. 1 he anti-venin is destroyed by heating to 68° C for ten minutes, while the toxic properties of the venom are unaltered by this treatment. By making mixtures of venom and anti-venin, and, after a certain time has elapsed for the interaction to take place, heating to M U for ten minutes, it was found that the mixture is non-toxic pointing to the combination of the toxin (venom) with the antitoxin (anti-venin). Calmette had performed the same experiment but with a different result finding his mixtures still kmc after heating. Calmette however, treated his solutions almost imme- diately after nnxing, and Martin and Cherry point out > Joum. of Path, and Bad., 1897, p. 460. 1'roc. Roy. Soc. Lond., vol. lxiii, 1898, p. 420. 172 Manual of Bacteriology that a certain time must be allowed to elapse for the interaction to take place, and noted that moderate warming hastens it, as is the case with all chemical interactions. For instance, they found that one mixture of venom and anti-venin allowed to interact for two minutes, five minutes, and ten minutes before heating, killed the animals in thirteen hours, fifteen hours, and twenty-three hours respectively (the control animal with the same dose of venom died in nine hours), but after fifteen minutes the same mixture rendered the animal ill but it survived, while after thirty minutes no toxic symptoms ensued. At one time it was stated that by electrolysis of toxin small amounts of antitoxin are formed, but this is very questionable. Electrolysis destroys the toxicity of toxins by the production of acids, chlorine, and hypochlorites. Ehrlich's views have been opposed, principally on physico- chemical grounds. Thus, Danysz observed that if ricin or diphtheria -toxin be brought into contact with its corresponding anti-body, the degree of neutralisation depends on the manner of mixture. If the toxin be added to the antitoxin in two fractions, allowing a considerable time to elapse between the additions, the mixture contains a much larger amount of free toxin than is the case when the whole (and same) amount of toxin is added at once to the antitoxin. This phenomenon, known as the " Danysz or toxone effect," seems inexplicable if toxin and antitoxin have relations the same as a strong base and a strong acid. Arrhenius, Dreyer, and Madsen maintain that the pheno- mena observed in the toxin-antitoxin reaction are explicable on the hypothesis that the rate of reaction— avidity— of the toxin decreases as antitoxin is added, that the interaction is a slow one, and that different fractions of the toxm are pro- gressively neutralised by the added antitoxin, but more and more slowly. On these grounds they consider that there is no reason to regard the diphtheria poison as a highly com- Toxin— Antitoxin Reaction 173 plicated body. Whereas Ehrlich considei*s the toxin and antitoxin to combine with great avidity, analogous to the combination of a strong base with a strong acid, e. g. NaOH with HC1, these critics believe the avidity of antitoxin for toxin to be feeble, analogous to the combination of ammonia with boric acid, in which as more and more acid is added, the amount of free ammonia decreases, but more and more slowly, in correspondence with a hyperbolic curve. The phenomena can be calculated according to the law of " mass action," there being an equilibrium between — Free KH3 Free H:AB _ K (N~H_i~H.2Q.iBy~ vol. vol. vol. where K is the constant of dissociation. The curve of the neutralisation of tetanolysin by anti-tetanolysin corresponds almost exactly to the ammonia-boric-acid curve. Whereas on Ehrlich's views the combination of toxin and antitoxin would be represented by a straight line, and the crude toxin seems to be composed of a whole series of different toxins and substances having an avidity for anti- toxin, on this hypothesis, although the greater part of the toxicity of toxin is removed by the antitoxin, the latter must be added in large excess before the toxicity completely dis- appears, and the course of neutralisation would be represented by a hyperbolic curve. In fact, as the antitoxin is added, the amount of free toxin diminishes but never completely dis- appears. There conies a point, of course, when the amount of free toxin is so small as to be negligible and cannot be recognised by the ordinary indicators (blood-corpuscles, animal tests, etc.). This hypothesis would explain the fact that while a certain amount, V, of a mixture of toxin and antitoxin is innocuous to an animal, a multiple of the dose, n V, of the same mixture may be toxic ; it would also explain Buchner's experiments alluded to above (p. 169), and Eoux's experiments in which a toxin-antitoxin mixture in- nocuous to normal guinea pigs was toxic to guinea-pigs whose resistance had been reduced by injections of the Massowah vibrio. 174 Manual of Bacteriology Nernst has questioned from the mathematical standpoint the validity of the views of Arrhenius, and so has Craw from much experimental work on agglutination and on the inter- action between megateriolysin and anti-megateriolysin ; Craw also considers that there is some doubt attaching to Arrhenius 's calculations. According to Craw, the two sub- stances most thoroughly investigated by Arrhenius and Madsen, diphtheria toxin and tetanolysin, do not admit of sufficiently exact determination, the former because of the uncertainty attaching to animal experiments, the latter because tetanolysin is a most unstable body. Woi-king with a more stable substance, megateriolysin, he holds that the Arrhenius and Madsen equation does not apply. Again, on the addition of a small amount of antitoxin to toxin there is no decrease in toxicity (as noted by Ehrlich and attributed by him to the presence of toxoid) as there should be, and Arrhenius was thus forced to the conclusion that a second substance, epitoxonoid, is present with the toxin in diphtheria toxin. Craw denies that the toxin -antitoxin reaction is reversible, believes that antitoxin must be regarded as a colloid (and is not in true solution), that the mixture therefore is heterogeneous, not homogeneous, and that the chemical law of mass action is not applicable. On the other hand, Craw maintains that the phenomena of the toxin-antitoxin reaction, including the Danysz effect, have their counterpart in adsorption phenomena, such as occur in the staining of paper, porcelain, etc., with anilin dyes, in the absorption of substances by colloids, etc. Thus, when solutions of arsenious acid are shaken up with colloidal ferric hydroxide, a portion of the arsenic is taken up by the ferric hydroxide and a portion remains in solution. Moreover, more arsenious oxide is taken up by the ferric hydroxide from dilute than from concentrated solutions ; this has its counterpart in agglutination. Again, when an antitoxin is added to a toxin in just sufficient amount to produce a non-toxic solution, the amount of toxin which must then be added to constitute a fatal dose is greater than the minimum lethal dose without antitoxin. This is also found to be the case with ferric Concentration of Antitoxin 175 hydroxide and arsenious acid ; if ferric hydroxide and arsenious acid are mixed so as to form just a non-toxic mixture, the amount of arsenious acid which must then he added to render the mixture toxic is greater than the toxic dose of arsenious acid.1 Arrhenius2 replied to Craw's criticisms maintaining the correctness of his own interpretation, and Craw3 has again maintained the validity of his views, so that the final settle- ment of these divergent opinions must be left for future research. The antitoxic constituent of antitoxin seems to be a protein body, probably allied to globulin, and, as already mentioned, the globulin content of the blood of an animal treated for antitoxin production increases in some cases. Tizzoni, by precipitating the antitoxic serum by saturation with magnesium sulphate at 30° C, obtained the antitoxin in the precipitate. By partial saturation of antitoxic serum with ammonium sulphate, the antitoxin is carried down with the second precipitate, that is, with the pseudo-globulin fraction. It is thus possible to concentrate antitoxic serum and to make use of a weak serum, which would otherwise be inconvenient on account of the volume necessary to inject in order to introduce the requisite amount of antitoxin. For this purpose various salts have been employed for saturation, ammonium sulphate (Pick and others), magnesium sulphate (Dieudonne), mixtures of sodium and potassium chlorides (Atkinson), etc. Dzergowski and Predt&chensky* have elaborated a very exact method by which they state that the whole of tlie anti- toxin can be concentrated and recovered from a comparatively weak serum by means of precipitation with ammonium sulphate. 1^ ^^y>/hysicf,l Chemistry and its Applications in Medical and Bxoloyical Science, 1905. 2 Journ. of Hygiene, vol. viii, 1908, p. 1. 3 Ibid., vol. ix, 1909, p. 46. 4 See Hewlett's Serum Therapy, 1910, p. 68. 176 Manual of Bacteriology Anaphylaxis. — An animal usually becomes more and more tolerantto injections of an antigen, e. g. to diph- theria and tetanus toxins in the preparation of the corresponding antitoxins. Sometimes, however, the opposite effect is produced, viz. increased sensitiveness. This has been noticed in the preparation of tetanus antitoxin ; after the animal has received_a, few dosps of the toxin without ill-effect, a smaller dose of toxin may cause tatai tetanus. ^"The tuberculin reaction (p. ^321) is another example; tubercle toxins circulating m the tuberculous individual render him peculiarly sensitive to a minute dose of tuberculin (i. e. tubercle toxin) which in a normal_rjerKon produces no effect. This condition of hypersensitiveness is known as ' ana- phylaxis ' (i. e. the opposite of ' prophylaxis '). Probably any antigen under pnrtirnln.r p»n^i*.;rm<=! may induce anaphylaxis, but the phenomenon has been especially studied in connexion with serum injections, though other proteins, e. g. egg-white, similarly cause it. The injection of an anti-serum usually produces no ill-effect other than the rashes, joint pains, and pyrexia already mentioned, even if large amounts of the serum be given extending over days or even two or three weeks, but a second injection of serum given after a first injection with an interval of twelve days or more between the two injections is Jia'BTeTo^beTollowed by effects which may be more or less serious, constituting the so-called " serum disease/' or immediate or accelerated reactions, " supersensitation," may ensue (see p. 168). r The symptoms of the serum disease are nausea and vomiting, small and rapid pulse, faintness or more serious heart failure, dyspnoea with rapid and shallow respiration and feeling of suffocation, collapse, rigors, convulsions, and even coma. The severity of the symptoms varies in different cases, and the symptoms Anaphylaxis 177 usually pass off in tlie course of an hour or two ; but a few fatal cases have been recorded. In the immediate reaction, rash, pyrexia, joint pains, vomiting-, rigors, and occasionally convulsions and collapse occur, generally within six hours after the second injection of serum. In the accelerated reaction, these phenomena appear between the eighteenth hour and the fifth day after the second injection of serum. The immediate and accelerated reactions may occur a long time after the first course of serum treatment if more serumjbe given. Groodall records one case in which over four years elapsed between serum treatments for first and second attacks of diphtheria, an accelerated reaction occurring after the reinoculation for the second attack. The amount of serum given does not definitely in- fluence the result The remarkable features of the phenomenon are— (1) they do not occur unless anjnterval of about twelve days or more elapses between the two injections of serum; (2) the long period which may intervene between the two injections of serum and still b5-2££ouirjanjedjDy^^ (3) the serious nature of the condition in some instances. The explanation of the phenomenon is difficult. Un- doubtedly the symptoms are due to some substance in the serum which has a toxic action, and have nothing to do with the antitoxic constituent, for normal serum produces the sams ftflW.s ' In experimental anaphylaxis produced in animals by the injection of normal serum, it is found that the con- dition only occurs if the two doses of serum are separated by an interval of about twelve days or moveS1*^ Moreover, the two injections must be of the same serum or other protein; thus a first injection of horse, serum followed by a second injection of rabbit^eriuxu would ^producejt. Extremely small doses of serum will 12 178 Manual of Bacteriology also bring it about; and lastly, anEesthetisation. when the second dose of_aeiJiiaia^3yen,23^vgiits the develop- ment of tlia-ayB4>toms— a. very extraordinary result. The Arthus phenomenon occurs when a guinea-pig receives several doses of normal horse serum at intervals of some days. Another injection of horse serum then causes an (edematous mass, an aseptic abscess, or an area of necrosis at the site of the new inoculation, which may be far removed from the region of the previous inoculations, and the animal becomes cachectic and dies. The ^Theobald Smith ph enomenon occurs when a guinea-pig has been sensitised hj a very small single dose_of normal horse serum, 0*01 c.c., O'OOl c.c., or even O'OOOOOl c.c. ; if, then, after an interval of twelve to fourteen days a somewhat larger dose of serum, 0T c.c, be given, the serious symptoms of hypersensi- tiveness develop within a few minutes, viz. respiratory failure, paralysis, clonic spasms, and frequently death. The symptoms are generally much more serious when the primary dose of serum is minute than when it is larger, e. g. one or more cubic centimetres. Various hypotheses have been advanced to account for anaphylaxis. Besredka believes that anaphylaxis is caused by the presence of two substances in the serum, one ther- mostable and having the properties of an antigen (see ]). 157), which he terms " sensibilisogen." and which on injection produces its anti-body, " sensibilisin." The other substance is thei'molabile^ and is termed "anti^ sensibilisin," and combines with sensibilisin whenever it meets with the latter. Sensibilisin is particularly fixed by the cells of the nervous system, and, according to Besredka, it is the violent reaction between anti- sensibilisin and sensibilisin in the nerve tissues which causes the serious disturbance characteristic of ana- Anti-Microbic Sera 179 phylaxis. When, therefore, a small close of serum (y-j-y-g-'y c.c.) is administered, the sensibilisogen slowly forms sensibilisin. If a second dose of serum is given twelve days or more after the first injection, the anti- sensibilisin in it combines with the sensibilisin formed by the first injection, and disturbance results. The reason why a large primary injection of serum (e. g. 3-5 c.c.) gives rise to much less disturbance with the second dose of serum than a small primary injection does is that the large amount of antisensibilisin present in the serum combines gradually with the sensibilisin as this is in process of being formed (i. e. in the pre-anaphylactic stage), and therefore there is compara- tively little sensibilisin left for the antisensibilisin present in the second dose of serum to combine with, hence the disturbance caused is much less. The j-eason Avhy anaesthetisatioii with ether when the second injection is given prevents the symptoms of anaphylaxis developing is, according to Besredka, that the anEesthetic renders the nerve cells insensitive tn the reaction between the sensibilisin and antisensibilisin. Anaphylaxis, supersensitation, or hypersensitation may be of considerable importance in serum treatment. On the serum disease, supersensitation, and anaphylaxis, see Hewlett Serum Therapy, ed. 2, 1910 ; Eosenau and Anderson Journ Amer. Med. Assoc., 1906, p. 1007; Yon Pirquet and Schick, Die Serum-Krankheit, 1905; Eichet, Ann. cle VInst Pasteur xxi, P. 497; Besredka, ibid., p. 950, and Bull, de lIf-P^eur,,n 1909, p. 721; Currie, Journ. of Hygiene, voL vn, 1907, pp 35, 61, and vol. viii, 1908, p. 457; (Lnbauni tbid., vol. vm, 1908, p. 9; Goodall,^., vol. vii, 1907, p. 607 --J^MmoBj^^.^H an animal be . -ected wifch increasing doses of bacteria, care being taken to keep below a lethal one, the animal gradually becomes ycuBtomedJo the microbe, and ultimately acquires a 1 "Pi If t Jatt stit 180 Manual of Bacteriology high degree of immunity, so that it is unaffected by amounts which would infallibly kill an untreated animal. Moreover, the blood-serum of such a treated animal, if injected into a second animal, will protect the latter against a few lethal doses of the microbe, but not against a large amount. Nor is the protection afforded pi'oportional to the amount of serum injected ; for example, if 0-005 c.c. of anti-cholera serum will protect against 5 mgrm. of living cholera culture, three times as much, or 0-015 c.c. of the serum, will not protect against 15 mgrm. of cholera culture, and when a certain _ dose of the culture is reached no amount of serumjvilL save_the animal. The mode in which the serum acts may be studied microscopically. If cholera anti-serum 1, and cholera, culture be injecJ^ejlJn^o_J^ cavity of a guinea-pig, and if the peritoneal contents be examined at short intervals afterwards, it will be found that the vibrios first lose their motility, then become distorted and globular, undergo, solution, and finally disappear. The protection afforded by the anti-serum is therefore due to the destruction of the microbes by solution, the process being known as h^mjnlysisfj and the bodies which bring it about being "to^Td " baoterioly sins." The reaction is known as "Pfeiffer's phenomenon" or reaction, from its discoverer. If the serum and the microbes be mixed in. vitro the latter are unaffected ; apparently, therefore, some con- S^Tof the Jivivg body in addition to the anti-serum Ts"7e7e~ssary for the solution of the microbes. But m 1895 Metchnikoff showed that the reaction will take place in vitro provided that some of thejre^eritoneal exudate of a normal guinea-pig be added to the Sure of anti-serum and microbes. The same year Bordet found that the addition of the peritoneal exudate i See Gruber, "Harben Lectures," Jown. State Med., 1902. / Amboceptor and Complement 181 is unnecessary provided the anti-serum be perfectly fresh. These experiments prove that the solution of the microbes is brought about by the interaction of at least two substances, one of which is present in all fresh serum and in the living body, but is unstable, disappearing on keeping or heating the serum, the other is a relatively stable body produced during the process of inoculation. The former, the ^unstable normal body present in all ./Y ^f} animals, is usually termed " complement" (Ehrlichf tiM^U^*^** and Morgenroth), "alexin" (Buchnerand Bordet), ovl QtJ^X/^iM' " addiment" ; while the stable constituent produced byl^jp^oitti^ immunisation is known as the " amboceptor " (Ehrlioh),** " immune body," "intermediary," "preparer" (Gr'uber), "fixate lit" (Metchnikoff), or "substance sensibilisatrice" (Bordet). These considerations suggest an explanation why anti-microbic serum neutralises but a limited amount of living culture, viz. the_ amount of complement present in the body ar, one time is limited, and when this has been used up bacteriolysis ceases. Anti- microbic sera are relatively inefficient in practice, insufficiency of complement being suggested as the reason. Attempts have been made to supplement the complement present by injecting Jj-esh normal serum . with the anti-sm-mn, Jbut without success, and some anti-microbic sera, e. g. anthrax serum, are not bac- ^ teriolytic ; this explanation is, therefore, unsatisfactory. Deflection of complement* (p. 185) may occur in some instances, or the complement may not be of the right kind. In other cases, the organisms in certain situations may be inaccessible to the blood-stream and to the anti-serum, e. g. in the bowel in cholera. The amboceptor or immune body seems to link the complement to the bacterium (Fig. 33); complement re- mains Free if the appropriate amboceptor or immune body is not present, and bacteriolysis does not ensue (see also p. 1 182 Manual of Bacteriology 188). Complement is thermolabile. i.e. it is jlcstroyed by heating* f.o nl>0C.for thirty minutes; while the amboceptor is thermostable, i. e. it is not destroyed by this treatment. According* to Ehrlich, fresh serum contains numerous complements which are more or less specific for different amboceptors (see also note, p. 189). When the com- plement is destroyed by heating* it is converted into " p.rmiplftTppntnid " (analogous to toxoid). Both com- plement and complementoid on injection give rise to anti-complement. The amount of complement in different sera varies considerably; horse serum contains very little, ffuinea^ pig serum much. Complement itself probably" consists of two drrft^^ •! RJ portions. f^*5 J — 1 I ! Pfeiffer'a reaction is of con- siderable value in practical bacteriology for the exact re- cognition of bacterial species. A mixture of an emulsion of the organism to be tested with a Fig ,33.-Diagramtoshow }] ntity of serum from the union between com- 4ucl"1 J _ plement (black) and a highly immunised animal is protoplasm of call by iniected into the peritoneal cavity means of the amboceptor ' J -U. — ; — — (white). (After Ehrlich.) of a normal guinea-pig, lhe fluid in the peritoneal cavity is then examined microscopically half to one hour after the in- jection, and if the reaction be positive the organisms will be found in all stages of degeneration, being mostly con- verted into spherules. In this case, according to Pfeiffer, the organism is to be regarded as belonging to the same_ specTeTas that by means of whighjhe immunisation of the iuiiiaal, from which the blood-serum was obtained, was carried out. If, on the other hand, the reaction be negative, the organisms are ujiafiEected after being in the Pfeiffer's Reaction 183 peritoneal cavity for an hour or so, and the organism is then considered to be a species different from that used for the immunisation. Thus, Pfeiffer's reaction may be made use of to differentiate the cholera-like vibrios from true cholera vibrios and the members of the typhoid-colon group~~from one another. The destruction of the bacteria by bacteriolysis is regarded by some as being brought about by osmotic - changes, by others by processes analogous to digestion. ' During bacteriolysis the specific immunising substances and anti-bodies are used up, and for the lysis of a given quantity of bacteria a certain amount of immune serum is necessary, while after lysis has taken place the latter loses the power of dissolving bacteria. The /L-A JLC/O^" same holds good for hemolysis, and the facts relating to bacteriolysis and haemolysis are almost interchangeable. Anti-endotoxic sera. — The comparative inefficiency of anti- microbic sera, particularly typhoid, led Macfadyen to attempt to prepare sera with microbial endotoxins, and. the work has been continued by Siidmersen and the writer. The method was to immunise horses with the endotoxin obtained by the method described at p. 40. With a typhoid serum so pre- pared G-oodall and the writer obtained promising results.1 Method of applying Pfeiffer's reaction.— For Pfeiffer's test, the organism must be virulent, and a high-grade immune serum is necessary. If the organism is_not virulent, it is spontaneously destroyed in the peritoneal cavity without the addition of immune serum. The method may be best explained m the case of a vibrio supposed to be the cholera vibrio. The cholera-immune serum (obtained from a horse repeatedly injected with cholera culture) should possess a titre of not less than 0-0002 c.c, i. e. this amount of serum mixed with one loop (2 mgrm.) of an eighteen-hour agar cholera culture (virulent), suspended in 1 c.c. of broth and injected into the peritoneal cavity of a small guinea-pig ' Proc. Boy. Soc. Med., vol. ii, 1907-8, Med. Sect., p. 245 et seq. 1 184 Manual of Bacteriology should cause granular defeneration and bacteriolysis of the vibrios^within one hour. Four mixtures are made — (a) one loop of an eigli teen-hour agar culture of the vibrio to be tested, O'OOl c.c. cholera- immune serum, suspended in 1 c.c. of broth ; (b) the same as (a), but 0-002 c.c. cholera serum; (c) the same as («.), but O001 normal serum of an animal of the same species as that furnishing the cholera serum ; (d) one quarter loop of the vibrio in 1 c.c. of broth, as a control of the virulence of the culture. These mixtures are then injected into the peritoneal cavities of four guinea-pigs each of about 250 grm. weight. At intervals of thirty and sixty minutes hanging-drop pre- parations are made of the peritoneal fluid of each animal, the fluid being obtained by inserting a capillary pipette through a minute incision in the skin. In the guinea-pigs injected with (a) and (b), if the organism be cholera, the vibrios should show marked degenerative changes within sixty minutes, while (c) and (d) will show plenty of active vibrios. If the organism be noii-virulent. two methods may be adopted for applying the Pfeiffer reaction. The first,_a micro- scopical or direct method, is carried out by microscopical examination of hanging-drop specimens of the organjsm_ suspended in a drop of the immune serum to which a trace of fresh peritoneal fluid (complement) is added. If the organism is homologous with the immune serum, the bacteria are soon transformed into granules. Controls are put up at the same time with a known strain of the organism with (1) its homologous immune serum + complement; (2) non- immune serum of the same animal + complement ; also of the organism being tested with non-immune serum of the same animal + complement. The peritoneal fluid may be obtained by injecting 3-4 c.c. of broth into the peritoneal fluid of a o-uinea-pig and four hours later withdrawing the fluid (now turbid with leucocytes) and centrifugalising, or allowing it to stand on ice for twenty-four hours. In the second, or indirect, method, the organism is used to prepare an immune serum by injecting an, ajdmal (e. g. a rabbit) with it, and the immune serum so prepared is tested Deflection of Complement 185 on a know" vimh?nt stain in the peritonea^ cavity of guinea- pigs in order to ascertain whether or no it brings abouT Dacteriolysis, i. e. the Pfeiffer phenomenon. Deflection, deviation, diversion or blanking of complement. — Pfeift'er in 1895 observed that a large amount of immune serum might not protect an animal from the cholera vibrio, while a smaller amount with the same dose of vibrio did so. In 1901 Neisser and Weehsberg demonstrated an analogous reaction in vitro. They studied the effect of a bacteriolytic immune serum when varying amounts of the inactivated serum were employed. The quantity ranged from 0-0005 c.c. to 1 c.c. To each of these amounts constant volumes of normal serum and bacterial suspension were added. No bacteriolysis occurred when large and small amounts of immune serum were used, b\it withTmediuni amounts bacteriolysis yvas com- plete. They explained this anomalous reaction, the absence of bacteriolvsis with Fig large. Diagram to represent amounts of immune- serum, as follows: When the ambocerj- tors are in large excess, a portion combines with the com- plement, leaving some ambo- ceptors free, and these free the condition of the blood in which there is an excess of amboceptors. The ambocep- tors (white) unite with both complement (black) and re- ceptors (dotted), so that the receptors cannot combine with the amboceptor-complemcnt groups. amboceptors then unite with the receptors before the, activated amboceptors (ambo- ceptors + complement) do, andthus the complem"^^^ ceptor groups are rendered inert. The reaction is repre- sented diagrammatical^ in Fig. 34. Arrhenius, however does not accept this explanation. He says : » If we have the compounds ea and ab which may combine to form the com- pound eab, the formation of the latter depends wholly upon whether e has a greater affinity for ab than for a. If noi then eab is not formed, even if a is not present in excess." \a = 186 Manual of Bacteriology amboceptor, e = microbe, b = complement.) The phenomenon may be quite analogous with the inhibition met with in agglutinat ion (p. 1 96). Aggressins. Bail has discussed the question of the relationship between bacteriolysis and immunity. He argues that there is apparently little relationship between the bactericidal properties of the body fluids and the immunity of an animal to infection through bacteriolytic processes; and points out that in rabbits immunised against anthrax thei-e is no bacteriolytic power, the bacteria disappearing gradually as the result of phagocytic action of cells, chiefly marrow-cells ; that a com- parison of the sera of sheep, rabbits, and cattle shows great variation in their content of immune body, though the animals arc almost equally susceptible to anthrax; and that in test- tube experiments a, bacteriolytic serum is blocked when the conditions are approximated to those in the body by the addi- tion of body colls to the mixture; the bactericidal properties of the serum disappear or are greatly inhibited. Kruse suggested that for infection to take place the invading wtpria. ult.Wn.tP f1)emical substances which SO act on the cells and fluids of the invaded animal that tliej_oy^rcome_ jjs" natural resistance against infection. . These substances are T.,„„;,iovQ,1 iwhim n,nr1 ttn.il to be distinct from the toxins, and are termed by these writer sJ^a^ggsilLsJ'1 The aggressins are supposed to be secreted by the living uninjured bacteria and not to be extracts, nor derived by solution, of the bacteria ; they occur particularly in the fluids of pathological oedemas and exudates, and may be obtained from these by centri- fugalisation and sterilisation at low temperatures. Bail believes that the aggressins cannot be anti-complements, anti-immune bodies, etc., but are substances heretofore unrecognised and the active substances of the infection, and i See Centr. f. Bakt, Orig., xlii, 1806, pp. 51, 139, 241, 335, 437, and 546. Also an excellent summary by Marshall, Philippine Jown. of Science, vol. ii, 1907, p. 352. Hemolysis 187 he considers that in order to produce true immunity in disease jtnti-aggressin sera must be prepared. The following are some of the properties of these supposed aggressins : (1) Sterilised aggressin with a non-lethal dose of the corresponding orga- nism renders the latter fatal ; (2)aggressiii alone is only slowly Jx>xic. producing a prolonged illness with emaciation preceding death ; (3) inoculation of aggressin with bacteriolytic serum into the peritoneal cavity suspendsthe action of the latter; (4) aggressin with bacteria~blocks phagocytosis. Bail believes that the aggressms promote infection by interfering with the protective mechanism of the infected animal, particularly, if not solely, by inhibiting phagocytosis. Upon the power to produce aggressin Bail has classified bacteria into (1) true parasites which always produce aggressin, e. g. anthrax and chicken cholera ; (2) half -parasites, the aggressin-producing power of which is variable, e. g. typhoid, cholera, dysentery, and plague ; (3) saprophytes. The virulence of an organism does not coincide with aggressivity, and extremely virulent bacteria may be half-parasites. Bail's hypotheses have been much criticised, and Wasser- mann and Citron believe that the ^u^posed aggressins are derivatives of the bacterial protoplasm' winch have the power of combining with the specific protective substances oFTEe" auimal and so inhibit the action of the latter ; they are, in fact, endotoxins ofje^ble^ toxicity. Hemolysis.1— Some blood sera possess marked powers of dissolving the red h1nnH-P.mTnCcles of another species, and of §ettin£_free their contained hremofflobin. (e.g. ,^!lsenTrn_aMssoJves rabbits' and_guineacm^ and ^m^l-senmi usually dissolves sheep's corpuscles)! and jfan animal be injected_with the blood-corpascles of mipdJi£r_s£eciies its blood-serum generally acquires the P-IgP-erty of dissolving the blood-corpusd^yTtiT 1 See Bulloch Practitioner, December, 1900, p. 672, and Trans. Path. Sac. Land vol. In, Part 3, 1901, p. 208; Gruber, " Harben Lectures » Jonrn State Med., 1902, February, March, and April; Ehrlich, Collected Studies on Immunity ■ Mnir, Studies on Immunity. , <( 188 Manual of Bacteriology which it has been injected. For example, the serum of a normal rabbit has no hemolytic action upon the red corpuscles of the sheep ; but if a rabbit receive a Eew injections of defibrinated sheep's blood, its blood- serum acquires hemolytic properties and dissolves the red corpuscles of the sheep. This solution of tlie_ blood-corpuscles Jsjermed " hsemolysis^ and the_suh=_ stances"whlch produce haemolysis are " hannolysins." If the active serum be heated to 56° C. it loses its haemolysing power, but can again be rendered hemolytic or" 11 activated " by the addition otjresh normal serum ; normal serum, however, rapidly loses its activating properties on keeping. It will thus be seen that there is an almost complete analogy between bacteriolysis and, haemolysis, the latter being brought about by the inter- action of two substances, one specific and stable produced by the injections, the hemolytic "amboceptor " or "immune body," and the other an unstable body present in fresh normal serum, the " complement" or "alexin." ifffimoTysin formed by the injection of corpuscles of another species is termed " heterolysin." If corpuscles of the same species be injected, hemolysin is formed ("isolysin"), but the injection of the animal's own corpuscles does not _give rise to hemolysin, i. e. " autolysin " is noflSrmedT Blood-corpuscles are more tangible entities than bac- teria, and are far easier to work with than the latter, and haemolysis has been the subject of a large amount of experimental work by Bor^t and Gengou, Ehrlich, Morgenroth, Gruber, Bulloch, Muir, and others, and the results obtained have shed considerable light upon the complex phenomena of immunity and of the actions of anti-bodies in general. Moreover, the globuhcidal material in haemolysis seems to be identical with the bactericidal one in bacteriolysis— that is to say, it is Haemolysis 189 the complement or alexin.1 According to Ehrlich's view, whether it be normal or " immune " serum (i. e. serum of a treated animal), bacteriolysis or haemolysis takes place only when the complement and amboceptor unite^ (Fig. 33, p. 182), complement by itself having Tittle" affinity for the bacterium or erythrocyte, the combination forming the " lysin/' which then acts. According to G ruber, however, neither bacteriolysin nor hemolysin exist as a chemical entity, the specific bacteriolytic or hemolytic action being due to the fact that the cells first absorb the amboceptor and so become accessible to the complement, for the two substances do not combine in definite proportions — the more the blood-corpuscles are laden with the amboceptor the smaller the quantity of complement required to bring about their solution. Many bacteria — e. g. B. pyocyanens, B. typhosus, staphylococci and streptococci — produce liaamo^ysmR~" and the hemoglobin staining occurring in septic dis- eases, etc., is probably partly due to the antinn of hnrlu^ of tin's nature elaborated by the infecting organisms. Practical Uses of Haemolysis, etc. 1. Haemolysis test— Some micro-organisms produce non- specific hemolysins, others do not; this may constitute a difference between allied organisms. For instance, as a rule true cholera vibrio do not hsemolyse, while many cholera-like vibrios do so. The test can be applied in two ways : (a) 1 As previously stated (p. 181), numerous complements undoubtedly exist, yet bacteria will absorb both bacteriolytic and hemolytic com- plements. Bordet and Gengou suppose that while a particular ambo- ceptor has a maximum avidity for its homologous complement (which may be termed dominant), it is also able to take up other "non- dominant" complements, and thus bacteriolytic amboceptor is able to absorb both bacteriolytic (dominant) and hemolytic (non-dominant) complements. ' 190 Manual of Bacteriology Defibrillator rabbits' blood may be mixed with melted agar cooled to 45° C. The mixture is poured into Petri dishes, allowed to set, and when cool inoculated with the organism to be tested in such a manner that separate, well-defined colonies are obtained. After twenty-four hours' incubation at 37° C, colonies when hsemolytic are surrounded with a clear, well- defined halo contrasting sharply with the dark opaque colour of the agar. If blood-agar is not available, a substitute may be devised by smearing some sterile human or rabbits' blood on a sterile agar plate, (b) A young agar culture is emulsi- fied in 4-5 c.c. of physiological salt solution ; 0T c.c. of this suspension is mixed in a tiny test-tube with 0"9 c.c. of sterile salt solution and one drop of a sterile suspension of well- washed rabbit or other corpuscles. After twelve to twenty- four hours haemolysis will be apparent if the organism forms hemolysins. 2. Fixation or absorption test) — A hsemolytic serum may be used as a delicate reagent for complement, and may thus serve as a test for an organism or an immune serum. As an example take the case of a supposed cholera vibrio. If an immune serum (previously heated to 56° C. so as to destroy complement) — hsemolytic for the corpuscles of an animal, or bacteriolytic for a given micro-organism, e. g. cholera vibrio- be mixed with the red corpuscles of the same animal, or with the cholera vibrio, the corpuscles or the vibrios respectively absorb the corresponding amboceptor or immune body. Bordet showed that if corpuscles or microbes that have absorbed the corresponding amboceptor be added to fresh non-heated complement (e. cj. fresh guinea-pig serum), the corpuscles or the microbes absorb the complement, so that none remains free in the liquid. But if fresh guinea-pigs' serum be added to cholera vibrios which have not absorbed any cholera amboceptor, the comple- ment will not be absorbed and remains free in the liquid. The proof of this is that if "sensitised" corpuscles corpuscles which have taken up hsemolytic amboceptor) be 1 Often termed " deviation of complement" test. Haemolysis 191 added to such a mixture, the globules are quickly hsemolysed. If, ou the other hand, vibrios which have already taken up the cholera amboceptor be added to the same quantity of fresh serum, the microbe-amboceptor complex absorbs the complement ; and provided the amount of fresh serum is not too great, the complement is absorbed so completely that " sensitised " corpuscles when added to the mixture are not dissolved. If vibrios other than cholera be added to cholera serum, the amboceptor js not fixed, the complement added remains free, and the sensitized corpuscles are dissolved. These facts constitute the " Bordet-Gengou phenomenon." The mixture of an inactivated hsemolytic serum (i. e. heated to 56° C.) with the homologous corpuscles (i. e. those with which the hsemolytic serum was prepared) is known as a "hsemolytic system." The method of carrying out the test is as follows : The cholera-immune serum is heated to 56° C. .for half an hour. An_eighfceen hours old agar culture, of the organism to be tested is suspended in 2 c.c. of sterile physio- logioaLsait, solution. The complement is fresh rabbit or guinea-pig serum; a portion of this is also heated to 56° C. "(= non-immune 'serum). The following mixtures are pre- pared in three small test-tubes : Tubes 1 and 2 each contain 0-2 c.c. microbic suspension + 0-6 c.c. heated immune serum + 0T c.c. complement. Tube 3 contains 02 c.c. microbic suspension -f 0-6 c.c. heated non-immune serum + 01 c.c. complement. These are well shaken to mix their contents, and are kept for half to one hour at 37° C. At the end of this time Ol c c of the following mixture is added to tubes 1 and 3 • two volumes of heated (to 56° C. for half an hour) serum haano- Jysmg sheep's red corpuscles + one volume of washed sheep's corpuscles. To tube 2 is added Ol c.c. of a mixture of two volumes of physiological salt solution + one volume of washed sheep s corpuscles. The tubes are kept for a further hour or so at 37 0, and at the end of that time the occurrence of hemolysis is noted. If the organism is homologous with the immune serum, the immune body will fix the complement in 192 Manual of Bacteriology tube 1 and no haemolysis will occur ; in tube 3 haemolysis will occur because the complement remaius free. Tube 2 serves as a control, and should show no haemolysis in three hours (though if kept for eighteen to twenty-four hours haemolysis will occur if the organism produces hiemolysins, apart from any action of complement). If the organism is not homologous with the immune serum, haemolysis will occur in tube 1, because the complement does not become fixed, tubes 2 and 3 being the same as before. The haemolytic serum may be obtained by injecting rabbits with a 10 per cent, suspension of well-washed sheep's red corpuscles. Four doses of 5 c.c. intra-peritoneally or 2 c.c. intra- venously are given at intervals of a week, at the end of which period the rabbit's serum should be strongly haemolytic. The sheep's blood should be obtained as aseptically as possible from the slaughterhouse ; the blood, as it runs, is caught in a sterile wide-mouthed bottle containing a coil of fine wire with which it is defibrinated by shaking. The fluid blood is then mixed with sterile physiological salt solution (0-9-O95 per cent, ) and eentrif ugalised, and the deposited corpuscles are a°uin washed with salt solution two or three times. °3. Antigen Test.See "Syphilis." HytotoxinS.1— Anti-sera, analogous to the hemolysins or ' hsemotoxins.iuay be prepared which have a destructive action upon cellular elements ; these are termed " cytotoxics." If a rabbit be injected with bull's semen, itsjiexiim. (" spermotoxin") "acquires the property of immobilismgJlHj_£perma^^ iuiLL_ The reaction is specific, but spermatolysis does not seem to occur. Similarly, by injecting ciliated epithelium into the peritoneum of a guinea-pig an anti-epithelial serum, or « trichotoxin," is developed. With liver, kidney, and nerve cells anti-bodies having a destructive action upon these cells are developed as a result of their injection. Nephrotoxic the serum of an animal inoculated with an emulsion of kidney, when injected into a second untreated animal, produces* albuminuria and uraemia with disintegration of the epithelium - of the convoluted tubules ; hepatotoxin, the serum of an i See Bulloch, Practitioner, May, 1901, p. 499. (Bibliog.) Agglutination 193 animal treated with emulsions of liver, produces fatty and inflammatory changes in the liver resembling phosphorus poisoning ; neurotoxin, the serum of an animal treated with emulsions of nerve tissues, produces paresis, paralysis, depression, convulsions, etc. ; a leucotoxic serum obtained by injecting leucocytes agglutinates and dissolves the leucocytes, and so on. The formation and mode of action of these cytotoxins resemble those of the hemolysins. It was hoped that the study and preparation of cytotoxins would open up possibilities in the way of treating such diseases as carcinoma and sarcoma, but so far this hope has not been realised. _ActGlutination.— Tf an animal be injected with cul- tures of typhoid or cholera, its serum soon acquires the property of agglutinating or of aggregating into clumps the typhoid bacilli or cholera vibrios re- spectively when mixed with a broth culture of these organisms. The reaction may be observed micro- scopically in a hanging-drop preparation ; the organisms first lose their motility and soon become aggregated into large masses or clumps. Microscopically, the reaction may be followed in a narrow test-tube into which the mixture of culture and serum has been introduced ■ after some hours the micro-organisms become ago-re- gated into masses so large as to form visible flooculi the substances which bring about this agglutination are known as agglutinins. Aggju^inins seem to be present m small amount in no^T^Tl^rT^^^ most normal human sera up to a dilution of 1 in 2 or 1 m 4 wil agglutinate the typhoid bacillus and still more powerfully the glanders bacillus They are also present ^genaUuh^; .ifjmold broth culture~of typhoid "u „ £frr — T rr >m» - r '"^^nuitiux bvpnoid be filtered thTnTTra— g]utinates thel^li in a fresh broth culture ; hence ^ ,„u„^ ^d a] Wsedjor agglutination" tests. Agg]ut^n^d 13 194 Manual of Bacteriology by the action of antigen derived from the bacterial cell,but may also be naturally present. Agglutination is brought about by the action of the agglutinin on the antigen ; the agglutinin first unites with the antigen, and tins may occur at 0°C, and afterwards exerts its specific action, which takes place only at higher temperatures and in the presence of certain salts. The agghitmable sub- stance is known as agglutinogen. Agglutinin is converted into agglutinoid at 70°-75° 0. ; the latter does not agglutinate, though it unites with bacteria and then prevents the subsequent action of agglutinin. The agglutination of organisms by anti-sera, though hardly specific, is usually very special ; given proper precautions as to dilution, time-limit, condition of test culture, etc., an anti-serum will generally only agglu- tinate the homologous organism or closely allied species -that is, it is a group reaction. The antiserum may agglutinate both the organism with which it has been prepared, and also allied species, though usually not toPthe same extent ; anti-typhoid serum or example may agglutinate not only the typhoid bacillus, but also Zugh to a less degree, members of the paratyphoid group. As the result of infection or of inoculation w, h Lirganisni J^^^^^, agglutinate the B. coli as well as the B. typhosus and W us serum B. typhosus and M. mehtenns. I he acting of the infecting o,-^ turned primary or homologous those acting ^ on ^other organisms secondary or heterologous. In a ase o± aoublo infection ^^Z^^^i Agglutination 195 _ agglutinins^ Castellan i,1 by applying the saturation test (p. 201), found that an organism would absorb both its primary and secondary agglutinins, but would not absorb two different primary agglutinins. This test, therefore, would distinguish a double infection from a single one. Thus, if a typhoid serum agglutinated both the B. typhosus and the B. coli, and the serum after saturation with typhoid culture still agglu- tinated the B. coli, this would point to an infection with the latter as well as with typhoid. The formation of primary and secondary agglutinins may be brought about as follows: In the bacterial cell there are several substances, each of which forms its own agglutinin. The cells of two bacterial species we can imagine both contain three or four substances capable of producing agglutinins, and it may happen that one of these in each species is the same and will produce the same agglutinin— the secondary agglutinin —and, therefore, the serum produced by each bacterium will agglutinate the other. I he agglutination reaction is made use of in bacterio- logical tests and in clinical diagnosis. The "Bordet J*™™l^aotion con^ts in^testin^annn1,.nWn becomes agglutinated, it is regarded as being of the same species as that with which the anti-sefum was WIth °ertain Potions the "Bordet- Dnrham reaction is one of the most delicate and certain for the recognition of bacterial species. The converse I V „ unknown serum upon a known microbe. It is especially used m the diagnosis of microbial diseases ; for exlmple" Zextschr.f. Hyg.,xl, 1902, p. 1. ' L96 Manual of Bacteriology in typhoid fever the blood of the typhoid patient powerfully agglutinates the typhoid bacillus, that of Malta fever the Micrococcus melitensis, that of bacillary dysentery the dysentery bacillus, etc. A remarkable phenomenon observed in connection with agglutination, which the writer has particularly ^f^orl int.liB P.ase of Malta fever, is the occurrence of what may be teamed a zone of no reaction or of inhibition with some particular diluticnT Thus, dilutions of 1 in 10 and 1 in 20 may agglutinate strongly, a 1 in 30, however, may hardly agglutinate at all, while dilutions of 1 in 40 and upwards to 1 in 100 or more may agglutinate well. A similar phenomenon has been observed with non-specific agglutinating agents, and also in the action of coagulating agents on colloid emulsions. Thus orthophosphoric acid agglutinates a certain volume of a suspension of B. coli when present to the extent of between 118cgrm. and 4 cgrm., and between M mgrm. and 0-001 mgrm., but not in intermediate amounts between 40 and M mgrm. Anti-serum, prepared by injecting erythrocytes, also agglutinates the red blood-corpuscles, and in certain diseases, e. g. pneumonia, chromocyte clumping may be a marked feature. Various theories have been propounded to account for the phenomena of agglutination : 1 Pfeiffer and Emmerich and Loew regarded agglu- tination as a vital paralysis of the bacilli due to the action of a bacteriolytic enzyme. Agglutination, how- ever is not a vital phenomenon, for dead bacilli agglu- tinate, and bacteriolytic enzymes seem to be destroyed L temperatures at which agglutinins remain unaffected. Gruber, Dineur and Nicolle supposed thata ,lutinous substance, « glabrificin/' is absorbed from he I by the bacilli causing the cell membranes or the Agglutination 197 flagella to become adhesive ; but this explanation will hardly account for the aggregation of non-motile organisms. 3. Paltauf and Duclaux considered that a precipitate is produced in the medium, which during flocculation mechanically carries the bacilli with it ; but there is no demonstrable evidence that such precipitation occurs. 4. Bordet separated the mechanism of agglutination into two stages — (1) fixation of agglutinin, and (2) aggregation. The fixation of agglutinin by the organisms he considers to be analogous to the adsorption of a dye by a tissue ; and once the agglutinin is fixed, the organisms obey the laws of inert particles, aggregation being caused by changes in surface tension, in the mole- cular attraction, between the organisms and the surrounding medium, a view supported by Craw.1 Ohno2, however, believes that the union of agglutinin and agglutinable substance is not analogous to the fixation of a dye by a tissue, but that it is a chemical combination, as maintained by Ehrlich. Agglutinated bacteria are not iniured bv ag-o-lutina- tion; they will, in fact, grow and multiply in an agglutinating serum. The amount of agglutination does not bear any constant ratio to the intensity of an infection; on the whole, if the patient is reacting satisfactorily to an infection, the agglutination reaction tends to be marked • if not, it may be feeble or absent. Thus, in severe typhoid infections with fatal issue, agglutination may be absent. Buffer and Crendiropoulo3 regard the agglutinins as being formed in the poly- morphonuclear leucocytes. ' Journ. of Hygiene, vol. v, 1905, p. 113. See also Joos, Zeitschr. f Hyg., xxxvi, p. 422, and ibid., xl, p. 203. 2 Philippine Journ. of Science, vol. iii, 1908, p. 47. :' Brit. Med. Journ., 1902, vol. i, p. 821. (Bibliog.) 198 Manual of Bacteriology The Agglutination Reaction. a. For Clinical Diagnosis (" WidaV Reaction). This is principally made use of in typhoid aud paratyphoid fevers, Malta fever, and hacillary dysentery. Collection of Mood.— Blood is collected (p. 131), preferably in a Wright's capsule (Fig. 35, d, p. 225), or in a capillary bulbous pipette (Fig. 7, p. 51), or in a vaccine tube. The ends of the tube are sealed, the dry end always being sealed first ; the blood is allowed to coagulate (which may be hastened by placing in the blood-heat incubator), aud then centri- f ugalised to separate the serum, care beiug taken that the dry sealed end of the tube, which will be perfectly sealed, is distal when spinning. If tubes are not available, the blood may be spotted on to a piece of glass, cover-glass, or slide, glazed paper, tinfoil, etc., and allowed to dry. For use, a drop of distilled water is placed on the dry blood to dissolve it, and the solution used like serum. The culture.— For the microscopic test a young broth culture is to be preferred. A hanging drop should be examined to ascertain that clumps are absent ; this specimen is kept as a control. If clumps are present they may be removed (in the case of typhoid) by filtering the culture through filter-paper. A suspension of an agar culture may also be used, likewise dead cultures : a broth culture or suspension of an agar one being heated to 65° C for ten minutes and preserved in sterilised e put up with iormal serum of an animal of the same species as that from which the immune serum has been obtained. A series of dilutions of both sera is made with salt solution and a twenty-four-hour agar culture of the organism to be tested used. Both the macroscopic and microscopic methods should be employed. The dilutions may be made with a 1 c.c. pipette graduated in hundredths, with the hsemocytometer pipettes, or by the method used clinically. 2._8atur_ation ferf.— Bordet, first noticed that a suspension ot a microbe added to the homologous agglutinating serum absorbs most, if not all, the specific agglutinin, whereas an organism not homologous with the serum absorbs little or only a portion of the agglutinin. The test may be carried out as follows : (a) Ten loopfuls of a young agar culture of the organism to be tested are mixed with 10 c.c. of a 5 per cent, solution of a highly agglutinating serum. After standing for two or 202 Manual of Bacteriology three hours at room temperature, the mixture is centrifugalised and the clear supernatant fluid decanted. (6) The agglutinating power of the decanted liquid is then tested on the organism with which the serum was prepared. If the organism treated in (a) is homologous with the organism with which the agglutinating serum was prepared, the decanted fluid will have lost most, or a considerable proportion, of its agglutinating power for the latter. The Meiostagmin Beaction. — Ascoli has found that if an immune serum be mixed with an alcoholic extract of the homologous antigen and the mixture incubated at 37° C. for two hours the surface tension is reduced ; if the serum and antigen extract are not homologous the surface tension is unaltered. For example, in the case of typhoid the following is the procedure. An alcoholic extract of typhoid bacilli is prepared; this is diluted with saline solution to 1-1000— 1-1 000 000. The typhoid serum is similarly diluted, 1-10. To 9 c.o. of' the diluted serum 1 cc.of the diluted antigen extract is added By means of some form of viscosimeter or stalag- mometer the number of drops yielded by a given volume of the mixture is ascertained, immediately after the mixture is made and after the mixture has been incubated at 37° 0. for two hours. If the surface tension has been reduced t ie number of drops counted in the second determination will be greater than in the first.1 _ Anti-ferments.2 — Bv the injection of rennin or othei enzyme the blood-serum of the treated animal acquires the property of neutralising the action of the enzyme with which S inoculation has been performed. Thus if rennm and anti-rennin (the serum of an animal injected with rennm) be nled with milk no curdling takes place. Sum arly he urn of an animal inoculated with pancreatm inhibits the action of this ferment, and if coagulated egg-albumen, pancreatin, and anti-pancreatin be mixed, the egg-albumen undergoes no ai i See Ascoli and Izar, Miincl, me*. Wool,, Ivii, 1910, pp. 62, 182, ^ Dean, Tran, PatK. Soc, Lond., vol. lii, 1901, Part 2, p. 127. Precipitins 203 Precipitins.1 — Kraus was the first to demonstrate the presence of specific precipitins in blood by adding typhoid, cholera, and plague anti-sera to filtrates of the cultures of the corresponding microbes. If to such a filtrate in a test-tube a little of the corresponding anti-serum be added by running in carefully, so that it forms a layer at the bottom, an opalescent ring makes its appearance at the line of junction of the two fluids. So also if an animal be injected with milk, its serum, jvhen added to milk of the same kind as that with which it ^as been injected, causes precipitation of_the casein- This reaction is specific, and it is thus possible— to— distfflgui.sh , various milks jrom one another. Similarly, anti-sera wTiich produce precipitates, each with the homologous substance, are obtained by the injection of peptone, of egg-albumen, blood- serum, and other proteins. The latter reaction has an important medico-legal application, for by means of it the blood and flesh of different species of aninmlscan be distin- ^guished^ Thus the presence of horseflesh in sausages can be detected. The method employed is to inject a rabbit intra- peritoneally with four to six injections of defibrinated blood or of blood-serum (or with a solution of the particular substance,. e.g. horseflesh), commencing with about 5 c.c. and increasing to 10 c.c. at intervals of a few clays. After treat- ment the animal is bled from an ear vein, and the serum is obtained. The blood to be tested may be dried on filter-paper, pieces are then cut up, a solution is made in P6 per cent, sodium chloride solution, and to this the specific serum is added. Tested in this way human blood anti-serum reacts— i. e. forms a precipitate— markedly with human blood, less so with ape's blood, not at all with other blood; ox blood anti-serum reacts with ox blood, less so with sheep, feebly with horse, hardly at all with dog. Mixtures of bloods may also be tested. Preci- pitins are also formed naturally in vivo. Thus the serum of a patient the subject of hydatid disease gives a precipitate with i See Nuttall, Journ. of Hyg., vol. i, 1901, p. 367 (Bibliog.V also Bn*. Med. Journ., 1902, vol. i, p. 825 ; Welsh and Chapman, Journ. of f?TZkl°n V91?' P- 177 ; ^ A™tralasian Med. Gazette, December ^lst, 1908 (hydatid disease;. 204 Manual of Bacteriology hydatid fluid, and the reaction may be used diagnostic ally. The production of the anti-body seems to be due to the globulin constituent of the injected serum. It will thus be seen that the anti-bodies which result from the injection into an animal of different substances are extremely numerous and have varied properties, their most notable characteristics being their extreme specificity and the extraordinary delicacy of the inter- actions produced by them. It is important to note that these anti-bodies are produced only as the result of inoculation with complex compounds allied to the pro- teins. The tolerance established by the ingestion or inoculation of simpler compounds, such as arsenious acid and morphine, is of a different nature, and is not coincident with the development of anti-bodies. Accord- ing to Ehrlich, the latter kind of tolerance may be due to the exhaustion or using up of certain receptors (" chemo-receptors") of the protoplasm (see p. 216). Immunity.1 No fact in biology is more striking than the differ- ences in susceptibility to infection exhibited by different races and different animals. For example, the natives in many parts of the world are com- paratively insusceptible to yellow and typhoid fevers and malaria, the dog and goat are rarely affected with tuberculosis, and tetanus is never met with in the fowl ; and to come nearer home, while some individuals are lucky enough to escape most of the commoner infectious fevers, others seem to See Metchnikoff, Immunity in Infective Diseases, 1905. Also Brit. Med Jonrn., 1902, vol. i, p. 784 ; 1904, vol. ii, pp. 557-5S2 ; and 1907, vol. ii, pp. 1409-1425; Journ. of Hygiene, vol. ii, 1902; Emery, Im- munity and Specific Therapy, 1909. Immunity 205 contract them on every possible occasion, and to suffer from all the ills that flesh is heir to. These instances show that there is often a natural insuscepti- bility to infective disease, or a natural immunity, as it is termed. This may be complete or partial, or it may appertain only to a race — " racial immunity " ; or, varying in different individuals and at different ages, it constitutes "individual immunity/' as in the case of diphtheria and scarlatina, which become more and more rare as age advances. Still more striking, perhaps, is the fact that an insusceptibility may be acquired after an attack of infective disease or be conferred in certain instances by inoculation. Thus second attacks of smallpox and ^scarlatina are rare, inoculated smallpox and vaccinia protect against variola, and bacterial vaccines confer considerable protection. With regard to the immunity of native races to certain diseases, this is ^probably due _J&_ natural selection and heredity ; during long periods of time, the individuals being all exposed to the same risks, the susceptible ones are weeded out, while the survivors transm^t_their insusceptibility to their descendants ; bu~T this, of course, does not explain the reason for the relatively greater immunity of the insusceptible indi- viduals. Immunity is generally not absolute either to infection or intoxication ; that is, infection can usually be induced under certain conditions. Thus fowls, which are highly refractory to tetanus and tolerate consider- able closes of tetanus toxin with impunity, can be tetanised with large doses of an active toxin ; white rats, which are insusceptible to anthrax, become susceptible after fatigue, or when fed on an exclusively vegetable diet. Immunity is therefore either (1) natural or (2) acquired, and it is evinced against either (a) toxins' 20G Manual of Bacteriology or (b) micro-organisms, and these different phases must be considered. 1. Natural immunity against toxins. — There are various non-specific reactions in the body by which toxins may be eliminated or destroyed. Thus the dilatation of the vessels and the acceleration of the blood-stream which take place in an inflamed area dilute and eliminate the toxin, and the proteolytic enzymes produced by the organisms and as a result of tissue disintegration may have a destructive action on the toxins. Oxidation, hydration and dehydration and various analytic and synthetic processes which go on in the body, and particularly in the liver, are other agencies whereby toxins may be destroyed. These non-specific processes by which toxin is destroyed or eliminated, though of the greatest importance, can probably deal with only small amounts of toxin ; if large amounts are present, specific reactions have to be evoked. Another cause of natural immunity to toxins may be the absence of suitable receptors for tlie_toxin. As already stated (p. 161), in order that a bacterial toxin or endotoxin may produce intoxication ,Jt must become anchored to the cells by its haptophore group, e protein nfain conned C^s"™111^ ^ ^ » B6hriDg a,'d ^ >— found that the serum 14 210 Manual of Bacteriology of the white rat, dog, and rabbit destroys the Bacillus anthracis, but serum fro... the mouse, sheep, guinea-pig, chicken, pigeon, and frog has no action. Thus, while the rabbit is highly susceptible to anthrax, its serum is germicidal; the chicken, on the other hand, is immune to anthrax, but its serum is inactive. Hence there is a considerable difference between the action of circu- lating and of extra-vascular blood. Vaughan, Novy and McClintock, in a series of papers, ascribed powerful bactericidal properties to the nuclems, and surmised that in serum the nucleins set free by he disintegration of leucocytes and other cells are the germicidal agents. Forrest and the writer^ found however, that all the germicidal properties ascribed by Vauglan to the nucleins are probably due to the weak alkali in which the nucleins were disso ved, and came to the conclusion that Vaughan's results are at least n° gCou ' also found that the plawa collected in v, ned tubes is often almost devoid ot bactericidal I^er whilst the corresponding « may be capable ot destroying large numbers of micro-organisms We ^therefore 'see that while the blood, lymph, and othTr fluuis and tissue juices undoubtedly exer more o less germicidal action on bacteria experimentally m .1 ;c often a marked difference m this respect ltro, there is < en - ^ ^ ^ in vitro andl m l ^tnLl if this factor is of great inl- and it may d f tural immunity. At portance m the p °ductao the same hme^t 8 b disintegration and liaS tudatTon occur, and thus the germicidal action TZ bodv fl'd b and tissues may be exerted n, of the body nuius vnf/her bv stimulating thouo-h such substances may act lathei oy i Joum. Roy. Army Med. Corps. Phagocytosis 211 the leucocytes or by rendering the bacteria more phagocytosable, as will be referred to later (p. 220). Thus Ivanthack and Hardy found that the coarsely granular oxyphile leucocytes in the frog are first attracted to the site of a bacterial invasion, there discharge their oxyphile granules, the bacteria then show signs of degeneration, and polymorphonuclear leucocytes and other "phagocytic" cells now approach and ingest the degenerate bacteria. The observations however, do not seem to have been confirmed. Wool- dridge also protected animals from anthrax by injections of " tissue fibrinogen » (nucleo-protein). For some micro- organisms a bacteriolytic mechanism exists, the ambo- ceptor-complement complex, whereby they may be digested and got rid of. Thus normal serum has a marked bacteriolytic action on B. typhosus and B coli In many cases, however, e. g. for staphylococci, such a bacteriolytic mechanism does not naturally exist but may be evoked as a result of infection The hypothesis which ascribes immunity to the sc:f r ,ba;tei;iolyfc;c action °f - ^ theory/' 7 b6en t6rmed the "Wad Another important theory of immunity is the doctrine vessels and oo„gla ! thTi T^" fr0m &e at the seat of a bacterial 7 P 7 eM,Sregate and engulf the LtT " ^ ^ a"d aPP™<* uactou m the same manner as they 212 Manual of Bacteriology do other foreign particles, and so rid the body of the unwelcome guests (Plate I., a and b). The migration of .the, leucocytes towards the scene of action {s explained" by Metchnikoff on the hypothesis t,W, the chemical substances elaboratedjjy^hs-baeteria attfScQSijS^r and exert what he termed " positive clu^nTol^xTs." In this case the bacteria are removed by the leucocytes, and general infection and death do not occur. But, unfortunately, in other cases the bacterial chemical products repel, or perhaps it is more correct to say do not attract, the leucocytes, and " negative^ cWiotaxis" occurs, so that the bacteria are free to T^w and multiply, and general infection ensues. Positive and negative chemotaxis can be shown to occur by a simple experiment. If a fine capillary tube con- taining some peptone solution be introduced into a suspension of bacilli, e. g. B. fluorescent hquefanens, under a cover-glass, and watched microscopically the bacilli will be attracted to the tube and soon invade its lumen If, however, a weak acid be substituted for the peptone water, the baeilli will be repelled The process L which the bacteria are ingested by the leucocytes can be similarly watched. The leucocytes which ac in this manner arc termed phagocytes, and they are of two classes-the macrophages, the large mononuclear leucocytes, and the smaller microphages or poly- ; I'honuclear leucocytes. Certain of the tissue ce Is ^endothelial cells also possess phagocytic property The importance of phagocytosis is also shown by the fact that, while in ordinary susceptible rabbits infection ™t i anthrax is followed by a feeble phagocytosis «d J animals succumb, in rabbits vaccinated against anthrax phagocytosis is very active. Moreover, m an Sla to^ to anthrax, such as the frog, anthrax WH grow and'multiply if they be enclosed m paper Phagocytosis 213 or collodion sacs, so as to prevent the access of the phagocytes. JPhagocytosis, in vitro, and probably also in the normal body, js extraordinarily active, so that it might be expected always to be sufficient to deal with any number of bacteria that might be introduced. If, however, the bacteria be virulent, negative chemotaxis^ will occur. Moreover, the presence of snW.n.rm00 which render the bacteria phap-ooytns^lp « opsonins," is necessary, and it seems likely that the^amounT of opsonin becomes diminished in infection (see p. 2Ziy. 1 v Mefcchnikoff admits that the destruction of bacteria m phagocytosis is brought about by chemical bacte- riolytic substances, which he terms " cytases " and which he regards as being derived from the leucocytes and as identical with the alexins. He believes that there are two kinds of cytases, one - macrocytase - obtainable from tissues, such as the spleen and lymph- glands, rich m macrophages, which acts specially on elements of animal origin, the other ■« microcytase » derived from the microphages, and which acts princi- pally on micro-organisms. He considers the alexic this10-* do ™ Tnre of a di^estive

this is doubtful), and as regards the complex of a cytolytic serum, which contains amboceptor and complement, believes that the amboceptor i formed within the macrophages in intra-cellular digestion and that a t of , from t|]em ff , and All the facts point to the leucocytes and leucocv c tissues being the great defensive mechanisms ^ fn parasitic invasion, either by the production of alexin or of bactenolysms, or by phagocytosis, or probably W a combination of these (the « cellulo-humoral »Z£ thesis of unmuinty). Jt is probab]e ^ ^° 214 Manual of Bacteriology pnrj^of phagocytosis takes place Jn the spleen. This organ acts as a sort ofJUtgr, and phagocytosis may be active in it when none can be discerned in the blood. Phagocytosis is also active in the bone^arrow. Experiments by Tizzoni and Cattani seemed to show that rabbits could not be rendered refractory to tetanus by injection of tetanus antitoxin after extirpation of the spleen ; and although Benario and other observers have not confirmed this, the manner in which the spleen is attacked in such diseases as tuberculosis, plague, etc., points to this conclusion. The discordant results obtained after splenectomy may be clue to the rapid regeneration of spleen tissue, and to other structures, such as the hamiolymph glands, taking on its functions after ablation. . Although small amounts of antitoxin may occasionally be met with in the normal animal (e. g. diphtheria antitoxin in man and in the horse, see pp. 160 and 286), this substance plays little or no part in natural immunity against either toxin or micro-organism lhus the blood-serum of the fowl, which is highly rejra^ory_ fn-ES^sT does not ej^tjM^t^i-^m^ DeiitraliBimga£^^Eifea§J^-; ., v . U^S^^^ immunity may be o O. induced in several ways : (1) By anattads of the disease ending m recovery 2 BT^cinating with a modified aid ess virulent form cftengTnfeotive agent (Pasteur's niet io (3) By trgatuu^wi^J^e"- enltures, 01 with or oxil of a dife^TTh«, B. «^»n« Md Ssmisliowed^ i Trans, rath. Soc. Land., 1893, p. 220. Active and Passive Immunity 215 KocVs comma, (2) Finkler-Prior's comma, (3) B. coli, (4) Proteus ■vulgaris, (5) B. prodigiosus, (6) B. typhosus — wil^ protect an animal against any one of tlie remaining five. He therefore concluded that there is an immunising agent common to all these six organisms, and that this substance is intra-cellular and a constituent of the bacterial cells themselves. In this case, however, the immunity is probably one against certain bacterial proteins and not against the specific endotoxins of the organisms. (5) By injection of the blood-serum derived from an animal treated or immunised by method 3 — that is to say, antitoxins or other anti-bodies (e. g. amboceptors) are introduced. The immunity acquired by methods 1-4 is known as "_active immunity," because the animal's cells and tissues are altered by the process, so that they~are no longer susceptible to the microbe or its toxin, The immunity conveyed by method 5— the injection of an immune serum, is known as " passive immunity," because the immunity lasts only so long as the anti- bodies remain ; there is no active participation of the animal's cells and tissues in the process. Active immunity is generally of long duration— some months at least— and ii^oT transmissible To the fetus ; but passive immunity^ of short duration— two* to ' four weeks— and is transmissible to the fetus and nursling Acquired immunity to toxins may be due to the elimination of the receptors concerned in the fixation ot the toxin by the cells, or to the production of the neutralising antitoxin. The leucocytes are probably the active agents in destroying and eliminating toxin whether neutralised by antitoxin or not. Various explanations have been given of the pro- duction of acqujredhnmunity against the organist 216 Manual of Bacteriology- Pasteur suggested that the organism, by its growth, in the body, exhausts some specific pabulum necessary^ f I for its development, so that it cannot again grow in Sfj^U^^^ the animal which has been attacked. This hypothesis, CftoLxAM.' therefore, presupposes that in the body there is some nutrient material necessary, for the growth of each species, which is ^difficult to believe, and is negatived by the fact that an organism will grow in the blood and tissues removed from an animal vaccinated against, and insusceptible to, the disease produced by itself. Pasteur's " exhaustion " theory has been revived by Ehrlich1 in a modified form, under the name of " atrepsy," to explain certain cases of immunity. Thus, for a chemical poison to act, Ehrlich assumes that particular receptors iu the protoplasm for binding the poison are necessary ; these he terms " chemo-receptors." Bird-pox, virulent lor both fowl and pigeon, if passed through the pigeon . becomes completely avirulent for the fowl. To explain this Ehrlich suggests that the parasite in passing through the pigeon has to assimilate substances different from those assimilated during its passage through the fowl ; therefore that, part of the receptors which deals with the nutritive substances of the fowl's organism is not in use during the passage through the pigeon, and may become atrophied, so that on the parasite being transferred back to the fowl it will not be able to thrive owing to the loss of the receptors necessary to assimilate the fowl's nutritive substances. Ehrlich suggests that the majority of non-pathogenic micro-organisms, it- introduced into the animal body, perish by this mechanism. In the case of mouse carcinoma inoculated into rats, the tumour-cells proliferate for a few days, then atrophy and disappear. Ehrlich suggests that some spec.fic substance is necelfary for the proliferation of mouse caivmoma-cells which is not present in the rat, and as soon as the traces of this specific substance carried over by the inoculatxon are > « IJavbcn Lecture/' ii, Jour*, Roy. hid. Public Eealih, 1907. Immunity 217 used up, the caucer-cells cease to prolifeiate aud finally atrophy and disappear. These are examples of Ehrlich's " atrepsy " aud "atreptic immunity." Ohauveau, in his retention theory, suggested that the Jjaateria during their growth iiijjis-tiasags form sub- stances which ultimately inhibit their growjj^fmr^ if the animal recovers, prevent a' subsequent deve- lopment of the organism. The same objections may be urged against this hypothesis as against Pasteur's exhaustion hypothesis. Bacteriolysis and phagocytosis are probably the two main factors which bring ab^uTthe refractory nnndif.^n in acquired immunity against bacteria, as well as recovery from an infection. After immunisation it inav be shown tlmtphagocytosis is increased, and thai_posi- tTvje_ciu3motaxTs takes place towards the organism whereas previously negative ehemoTaxis occurred ■ the jeucocytes have_been " educated," as it were to be attracted, instelToFrep^IIe^y the bacterial invasion According to Andrewes,1 the defence against the pyogenic cocci is not only essentially phagocytic, and dependent upon the polynuclear leucocytes, but is also, in the main opsonic In tuberculosis and syphilis the polynuclear leucocyte takes little part in bodily defence, which is essential y a function of the endothelial and fixed tissue- cells With the colon group of organisms certain humoral responses, notably agglutination and bacterio- lysis, are better marked than with most other bacteria and polynuclear phagocytosis seems subsidiary • Anfclt0XV1. fo^jglL^aJbl^layB little or no nart m acquired imm^^l^^^.y trom jfe- found until the disease has subsided. Possibly Tn 1 " Croonian Lectures," Lancet, Juno 25th et se2., 1910. ' 1 218 Manual of Bacteriology chronic infections, antitoxin formation does play a sub- sidiary role in recovery. To sum up, natural immunity is probably due to a number of factors, some or all of which may be operative in particular instances, and it is impossible to state with certainty any general law. Jn most cases phagocytosis Jsjjip, principal means of defence, the germicidal, inhibi- tory, or bacteriolytic acticms_oi_the body-fluids aiding, Though oT subslcliary importance ; in others the cells and tissues are unaffected by the bacterial toxins, sometimes because the cells are lacking in the particular side- chains or receptors which (ix the toxin ; sometimes because, for some unknown reason, the cells are unaffected by the toxophore group of the toxin. As regards the immunity acquired after an attack of disease, this may be due to the " education " of the leucocytes, whereby they are attracted, whereas formerly repelled, by the products of bacterial develop- ment, or to substances which stimulate the action of the leucocytes. The germicidal, inhibitory, and bacteriolytic actions of the body-fluids may also be enhanced. ^ It seems probable also in certain instances that the side- chains or receptors having an affinity for the toxin become in some way destroyed or used up, so that further fixation of the particular toxin cannot take place. . It is to be noted, as Metchnikoff has pointed out, that immunity is much more rapidly acquired against micro- organisms than against their toxins. In_Jiatuj^ii_ jsjrrincipally against jn^ro-o^gamsms^ thajJhe_bodjL requires protection . Adaptability seems to be one of the innate proper- ties of protoplasm, and immunity is but an instance o adaptability. It might be expected, therefore that immunity towards infection will become established, Role of the Serum 219 more or less completely, when the need for it arises ; and we find that this is the case, however difficult it may be to explain the mechanism by which it is attained. The Role of the Serum in Phagocytosis. 1 The fact that in an immunised animal, no sooner does the virulent organism gain access than the leucocytes migrate to the site of infection, surround the invaders, ingest and so destroy them, was at one time ascribed by Metchnikoff to " education, i. e. modification, of the leucocytes; but since the serum of the immunised animal injected into a non-immunised one causes the leucocytes in the latter to behave in the same manner as they do m the immunised animal, the effect must be clue to something in the plasma or serum, and Metchnikoff ascribed the action to substances, " stimu- lins," which heighten the activity of the leucocytes. Later work has not confirmed this view, and no certain proof of the existence of stimulins is forthcoming although Leishman attributed a stimulin action to thermostable substances in the serum in typhoid and Malta fevers. Subsequently Metchnikoff conceived thejserum as acting. noi_x>n the leucocytes, but on the microbe, causing it to become positively chemotactic and no longer to repel, but to attract the phagocytes. Considerable support was given to this view by the work of Wright and Douglas, who, by a modification of Leishman's ingenious method for quantitatively estimating phagocytosis, emphasised the importance of the serum in the mechanism of phagocytosis. Neufelcl and Rimpau also concluded that substances, " bacteriotropines," are produced in the course of 1 See Dean, BrU. Med. Journ., 1907, vol. ii, p. 1409. (Bibliog.) 1 220 Manual of Bacteriology immunisation winch promote the phagocytosis of bacteria. Leisjiman's method for estimating jri'-agocytosis.1 — A thin suspension of some micro-organism, e. g. M. pyogenes, is mixed with an equal volume of blood from tlie finger ; a droplet of this mixture is placed on a clean slide, and covered with a cover-glass, and the preparation is at once placed in a moist chamber in the incubator at 37° C. for half an hour. At the end of this time it is taken out, the cover-glass slipped off, and the films on slide and cover-glass are dried, fixed, stained and examined microscopically, and the number of microbes ingested by the polymorphonuclear leucocytes is counted. Wright and Douglas2 found that washed leucocytes without serum are non-phagocytic^ but/become m-aa_the- addition of normal serum. If, however, the serum be first" heated to 60°-6~ "J. before being- added to the mixture of leucocytes and microbes, phagocytosis does not take place ; but if the unheated serum is mixed with the bacteria, the mixture kept at 37° C. for fifteen minutes and then heated to 60° C. for fifteen minutes, phagocytosis can still take place, thus demonstrating that the serum acts in some way on the bacteria, rendering them suitable prey for the phagocytes. This thermolabile serum feast preparer is called by Wright and Douglas " opsonin " (from a Greek word meaning "to cater for"). They have also shown that during the process of active immunisation the opsonic value of the serum is increased, and they have succeeded in demonstrating this opsonic immunity for a number of infections, such 1 Brit. Med. Journ., 1902, vol. i, p. 73. 2 Proc. Boy. Soc. Land., B. lxxii, 1903, p. 357 ; B. lxxiii, 1904, p. 128; B.lxxiv, 1905, pp. 147,159; B.lxxvii, 1907, p. 211. Also vf. Practitioner, May. 190S ; various papers in Lancet and Brit. Med. Journ. ; A\ right, Studies in Immunity, 1909. Opsonins 221 as the staphylococcic, Malta fever, jmeumococcic, and tuberculous. If it be desired to measure the quantity of opsonins present, say in a case of f urunculosis, which is almost always caused by the M. pyogenes, the following are required : (1) a drop or so of the patient's serum • (2) a drop of serum from a normal person ; (3) a suspension in salt solution, of a culture of M. pyogenes preferably derived from the furuncle ; (4) leucocytes washed free from the plasma. Equal volumes of the patient's serum, leucocytes, and suspension are mixed, drawn up in a capillary tube, incubated for fifteen minutes at 37° C, and films are then prepared and stained. As a jxmtroT a similar mixture is prepared and treated in the same way, but using the normal serum instead of that of the patient. The films are * then examined, and the number of cocci taken up by, say, fifty leucocytes is counted in the two specimens, and a ratio obtained. Taking the figure for the normal serum as 1, that for the patient's serum will probably or 0'6,~d this is termed the " opsonic index" (see below, p. 229). In subacute and chronic local infections the opsonic value of the serum is usually diminished, occasionally increased. In acute infections the index will, as a rule, be low ; in chronic infections which are not strictly localised, e. g. tuberculosis, the index will sometimes be low, sometimes high. A low index generally indi- SSiea-afijnfectiaji, or_a_low power of resistance to the particular organism, or that a chronic but quiescent infection exists ; a high judex may indicate that the Bgisgnjuas had an infection but has overcome it, or has a quiescent infection. The normal index for healthy persons vanes only within narrow limits, from about 0-8 to 1-2 as extremes; an index above or below these values is therefore probably pathological. •222 Manual of Bacteriology By injecting small quantities of a varying consisting of a killed culture, tuberculin, etc., the opsonic index can be raised, and the infection thereby tends to be cured. The first effect of the injection is to cause a fall_ in the __opsojnc_jndex, the " jiegativej^luise^ ' of \\ right, which is usually afterwards followed by a rise, and by properly spacing the injections a considerable rise in the opsonic value may ultimately result. If too much vaccine be given the effect may be to per- manently depress the index_and cause harm instead of good, hence the desirability of controlling all injections by determinations of the opsonic index. This, however, renders the treatment very laborious, and generally by employing small doses and allowing at least a week to elapse between the doses, determinations of the opsonic index are unnecessary (for dosage, etc., see p. 232). By movement, massage, etc., applied at or about the seat of a local infection, bacterial products are dis- seminated which may alter the index ; a process of auto-inoculation may thus result. The opsonic index may_ be used for diagnostic pur- ^ji^ses ; a low or high opsonic value towards a particular organism suggests that an infection by this organism exists or has recently existed. Bulloch came to the conclusion that the blood con- tains a number of specific opsonins, one for tubercle another for M. pyogenes, and so on. Simon, Lamar, and Bispham,1 however, from a number of carefully devised experiments, conclude that specificity of opsonins does not exist, and suggest that opsonins may be a constant quantity, and that the number of organisms taken up by the leucocytes is influenced by a second unknown and variable factor. 1 Journ. Exjoer. Med., vol. viii, 1906, p. 651. Nature of Opsonins 223 Russell1 also concludes that in normal serum the opsonins are "common" and not specific, and can be removed by a number of bodies. In immune serum, on the other hand, both "common" and "immune" opsonins are present, the latter being- quite specific. That is to say, in the process of immunisation specific opsonins are formed, and the increase of opsonins following injection of a vaccine is probably due to the formation of immune opsonins which react specifi- cally. Muir and Martin2 believe that in immune serum a specific, immune, thermostable opsonin is present, and also a normal, thermolabile opsonin. Wright considers the opsonins to be substances distinct from all others, but Metchnikoff, Dean, and other observers suggest that they are identical with the "substance sensibilisatrice." It is doubtful if opsonins are present in more than traces in the unaltered blood plasma : like alexins, they seem to develop as a result of coagulation. The role of opsonins in immunity and in recovery from infection is therefore a complex problem. The opsonic method has been criticised of late. Thus Moss3 says : " None of the present methods of estimating the opsonic content of the blood seems sufficiently accurate to be of practical value"; Fitzgerald, Whiteman, and Strangeways,* m an elaborate investigation, concluded that the method is unreliable. Whereas Wright takes into account the serum only, Shattock and Dudgeon - state that "the cells (i e the phagocytes) vary in value like the serum." It may be granted ^ Johns Hopkins Hosp. Bull., vol. xviii, 1907, p. 252. 2 Proc. Roy. Soc. Land., B. lxxix, 1903, p 187 3 Johns Hopkins Hosp. Bull, vol. xviii, 1907, p 237 i, lm%oTlMeef0r StUdV (Cambridge), vol. 5 Proc. Roy. Soc. Med., vol. i, 1908, "Medical Section," p. 169. 22 I Manual of Bacteriology that the whole truth respecting the opsonic reaction and method is not yet fully known, but many of the criticisms have been based on au imperfect technique. On the whole, it may be said that Wright's method, with careful technique and in practised hands, gives information previously impos- sible to obtain, and the treatment by vaccines is of considerable value in particular cases. Method of Determining- the Opsonic Index.1 The requisites are : 1. The jserum of^frhe pafcjput to be tested. 2. The serum of a healthy person for a control. o. A suspension of the organism for which (lie deter- mination is to be made. 4. A suspension of living leucocytes. 5. Several Wright's pipettes with india-rubber teats or nipples. 1 and 2. The sera. — These two specimens should be taken al about the same time, and the determination should be made as soon as possible. The blood is preferably collected in a Wright's capsule (Fig. 35, d). Both cuds of the pipette arc broken off, and the blood is collected by immersing the bent end in the blood as it runs from a prick in the ear or finger. The capsule should be at least one third filled. For pricking, a flat-pointed needle of the Hagedorn type is preferable; a prick with an ordinary needle does not yield sufficient blood. After filling, the capsule is sealed in the name, the dry or straight end being sealed first. After coagulation the capsule is centrifngalised to obtain clear serum ; for this purpose the capsule is hung by the curved end m the centrifuge. 1 This section is largely taken from the excellent account given by Emery in his Clinical Patholoijy and Hwmaiology (Lewis, 1908). The Opsonic Index 225 Little change in the serum ensues for two to three days if the capsules are kept sealed. 3. Suspension, of the organism. — In the case of tubercle, suitable dead cultures can be purchased. To prepare the emulsion from these, take a small portion (about as big as a grain of rice) and place it in a small agate mortar and grind it up with the pestle ; then add l-o per cent, salt solution drop by drop until about 2 o.o. have been added, continuing to grind meanwhile. This gives an emulsion which contains isolated bacilli as well as clumps. These latter must be got rid of. Fig. 85.- a. Glass pipette, with india-rubber teat for opsonic and to do this it is necessary to centrifugalise for three or four mmutes. With the fcifb^^ gonococcus spontaneous phagocytosis is apt to occur if ordinary (0-8 per cent.) salt solution is used .flu ou£ococf emulsion is prepared hy taki"§- " agai culture not more than twenty-four hours old addmg salt solution (0-8 per cent.), and shaking gently so as to wash off the growth. When the emulsion is made rt must be plpetted off into a small tube and centnfugahsed for a few minutes. The emuls «t not be too th:ck, otherwise the leucocytes will take up an 226 Manual of Bacteriology uncountable number of cocci ; the proper density can be judged by experience alone, but the emulsion should be only faintly opalescent. Emulsions of pneumococci and other organisms are made in the same way. Variations in the number of bacteria ingested may occur according as recently isolated or old strains are employed. Instead of centrifugalising, the suspensions may be filtered through a double thickness of filter-paper. 4. Suspension of living leucocytes.— To prepare this, take about 10 c.c.of physiological salt solution containing Vpercen^oi^odi^^ "ofthe" blood" This must be freshly prepared (or kept sterile which is inconvenient), and the simplest method is to' use "soloids" prepared for the purpose by Burroughs & Wellcome; one of these dissolved m 10 c c of water will yield the solution required. This is put into a centrifugalising tube and warmed to blood- heat. A healthy person is then pricked m the ear or finder, and his blood is allowed to drop into the fluid until 1 c.c. or more has been collected. The tube is then put into the centrifuge, very exactly counter- balanced, and ^qentrifugalised until ajHhe have^Hne-iO-iho bottomland the supernatant fluid is ^^XaTlfthe deposit is closely examined the red_ corpuscles wilU^e^^ **** <**™ with a capillary pipette armed with an mdia-rubbe "ipple, or with a syringe, the whole of the clear fluid is Ze ted off as close as possible to the leucocyte layer, ^w thont disturbing the latter. The tube « then mied with saline solution, the blood and fluid are mixed S hi xture is centrifugalised, and the clear fluid again Zet ted off, and this j^gges^of washing^rep^ted. Next the eucocyte layer with the upper layer of red forpuscles (which also contains leucocytes) is pipetted The Opsonic Index 227 off into a small tube, and the whole is thoroughly mixed by repeatedly sucking into, and expelling from, the pipette. The result is a suspension of living leucocytes mixed with red corpuscles. 5. Wright's pipettes with india-ruhher teats.— These are made of glass tubing drawn out in the blowpipe flame into the form shown in a, Fig. 35, which is two thirds full size. The end of the fine extremity should be contracted as shown in b. Glass tubing must be chosen which properly fi is the teats. The process.— (1) Prepare a pipette by placing an mdia-rubber teat on the thick end. Then with a grease pencil or with pen and ink, make a transverse line about an inch from the pointed end. The volume of fluid contained in the tube between the point and this mark is spoken of as the unit. (2) Haying the patient's serum and the suspensions of leucocytes and of bacteria ready to hand, take the pipette between the index finger and thumb of the right hand and compress the nipple. Immerse the pomt eneath the surface of the suspension of bac 1H and relax the pressure on the nipple until the emuls i n has risen exactly to the mark so that one unit has anTretT^^' ^ ~ from the fin d and relax the pressure again so that a small volume of 1S sucked up. This will be quite easy if the poin - a good one, otherwise it will be difficult o/im baTteria^AhT1 ub'MeTv^ ^ ^ °f Lastly, draw up one unit of the serum Tl ■„ now bp in • u , seium. ihere will now be m the pipette (counting fr0m the nipple 228 Manual of Bacteriology towards the point) one unit of bacterial emulsion, a bubble of air, a unit of leucocytes, a bubble of air, and lastly a unit of serum (c, Fig. 35). (3) Put the point of the pipette on to a clean hollow- ground slide or an artist's porcelain sunk palette, and express the whole of its contents, and mix well to- gether, aspirating them repeatedly into the pipette and expelling without causing bubbles. If bubbles form, a hot wire brought near will quickly dispel them. When thoroughly mixed, aspirate the mixture into the pipette, suck up a short volume of air, and seal the tip in the flame. Then place the pipette point downwards in the incu- bator, or better, in a water-bath at 35° to 37 C°, noting the time exactly, and proceed to prepare a second pipette in precisely the same way, using the same suspensions of bacteria and leucocytes, but the control serum instead of the patient's. Place this, in the incubator or water-bath, by the side of the other, noting the time at which this is done. When each pipette has been incubated for a quarter of an hour it is removed from the incubator or water-bath, the end broken off and the nipple fitted to the thick end; then the contents are expelled on to a hollow slide or porcelain palette and mixed thoroughly together. Films are then prepared. This may be done by depositing a drop in the middle of a large cover-glass (1-inch squares, No. 2), dropping on to it another cover- glass and drawing the two apart. Or the films may be made on slides, for which Wright recommends roughing the slides with emery paper and spreading the film with the sharp edge of a broken slide (see next page). The films then have to be stained For staphylococci, streptococci, pneumococci B coh etc the fiL may be fixed with formalin and stained with The Opsonic Index 229 carbol-thionine or borax-methylene blue, or they may be stained without previous fixing with the Leishman stain. For tubercle, the films may be fixed in a saturated solution of mercuric chloride (one or two minutes), stained in warm carbol fuchsin, decolorised with 2\ per cent, sulphuric acid in methylated spirit, and counter- stained with methylene blue. Wright now uses the whole blood instead of the leucocyte layer only. After the blood has been drawn into the citrated salt solution it is centrifugalised, washed twice with salt solution, the fluid is pipetted off, and finally the corpuscles are well mixed. The various mixtures— washed corpuscles, bacterial suspension, and serum— are made and incubated as previously described. In order to make the film for staining and counting, the contents of the pipette are discharged on to one end of a slide roughed with fine emery paper and the mixture is spread by means of a slide "which has been broken across after notching with a file or glass cutter. The object is to obtain a broken edge having a very slight concavity, and many slides may have to be sacrificed to attain this. The film is spread by drawing (not pushing) along, the leucocytes adhere to the edo-e of the spreader, and finally are deposited mostly at the end of the preparation, the red corpuscles beino- ]effc behind. ° _ Lastly, the films are examined with the oil-immer- sion ens, preferably with the aid of a mechanical stage, and the number of organisms contained in not less than fifty polymorphonuclear leucocytes is counted. Parts of the film in which the cells are broken down or not well stained, or cells containing obvious clumps of organ.sms, should be avoided. The ratio between the number m the control and the number in the specimen prepared with the patient's serum gives the opsonic 230 Manual of Bacteriology index. Thus, if in the control there are 125, while in the patient's specimen there are 75, the index would be VW = 0"6, i. e. not much more than half the normal. Preparation of vaccines for treatment, etc. — The vaccine used for treatment is ajsterilised, standardised suspension of the infecting organism, except in the case of tuberculosis, for which tuberculin (TR or BE) or an analogous preparation is employed. In certain instances a mixture of organisms is used — e. g. M. pyogenes, var. aureus and var. albas, with or without the acne bacillus in some cases of acne — and the strain of organism isolated fiom the lesion is generally to be preferred. The vaccine is prepared by growing the organism under appropriate conditions, the staphylococcus on agar, the strepto- coccus, pneumococcus, and gonococcus on blood-agar, etc. The growth is then emulsified by adding a few drops of jsterile 0 ] per cent, sodium chloride solution and well rubbing up with a sterile glass or aluminium rod. Two or three tubes are treated in this way ; the emulsion is poured into a small sterile Erlenmeyer flask of stout glass, the tubes are rinsed out with a little more of the salt solution, and the washings added to the contents of the flask, two or three sterile glass beads are added, and the flask is shakenjigorously for some minutes thoroughly to break up the masses of organisms. The contents of the flask, which should measure 5 c.c. or thereabouts, are then centrifugalised for some minutes, and the emulsion is poured off from the deposit into a second sterile flask and is now ready for standardisation. ^StaudarMsation is carried out by Wright's method. Two_ or three volumes of citrate solution are sucked up into a pipette~liuch. as that used Tor opsonic^eterad~nations, the fingeris pricked and one volume of blood is taken irp in the pipette, separated from the citrate solution by an air-bubble, and finally one volume of the bacterial emulsion, also separated from the blood by an air-bubble, is taken up. The whole contents of the pipette are then well mixed by expelling on to a clean slide and sucking up three or four times. About one third of the mixture is then transferred to each of three clean Standardisation of Vaccine 231 slides, and the drops are spread with the edge of a slide so as to obtain thin uniform smears. These are allowed to dry, stained with Irishman's stain, and the number of red cor- puscles and bacteria is counted _in_a number of microscopical fields. Assuming that there are 5,000,000 red cells in a cubic millimetre of blood, it is easy to calculate approximately the number of bacteria contained in the emulsion. Suppose that 500 red cells have been counted, and with these 1500 bacteria are admixed. Since equal volumes of blood and emulsion have been taken, one cubic millimetre of bacterial emulsion . 5,000,000 x 1500 , will contain — 5QQ - = 15,000,000 bacteria. But one cubic centimetre contains 1000 cubic millimetres, there- fore the emulsion contains 15,000,000 x 1000 = 15,000,000,000 bacteria per cubic centimetre, and by appropriate dilution any bacterial content of the emulsion may be obtained. Thus, if 1,000,000,000 organisms per cubic centimetre is desired, 1 c.c. of the emulsion must be diluted with 14 c.c. of salt solution. _To the prepared dilution of the bacterial emulsion Q-5 per ~~cenr. of carbolic acid, or 02 per cent, of trikresol, is added, and the flask is placed in a water-bath at 56° to 60° CTfor" one or one and a half hours, according to the resistance of the organism. The stock solution may subsequently be intro- duced into small sterile glass serum bulbs of 1-2 c.c. capacity, and the bulbs, after sealing and standing for twenty-four hours, may again be sterilised for an hour at 60° C. to ensure the destruction of the organisms; cultures may be made from the sterilised vaccine to ascertain that this is the case-. The lower the temperatm-P flipjessthe heating^ consistent with sterilisation, the more active will be the vaccine. ~ The table 1 on the next page gives an idea of the doses of vaccines, their toxicity, and frequency of inoculation. The smaller doses are given at the commencement of the treatment, and the doses are gradually increased. The writer has employed endotoxin solutions as vaccines avid believes they are very efficient. 1 See Harris, Practitioner, May, 1908, p. 647. 232 Manual of Bacteriology Vaccine. Tuberculin B. coli Pneumococcic Streptococcic Staphylo- coccic ■ If. melitensis Gonococcic Relal ive toxicity. Doses. Very toxic Very toxic Less toxic than B. coli More toxic than pneumococcic Less toxic than streptococcic Slightly toxic i 2 000 mOIU 5 - L5 millions 10-50 millions 20-60 millions 100-1000 millions -jL sq. cm. of surface agar culture (because very difficult to couut) 100-5UO millions Frequency of inoculation. Every 10-14 days. Every 2, 5, or 10 days. Every 36-48 hours in pneu- monia ; every 10 days in chronic infections. Every 7-14 days. Every 10 days. Every 7-14 days Every 7-14 days. Prophylactic vaccine^- In addition to the therapeutic "v^nes for the treatment of the declared disease, vaccines are also employed iox^revention^ disease. The preventive or prophylactic vaccines may be : (1) ' Li vino-, bjij ntt,pniintlp4,_cnltures, e.g. anthrax and cholera^This method has also been proposed for plague, and vSia must be regarded as of this nature (this is the " Pasteurian method ") . (2) Kdledcujlures, autolysed cultures, and end^Ktoxins. The fost"~and second are used for typhoid, plague and dysentery, and Hewlett has suggested endo-toxms for typhoid, cholera, plague and diphtheria. (3) Immune sera give protection for a limited time. (4) Besredka has suggested "sensitised vaccines," i.e. killed cultures saturated with the homologous immune body derived from an immune serum. He claims that these give rise to much less reaction than the killed cultures. (For further particulars, see Hewlett's Serum Therapy, Ed. 2, J. & A.. Churchill, 1910.) Suppuration and Septic Conditions 233 CHAPTER VI. SUPPURATION AND SEPTIC CONDITIONS. The subjects of septic infection and of suppuration are of great practical importance, and a knowledge of their aetiology is one of the main factors which have conduced to the great advances that were made during the Victorian era in the treatment of wounds, whether accidental or made by the surgeon's knife. Ogston in 1881 and Bosenbach in 1884 demonstrated that micro-organisms are almost invariably present in the pus of acute abscesses, and these observations were repeatedly confirmed by subsequent investigators. A number of experiments were then initiated in order to ascertain whether these organisms bear a causal relation to the phenomena of suppuration or are merely accidentally present. These experiments showed that a large number of organisms can produce suppuration, and render it certain that in ninety-nine cases out of a hundred the suppurative and septic conditions met with spontaneously, or occurring after surgical inter- ference, are due to the action of micro-organisms, ine chief of these are several micrococci (commonly known as staphylococci, and the infections which they produce, as staphylococcic infections) and streptococci. Under the terms "suppuration" and "septic diseases" are included such varied conditions as abscesses, boils and carbuncles, cellulitis, osteomyelitis, erysipelas, gonor- 234 Manual of Bacteriology rhoea, infective endocarditis, pyasmia, septicaemia and sapraamia, puerperal fever, and hospital gangrene. As will be gathered from the descriptions of the individual organisms, suppuration may be set up by inoculation with several species, and a number of experiments by various observers, carried out by inunc- tion, subcutaneous inoculation, and inoculation in the serous cavities and circulation, have conclusively proved that this is the case, not only in animals, but also in man. A problem of great importance is whether micro- organisms are usually the cause of suppuration, or whether mechanical injury, chemical agents, etc , can also produce it. Mechanical injury alone does not seem to be capable of inducing pus production, but it is otherwise with regard to chemical agents. For a lung time considerable difference of opinion existed and discordant results were published. These dis- crepancies have now been explained, and are found to depend upon the method of experiment and the par- ticular animal and chemical agent employed. That chemical agents should produce suppuration was only to be expected, for it would be against analogy, derived from all other bacterial diseases, if the pyogenic organ- isms do not produce suppuration through the chemical substances formed by, or present within, their cells, and if these chemical substances act thus, why should not other chemical substances be found to act in a similar way ? In experiments with chemical agents the greatest care has to be taken to exclude the entrance of micro- organisms. This is best done by sealing the sterilised substance in sterilised fusiform glass tubes and intro- ducing these under the skin or into the tissues with strict aseptic precautions. When the wounds have completely healed the tubes are broken by pressure Saprsemia and Septicaemia 235 and their contents allowed to diffuse into the sur- rounding tissues. Sterilised cultures (above a certain amount) of the Micrococcus pyogenes and a crystalline body, phlogosin, obtained by Leber from its cultures, produce abscesses on inoculation. Mercury produces suppuration in the dog-, but not in the rabbit ; silver nitrate (5 per cent, solution) has a similar action. Ammonia fails to pro- duce pus ; it is either absorbed without damage, or if in stronger solution produces necrosis of the tissues. Turpentine produces large sterile abscesses in carnivore and Brieger's cadaverine is likewise stated to set up suppuration. Buchner was also able, by warming various bacteria with 0-o per cent, caustic potash, to obtain a solution containing protein which was powerfully pyogenic, and JVannotti found that sterilised pus had a similar pro- perty. It thus seems certain that a number of chemical substances can set up suppuration. At the same time, it must be clearly recognised that suppura- tion and suppurative complications, as they occur naturally, are to be regarded as due to the activity of micro-organisms in almost every instance Of so-called "septic" diseases, sapraBmia, septicemia, meantyTeia T^' * ''-P-W'i meant the constitutional condition arising from the absorption of the toxic products elaborate! bv micro organisms, the latter being localised and absent r 2 the general circulation. In the acute form it is not a "a^e ltl01\tL? ^ ^ing that wnth g uuD tne litems the symptoms rapidly abate In septmamna not only is there usually (though W necessanly) a local site of infection, but in addife m.oro-orgau.ms a,, present iu the general oloula ion. 1 236 Manual of Bacteriology It is true they are not abundant in the latter situation, and Cheyne1 believes that they are to a large extent arrested in the capillaries. Micrococci and strepto- cocci are the commonest forms. Pyasmia is charac- terised by the presence of micro-organisms, most frequently streptococci, in the general circulation, and in addition by the formation of abscesses in various situations. These arise usually from suppurative phlebitis with the formation of septic emboli and thrombi. The sequence of events, according to Cheyne (loc. cit., p. 831), is {a) phlebitis in direct connection with the wound ; (b) a thrombus impregnated with micro- organisms is formed in the vein ; (c) this softens and disintegrates, and particles or emboli are carried to distant parts ; (d) these lodge in the capillaries, with the formation of infarctions and abscesses. Suppurative pylephlebitis is a pyaemia affecting the portal system of vessels. As regards the so-called chronic pyaemia or multiple abscesses, Cheyne considers that it differs from true pyasmia in that embolism plays no part. Organisms gain access to the blood-stream, settle in any spot where the vitality of the tissues is depressed, grow and multiply, and there produce an abscess. The mere presence of micro-organisms does not always suffice, however, for they may be present without producing suppuration; and the same organism, tor example, the Streptococcus pyogenes, may at one time produce a localised abscess, at another diffuse cellulitis and at a third pyaemia ; a number of factors control and modify the occurrence and the particular form of septic disease. . . ^o As already mentioned (p. 208), many micro-organisms when iniected into the blood-stream are rapidly deposed 'of - so when moderate quantities of the ZUcrococcus i System of Medicine, Clifford AUbutt, ed. 2, vol. i, p. 876. Micrococcus Pyogenes 237 pyogenes are injected into the circulation of a rabbit, abscesses, as a rule, form only in the kidney. If, however, the organisms be attached to gross particles, so that they cannot pass through the capillaries, em- bolism occurs and abscesses form about the embolic foci. The virulence of the infecting organism, which varies much, is another factor of great importance. The effect of inflammation and injury in making a part "susceptible" is also very marked. Inject the M. pyogenes into animals in which the endocardium or a bone has been damaged, and in all probability an endo- carditis or an osteomyelitis will ensue. The dose and concentration of the organisms are also important factors. Watson Cheyne found that 250,000,000 cocci (M. pyogenes) injected into the muscles of a rabbit produced a circumscribed abscess, but 1,000,000,000 caused a general septicaemia and death. So, probably, while the cells in a healthy wound can dispose of a few organisms, if the latter are abundant or in masses they may gain the mastery. Micrococcus pyogenes, var. aureus (Staphylococcus ^pyogenes aureus). Morphology and biology. — A minute spherical organism measuring about 0'75 ft in diameter. It generally occurs in more or less irregular groups, but may be met with singly or in pairs (Plate I., c). It is fi J} I rS'm0fclle- doesnot_form spores, and stains well withiT^" aTTthe amlin dyes and also by Gram\s__metlind It isSjM^ aerobic and facultatively anaerobic, will develop m%2uij vacuo, and grows well and rapidly on all the usual culture media at temperatures from 18° to ;57° C On agar- agarjfc forms a jhickish, moist, shining growth, cTeW" Ct^C\ coloured at fir^buT^teT-a day or two"5e7el0pi^T ' ' 238 Manual of Bacteriology *fl¥i o s 93 •A Streptococcus' pyogenes . Streptococcus salivarius Streptococcus a nginosus Streptococcus fcecalis . Streptococcus equinus . Streptococcus pneumonia (pneuinococcus i + + + ± ± ± + + + + + + + + + + + + ± + 1 _ ± ± + + + + ± ± + + In li 1/ US hrevit longus brems brcvin hrevit + + + + 0 + (i "6 + = Positive or acid-production. - = Negative or no acid-production. ± = Acid-production sometimes present, sometimes absent. (These differences are not constant ; with various strains one or other reaction may lie lacking.) 1 Lancet, 1906, vol. ii, pp. 708, 775, 852. 2 Ibid., November lltli, 1905, and Rep. Med. Off. Lor. Gov, Board for 1903-04, p. 388. :l Proc. Roy. Soc. Land., b. vol. lxxxiii, 1911, p. 541, Bacillus Pyocyaneus 249 Bacillus pyocyaneus. This is the organism found in green and blue pus, and it also occurs on the surface of the body. Its presence in wounds greatly retards healing, and occa- sionally a general toxaemia may result from it. It has been met with in otitis media and in the green pus of the pleural and pericai'dial cavities. It is a slender bacillus measuring 3 to 4 /n, frequently united in pairs < and forming filaments. It is actively motile, does not Mw-^D/i -4- form spores, and is aerobic and facultatively anaerobic. SJtyf^j Q It does not stain by Gram's method"! Un gelatm it ^ grows freely with rapid liquefaction, a greenish, fluores- V^^Vv cent colour developing in the liquid, while whitish \$JP -I , flocculi of growth sink to the bottom. On ao-ar a whitish, moist layer develops, and the medium is stained a greenish colour. On potato the growth is a dirt}^ brown or sometimes greenish. Milk is coagulated and a greenish colour develops. £ry exposure to a temperature of 60° C. for ten minutes. Pathogenicity. — The gonococcus isa strictly parasitic__ organism, and seems exclusively to jattack man. From inoculation experiments on the human subjects it appears to be the specific organism of gonorrhoeal urethritis and vulvitis. In the female it is most frequent in the urethral or vulvar discharge, less so in that from the cervical canal, and is rarely or never seen in a purely vaginal one. It is generally, even at an early stage, associated with other organisms, particularly other diplococci (see table, p. 261) which have to be distinguished from the gonococcus. The features which serve to identify the latter are its shape and size, its non-staining by Gram's method, its arrangement in groups ^wit hin the pus-cells^ absence of growth on ordinary media, the characters of the colonies, and the < fermentation reactions. The gonococcus is associated with a variety of lesions besides those already mentioned, viz. epididymitis, ovaritis, salpingitis, cystitis, peritonitis, arthritis, and conjunctivitis. It has been met with in the blood, and occasionally produces endocarditis, pericarditis, and meningitis. The gonococcus is fatal to guinea-pigs and mice by intra-peritoneal inoculation. Toxin, anti-serum, and vaccine. — Christmas1 found that the blood-senimof the rabbity fluid or c^agilated, is an excellent_culture mediumjor the gonococcus. By culTrv^tinY~thT gonococcus "for ten days in an ascitic bouillon mixture he succeeded in obtaining a toxin which, when injected intra-venously into rabbits in large doses, caused death, in smaller doses fever and loss of weight while precipitated with alcohol and injected into the anterior chamber of the eye it produced severe i Ann. da I'Inst. Pasteur, xi, 1897, p. (309. Diagnosis of Gonorrhoea 259 inflammation. By injecting1 7-abbits with small closes of the toxin immunisation was produced, and the blood acquired antitoxic properties. _A vaccine may be pre- ^ pared by sterilising cultures with' heat,, and has proved") of service in chronic gonorrhceal infections, fay ^i£vtt^t\* Clinical Diagnosis. The diagnosis of gonorrhoea is very important, not only in clinical but also in medico-legal cases. For this purpose microscopical examination and culture methods are made use of. In a chronic gleet the material must be examined care- fully and repeatedly. (1) Microscopical examination. — Several thin smear speci- mens of the pus or discharge should be prepared. If the best results are desired the films should be _air dried, and then fixed by placing in a mixture of equal parts of alcohol and ether for fifteen minutes. After fixing, a couple of the films are stained in Loffler's blue for five to ten minutes, washed in water, dried and mounted. Irishman's stain also gives good results, the films being merely air-dried and not fixed. The preparations are then examined with a xV-inch oil-immersion ; a lower power lens is useless. The "ovoid cocci in pairs, and occasionally in tetrads, occurring within the pus-cells in groups of not less than four pairs are very characteristic. Diplococci situated outside the pnR-p.ePg J^]dbjLiH3^^ to bT^oted that The nuclei of the pus-cells are deeply, the cytoplasm only faintly, stained with methylene blue). The next step is to ascertain the staining reaction by Gram 'sjnethod ■ Stain two more films for fifteen minutes m amlin gentian violet, dip in water, place in Gram's iodine solution for two minutes, decolorise in absolute alcohol until the drainings fail to stain white filter paper and counter stain for forty.five seconds in a saturated aqueous so uW of Bjsjmrck^brovvn diluted with three times its volume of distilled water. The gonococci are decolorised 260 Manual of Bacteriology and take up the brown stain. In chronic urethritis the urine may be centrifugalised, and preparations are made from the deposit and threads and stained. (2) Culture methods. — Whenever a diagnosis is of great importance an attempt should be made to cultivate the organism. Plate cultures of agar smeared with_ blood as described (p. 257) and another set with agar only should be prepared and incubated at 87° C. In forty-eight hours colonies of the gonococcus should be recognisable on the blood-agar. but y^t, mi the plain agar. If cultures are obtained, the fermentation tests (p. 261) may be applied. N.B. — The greatest caution must be exercised in declaring a case free from infection on the ground of negative results of the examination. Micrococcus catarrhalis.1 This organism occurs in the nose and throat in cases of catarrh, and particularly in the "influenzal cold" (see " Influenza"), in bronchial catarrh, and occasionally in other conditions and in well people. Morphologically it occurs in pairs and tetrads, often within the polymorphonuclear leuco- cytes. It is Qram^ie^atiye^ The primary generation develops feebly on agar, but subsequent generations grow fairly well, forming whitish translucent colonies. Blood or ascitic media should be used for isolation. Some of the fermentation reactions and a comparison with other G-ram -negative cocci are given in the table on the next page. Micrococcus tetragenus. This organism is frequently met with in phthisical cavities and may be expectorated in the sputum, and has also been found in the pus of acute abscesses. The cells occur singly » See Gordon, Brit. Med. Journ., 1905, vol. ii, p. 423; Arkwriglit, Joum. of Hygiene, vol. vii, 1907, p. 145. Gram- Negative Cocci 261 The Characters of the Chief Gram-negative Cocci (Gordon), Organism or source. M. catarrhalis. Nasal and pharyngeal dis- charge Jlf. intracellularii (.meningococcus). Cerebro-spinal menin- gitis M. gonorrhtece fgono- coccus). Urethral discharge From nasal discharge from Hertford cnse of influenza-like epi- demic (see " Influ- enza ") lb From urethra M. neliteniit. Malta fever o Growth on Growth on Pathogenicity. a '/. O CO o u 3 nutrose ascitic gelatin at at o 43 O tn o A agar at 37° C. 20° C, a =1 5 ■z "5 43 ■~> a 1 w upaifiic, - - Pnsif.ivp i DTflWfi i yj in i > ' i^ i u vv o Alice and o 0 o 0 L; I )l [1 U III I on ordinary guinea-pigs bjr intra- peri toneal inoculation only agar at 37J C.) i^ieai, OLUUULU ^pcrn t,\ VP, XI I. 1^ (1 11 V KJ Tti SflmP pnspc • _l_ _{_ o mice and o*n i ii pft -ili trt; liv intra- pei'i toneal innpiilH t.i nn cn'flivtll ^PffH t,i VP XI GgdUl V l. lb"! + _i_ o o n*nlpCG lilnnfl added Clear, smooth, Negative at Mice find o _l_ o later becomes first, positive later (grows on guinea pigs by yellowish intra-peri toneal ordina» y ngar at 37J C.J inoculation Opaque, granu- Negative lb. + + + + lar Opaque, some- Positive + + + + what granular, smooth edges Creamy and Positive Monkeys. Also 0 0 0 slightly rabbits and guinea-pigs by intra-cerebral | yellowish inoculation + = acid. = alkali 0 = no action. (diameter 1 /*), in pairs, or in fours, and are enclosed within a capsule. It stains with the ordinary anilin dyes and also by G-ram's method. Ou gelatin it develops slowly, with the /•/ A f\ formation of a thick, white, shining growth without liqne- rX\ U* faction. On agar the growth has much the same characters, and on potato is white and viscous. Inoculated into animals, particularly mice, a local abscess may form, but usually it produces a fatal general infection, and the organism is found in the blood and organs. A few cases of general infection in man have been described. 262 Manual of Bacteriology Sarcina ventriculi. An organism occurring in the contents of the stomach, especially in cases of dilated stomach. Originally described by (roods ir in 1842. It occurs as a large ovoid cell, several of which are grouped together quadrilaterally so as to form more or less cubical masses, the so-called " woolpacks." According to Falkenheim, it forms on gelatin in thirty-six to forty-eight hours roundish, prominent colonies of a yellowish colour, and in neutral hay infusion a brownish film and flocculi. It produces an acid reaction. Other sarcinse also occur in the stomach. Clinical examination. — 1. The organism can be detected in the vomit, etc., most readily by examination in the fresh state, a little of the material being placed on a slide, diluted with water if necessary, irrigated or not with iodine solution, covered with a cover-glass, and examined. 2 Cover-glass preparations may be stained with weak carbol fuchsin, or by Gram's method. Other Organisms met with in Suppurative and Septic Conditions. Many other organisms may be met with in various suppura- tive and septic processes, e. g. : a. The B. coli in cystitis and pyelitis, ischio-rectal abscess, peritonitis associated with perforation and intestinal ob- struction, and puerperal fever (see Chapter X). b. The Diqjlococcus pneumoniw in abscesses, empyema, arthritis, meningitis, pericarditis, peritonitis, etc. (see Chapter XII). c. The B. typhosus in abscesses, cholecystitis, empyema, and osteomyelitis (see Chapter X). d. The B. cedematis and B. Welchii in foul, gangrenous wounds (see Chapter XIII). e. The B. tuberculosis and B. mallei (see Chapter IX). /. The actinomyces and *trejdothri% forms (see Chapter XV ) . Coley's Fluid 263 g. Blastomycetes, Sporotrichon (see Chapter XVI) and Hyphomycetes (see Chapter XVII). A. The Amoeba coli (see Chapter XVIII). i. Capsulated bacilli (see note, p. 271). Coley's Fluid. This preparation consists_o£ the toxins of the streptococcus of erysipelas and the B. prodigals. It was devised by W. B. Coley, of New York, as a cure for inoperable malignant tumours, particularly sarcoma^ The treatment is based on the undoubted fact that malignant growths may decrease or even disappear completely after an attack of erysipelas (p. 244). Originally prepared by growing a virulent_strepto^ coccus obtained from a fatal case of erysipelas in bouillon for about ten days; the culture is then inoculated with tlie_.R_ jyvdigiams- and the two are allowed to grow together for another week or ten days. The culture is finally heated to from 58° to 60° C. fovone hour, and a pJece_orthymol added to preserve it The fluid is now prepared by growing the organisms separately and then mixing the two sterilised cultures in proper proportions. The fluid is injected subcutaneouslv in the vicinity of the tumour. The primary dose recommended is i minim of the fluid. The dose is gradually increased each day until there is a temperature reaction of 103° to 104° F. Full particulars will be found in Coley's paper (Proc. Boy. Soc. Med., vol. in, 1909-10, Surg. Sect., p. 1). Manual of Bacteriology CHAPTER VII. ANTHRAX . Anthrax is essentially a disease of cattle known as splenic fever, and though occurring in England only sporadically, or in small outbreaks, in some parts of the world it assumes serious proportions— as in Siberia, where it has been termed the Siberian plague. In France also at one time it ravaged the sheep to such an extent as to threaten them with extinction. Man is also occasionally attacked. Anthrax was the first disease to be definitely asso- ciated with a specific micro-parasite, for the organism was observed as glassy homogeneous rods and filaments in the blood of infected animal so long ago as 1849 by Pollender and 1850 by Davaine, and the latter also claimed in 1863 to have demonstrated by inoculation experiments the causal relation of the -organism to tlie disease. Davaine's experiments were made by inocu- lating an animal directly with the blood from an infected animal, and were, therefore, hardly conclusive, as they did not comply with the second and third of Koch's postulates, which declare that the micro-organism must be cultivated outside the body, and the cultivated organism must produce the disease on inoculation, and the objection was raised that infection was due, not to the bacillus, but to something else in the blood. This objection was subsequently removed by the work of Pasteur and of Koch, who obtained pure cultures of the organism, the Bacillus anthracis, and with these PLATIi IV. a. Bacillus avthracis. Smear of blood of inoculated guinea-pig. x 750 7i' Manual of Bacteriology Sidney Martin,1 by growing the anthrax bacillus in alkali albumen for ten days, obtained from the culture albumoses and an alkaloidal substance. Prom the bodies of animals which had died of the disease, chiefly from the spleen and blood, he obtained similar sub- stances, the amount of alkaloid being more than double that of albumose. The mixed products produced fever in animals followed by coma and death. The albumose was proved to be the fever, and the alkaloid the coma, producer; the latter also caused a spreading oedema at the seat of inoculation. Anti-serum . — An anti-serum for anthrax was pre- pared by Marchoux by immunising sheep by vaccination a nd then inoculating with progressively increasing doses of virulent anthrax cultures. Sclavo has prepared an anti-serum by first immunising asses with a vaccine and then inoculating them with increasing doses of virulent cultures over a prolonged period. This serum has been used successfully in many cases of anthrax in man. Vaccine. — An attenuated virus has been extensively employed for the 'prophylactic vaccination of cattle and sheep. Cultures are attenuated by growing at 42°— 43° C. (Pasteur, Chamberland, and Roux). A weak vaccine is first injp.c.tpdJ followed after ten to twelve days by an injection of a stronger vaccine. The mortality as a result of the vaccination is small and the animals are subsequently protected for some months against the virulent disease. Sobernheim has applied a combined method, o-15 c.c. of anti-anthrax serum beingjnocu- "lated on one side of the animal, and the vaccine on the_ other. This practically eliminates all danger from the vaccine. 1 Brit. Med. Joum., 18V2, vol. i, p. 641. Diagnosis of Anthrax 27 ■> Clinical Examination. (1) In veterinary practice. — If au animal is suspected to have died from spleuic fever, an extensive post-mortem is in- advisable because of the risk of distribution of material contain- ing bacilli with subsequent development and dissemination of spores, with infection of pasture, etc. The abdomen should be - . opened and the spleen examined. If this is found to be much "^^-CC^ enlarged, and so soft that it can hardly be handled without rupture, there is a high probability of splenic fever, which the history of sudden death, with or without symptoms, coupled with a sanguineous discharge, increases. To confirm the diagnosis, some smear preparations should be made, from the spleen and blood, and can be stained and examined on arriving home. If slides or cover-glasses are not available, the ear or a small piece of the spleen may be removed and taken home, where the specimen may be examined. When material is sent from a distance for examination the ear should be forwarded. The smears may be stained with Loffler's blue and by Gram's method with eosin. Methylene-blue staining gives the most characteristic appearances, which are numbers of large bacilli forming chains of five or six segments with a pale halo round them resembling a capsule, and if the post-mortem has been made shortly after death no spores are visible. Unless the material be quite fresh large saprophytic bacteria somewhat resembling anthrax are always present and must not be mistalcenfor that organism. If a hanging-drop preparation can be made, a characteristic is the^on-motility of the bacilli. The stained preparations can be kept and produced uTa court of law if necessary. Cultivations can also be made from the spleen, but the necessary culture media are not of course usually forthcoming. Finally, a guinea-pig or mouse may be inoculated subcutaneously in the abdomen with a particle of the spleen, and after death examined microscopically and by culture methods. As regards the disposal of the carcase of an animal dead from anthrax, this should be Ju^ed if possible, but, failing 276 Manual of Bacteriology this, it may5 be buried in a deep pit, preferably with plenty of lime. All graces of blood and discharge, must be carefully mopped up with a strong lime-A^dlsh or solution of chloride of lime, or other reliable disinfectant. (2) In man. — In malignant pustule, smear specimens should TSe prepared from the fluid of the vesicles or with the scrapings from the incised pustule, or sections of the excised pustule may be made, and stained, some with Loffler's blue, others by Gram's method with eosin. The bacilli are not often met with in the blood, except shortly before death. At the same time cultivations on agar and gelatin should be prepared, and may yield positive results when the microscopical examination has been negative. In the later stages of the disease the bacilli may be difficult to find, even in sections. In all cases of doubt a giiinea-pig__or mrmsp shpnld he bmculated subcutaneouslv with the material, and if the animal dies the diagnosis of anthrax may be confirmed by the characteristic appearances, by a microscopical examination, and by cultivation. Thg_animal experi m en t, i s by far the most _ certain method of diagnosis, a negatiygxesult being nearly as valuable as a positive one. N.B. — It must be noted that both cultivation and inocula- tion experiments may fail to give positive results if the material be old or putrid. Diphtheria t CHAPTER VIII. DIPHTHERIA.1 Diphtheria in England-The Diphtheria Bacillus-The Pseudo- Diphtheria Bacillus-Clinical Diagnosis-The Xerosis Bacillus- Diphtheritic Affections of Birds and Animals. Diphtheria seems to have been known from the earliest ages, being recognised by the classical (medical) writers, and it was epidemic in England and on the Continent during the Middle Ages. Bretonneau 2 experienced an outbreak at Tours, 1818-1821, and gave to the disease the name " Diphterite " (afterwards changed to " Diph- terie") from the formation of membranes which is so marked a feature in it. In England the diphtheria deaths have only been separately scheduled since 1855. Since 1881 until recently there has been a steady increase in the prevalence of diphtheria, particularly in the large towns, but latterly the prevalence seems to be decreasing. As regards croup, it it universally admitted that the vast majority of cases of membranous croup are cases of diphtheria. Diphtheria is distinctly a disease of the young, espe- cially at the ages from two to ten, and this holds good both for London and for England and "Wales. That diphtheria is an infective disease is amply 1 See The Bacteriology of Diphtheria, Cambridge University Press, 1908. 2 See Memoirs on Diphtheria, New Sydenham Soc, 1859. 1 278 Manual of Bacteriology proved by the history of epidemics, and by the recorded cases where the disease has been conveyed from one individual to another. The disease occurs in all grades of severity, from the classical ones with wash-leather-like membrane and great prostration, to those which present a mild tonsil- litis or angina. The bacteriological study of diphtheria was com- menced as long ago as 1882 by two German investi- gators,,Klebs and LiiffW Klebs_especially investigated the pathological hjstology, and ascribed the disease to small rod-shaped organisms, which he observed in the membrane. It was reserved for Loffler to place this observation of Klebs on a firmer basis by the isolation and cultivation of the bacillus from the membrane, and by the production of certain phases of the disease by inoculation with the isolated organism. The cause of diphtheria is, therefore, this diphtheria bacillus, which, from its discoverers, is frequently known as the Klebs- Loffler bacillus. The isolation of the specific organism was by no means an easy matter, as a number of other species of bacteria is frequently associated with it in the membrane, but was accomplished by Loffler by the use of a special culture medium now known as Loffler's blood-serum, which consists of a mixture of blood-serum (ox serum was that originally used) 8 parts and glucose bouillon 1 part, the whole being coagulated (see p. 61). On this medium the diphtheria bacillus grows and multiplies exceedingly well, while the other organisms associated with it in the membrane are to a large extent inhibited in their growth. By rubbing a small piece of membrane from a case of diphtheria over the surface of two or three tubes, or of a plate of Loffler's serum, and incubating at 37° C. for twenty to twenty-four hours, colonies of the The Diphtheria Bacillus 279 diphtheria bacillus will be found more or less isolated according to the number of organisms present m the membrane, and by subculturing from these pure cultures may be obtained. Characters of the Diphtheria Bacillus. Morphology.— The B. diphtheria is a small, delicate bacillus, with rounded ends, measuring 3 or 4 in - length. It is non-motile, does notjknmj^spores, and is Oh(/L£y1 aerobic and facultatively anaerobic, The size varies /f/^J-* somewhat even on the same medium, and three varieties of the bacillus have been described, viz. long, medium, and short, according to the length. These varieties tend to be constant and to breed true. Some of the rods both in cultures and in the membrane have a swollen end, the so-called clubbing, and parallel grouping, both in the membrane and in cultures, is almost universal, the bacilli lying parallel side by side (Plate VI., a). This parallel arrangement arises from the peculiar mode of division of the bacillus. If a cell be observed upon a warm stage it first elongates, then becomes constricted at about its middle, and then suddenly one side of the cell-membrane seems to rupture and one half of the cell bends over to the other, so that the two halves form a V. This mode of division, occurring in contiguous cells and being repeated, and the cells thus becoming more and more crowded together, leads to the arrangement in parallel series. The bacilli are generally joined end to end in pairs, and distinct thread and branching forms, though of rare occurrence, may be met with. On different media the same strain exhibits considerable variation in size. On blood-serum and on gelatin the bacilli are of medium length and on the whole fairly regular in 280 Manual of Bacteriology shape ; in broth they tend to be short and stunted • while on agar, especially glycerine agar, they are much larger than on the former media, and long club-shaped spindle-shaped and barred or segmented involution forms are abundant ; on blood-serum club-shaped involu- tion forms also occur, but sparsely in a young, eighteen to twenty hours' culture, in a forty-eight hours' culture more numerously. Staining reactions.— -The B. diphtheria stains well with the ordinary anilin dyes and is Jxram-positive. YUUAA^ Wlth L°ffler's methylene blue the coloration is usually *V » somewhat irregular, more deeply stained portions alter- nating with paler intervals, the so-called segmentation, and especially marked with agar cultures. The ends of the organisms are also frequently deeply stained, the so-called polar staining while the phenomenon known as " metachromatism ,5Ts often marked both at the poles and also in the rod, appearing as granules of a purplish tint and contrasting with the blue of the methylene 1 blue. With Neisser's stain (p. 308) deep inky coloured (- fiats, appearing somewhat larger in diameter than the rods, occur at the poles of the organism and occasionally at the centre. Cultural reactions. — The diphtheria bacillus is an aerobic and also a facultatively anaerobic organism, and Ct^4^;-* grows well on all the ordinary culture media, forming . cream-coloured growths or colonies.the latter on serum tending to be somewhat flattened, with regular margins. It grows slowly on gelatin, forming a raised whitish growth without liquefaction of the medium, and flourishes in milk, with the production of an acid reaction, but without curdling. In broth some strains give a granular growth on the sides and at the bottom of the tube, the broth remaining clear, sometimes with n thin surface pellicle; other strains may render the PLATE VI. a. The Klebs-Loffler or diphtheria bacillus. Cover-glass preparation of a serum culture, x 1500. b. Section of diphtheritic membrane with Klebs-Loffler bacilli. Gram and eosin. x 750. To face page 280. t WtODU c) 0 *~cXuxf eiuM.A^ ^<^1I^; ^ The Diphtheria Bacillus 281 broth turbid throughout. On potato the growth is slight and invisible. The indole reaction can be obtained in peptone-watery cultures either with or without a nitrite, but the writer has shown that this reaction is due, not to indole, but tn slcatolecarboxylic acid (see below, p. 301). ^ he diphtheria bacillus attacks glucose and lactose ^ < / J y with the formation of acid only, nogas (see Table, p. ^^r^ 306). As regards the production of acid, Neisser \ found that during the first nine hours there is little or^-> . — ^ none ; at the end of twenty-four hours a considerable*-*"*'2*''** quantity has been formed, and the amount increases until the end of the second day, after which the production ceases. The B. diphtherias is agglutinated by the serum of patients and by a diphtheria serum, but the test is difficult to apply on account of the coherence of the growth, is somewhat erratic with different strains, and is of no practical value in the diagnosis of the disease. For the same reasons, the agglutination reaction is of little use for the recognition of the organism and for distinguishing it from the so-called " pseudo-diphtheria " bacilli. The Klebs-Loffler bacillus rptains itsvitality in cultures for a month, and when dried for three or four weeks. According to Welch and Abbot, it is destroyed in ten minutes by a temperature of 58° C. It is readily destroyed by antiseptics when in culture, but in the membrane it is difficult to find an agent which will penetrate and kill the bacilli beneath the surface. The diphtheria bacillus and its characters under cultivation have been described somewhat fully, because of the importance of the identification of the organism as a means of clinical diagnosis. As mentioned at the commencement of this chapter, the clinical dia- 282 Manual of Bacteriology gnosis of diphtheria presents many difficulties, and considerable assistance may be derived from a bacterio- logical examination. The diagnosis is ba,spd nn t.liP presence or absence of the Klebs-Loffler bacillus, either in smears, or in cultivations, made from the membrane or secretion (see p. 306). This method is of very real assistance in doubtful, and especially in mild, cases, which clinically it may be very difficult to decide whether they be diphtheritic or no. The mild cases are those which it is of the greatest importance to identify, especially in schools, for if not recognised the patients may go about and prove a source of infection to all around. The method also affords valuable evidence as to when a case can be considered free from infection ; so long as bacilli are present in the throat infection must be possible, and the length of time for which they may occasionally persist is remarkable. ^In half the cases the bacilli disappear within three days oJL the disappeai-ance of the membrane, in a few cases they linger for as long as three weeks, but occasionally they persist much longer. The writer isolated them for so long as five months' (and virulent to the last) ; and a case is recorded in which they persisted for no less than fifteen months after the attack. In all cases two or three examinations should be made at short intervals with negative results before the bacilli can be pronounced to be absent, and no case should be discharged from hospital until the absence of bacilli has thus been proved. When bacilli persist, treatment with anti- septic sprays or gargles, combined with syringing the nose, may be tried. Syringing the nose is important, for the bacilli probably extend to the post^ia^J^paSeV- where they are untouched bv a throat spray or gargle. Another mode of treatment has also been adopted. A polyvalent anti-mierobic agglutinating anti-diphtheria Diagnosis of Diphtheria ■2M serum lias been prepared, dried, and compressed into tablets, one of which is dissolved in the mouth every two hours, and fifteen minutes after solution the naso- pharynx is flushed with physiological salt solution. While this treatment sometimes succeeds, it often fails. The writer has tried the use of subcutaneous inocula- tions of diphtheria endotoxin (0-2-1 -0-2'0 mgrm.) at intervals of seven to ten days. About half the cases seem to clear after one to three injections. With regard to the value to be attached to the bacteriological examination for diphtheria, while the finding of the bacilli is proof positive of the diphtheritic nature of the affection and its infective nature, their apparent absence is not of so much value, as various circumstances modify the result. For 'example, an unskilled person may not happen to touch the right spot with the swab, or from struggling, etc , on the part of the patient even a skilled operator may fail to reach any but a small portion of the mucous membrane, instead of obtaining a good mop from all over, especially when there are no definite patches of membrane. The use of antiseptic gargles or paints shortly before the swabbing is taken will likewise prevent the growth of the bacilli. It sometimes happens that a very mixed growth is obtained in the cultures, and in such cases the Klebs-Loffler bacillus may be missed. Bearing such sources of fallacy in mind, and making due allowances for them, the negative result of a bacteriological examination may have considerable value in those cases which clinically are doubtful. In no case wlip.ru there, it? r\ rorif^H^h- suspicion of diphtheria should treatment, with antitoxin be delayed mini tlie~baderiological report is obtained. The bacilli from the throat are frequently associated with other organisms, especially micrococci and torn he"; 284 Manual of Bacteriology and those cases in which the temperature tends to be high and the throat fetid are usually a mixed infection of diphtheria bacilli with the Streptococcus pyogenes or Micrococcus miogenes, var. aureus. The fact of such mixed infection cannot, however, be definitely decided from the cultures, as tlese organisms may be present in the mouth or throat without necessarily taking part in the infective process. Nor can the severity of the disease be gauged from the charactei's or numbers of the diphtheria bacilli and other organisms present, though perhaps in a number of cases those which yield practically pure cultures will probably be more severe than the cases which yield cultures with few bacilli. It has been stated that the long form of the diphtheria bacillus is the most, and the short form the least, virulent, the medium being intermediate, but this is by no means a universal mile. Westbrook1 has divided^all forms of the diphtheria bacillus into three groups, .^i^nm^pd hj t.TiP.ir staining reactions with methylene blue. Those with deeply staining granules he calls " granular fornis," those with transverse bands " harred forms," and those staining evenly " solid^forvis.'' Each group is further divided into seven types according to shape and size, the types being designated by the letters A to G and being progressively smaller from A to G. It is sometimes stated that a microscopical examina- tion, unless controlled by inoculation of the isolated bacteria, is unreliable. Such a statement is extremely misleading. If the bacilli which have been cultivated from a suspicious throat possess all the characters of diphtheria bacilli, inoculation experiments are not needed, and if they were performed with a negative result {i.e. the bacteria are not virulent) would prove little, for the bacilli from different parts of a culture i Rep. Minnesota State Board of Health, 1899-1900. Pathogenicity of the Diphtheria Bacillus 285 from a throat often possess different degrees of virulence. Occasionally, it is true, even the expert may he in doubt about a particular bacillus, but such cases are the exception. Here an inoculation experiment may help, but would be of no value if a negative result were obtained. It is absolutely essential in the microscopical examination for diphtheria to use a good lens, proper illumination, and sufficient amplification, not less than 800-1000 diameters. Pathogenicity. — The diphtheria bacillusjs pathogenic for man, fh^JinrsA^y, rabbit, guinea-pig, cat, chicken, ' jjjgelm, and finches, all of which are more or less susceptible, (while mice and rats are immune.^ In man the respiratory tract is usually affected, though the conjunctiva and other mucous membranes, as of the vagina and stomach, and wounds may be attacked. A pseudo-membrane usually forms, consisting of laminae of fibrin entangling a few leucocytes and other cells, and here and there small effusions of blood, together with coagulative necrosis of the underlying mucous membrane, and the bacilli are for the most part located in the superficial layers of this pseudo-membrane (Plate VI., b), though in all cases in which the disease has lasted for any time they are found in the lungs, spleen, and kidneys, and may occur even in the blood. If the patient recovers from the diphtheritic attack, paralytic sequela? are not uncommon and are due to a peripheral neuritis. Pseudo-membranes may be formed by other organisms, e. g. by the streptococus and rjneumococcus "also by the pneumobacillus, Imd occur in" Vincent's j&gina (p. 311), but it" is doubtful whether paralytic" sequelas follow any but a diphtheritic infection. Some remarkable skin affections of an eczematous or ichthyomatous nature nave been found by Hare1 and others to be due to the diphtheria bacillus. 1 Lancet, 1908, vol. i, p. 282. 286 Manual of Bacteriology Another affection which seems to be generally diphtheritic is membranous rhinitis Whereas true nasal diphtheria is a serious condition, membranous rhinitis is seldom, if ever, attended with any risk to life, sequelae do not occur, and it is rare to obtain a history of infection from cases of it. This is extra- ordinary and very difficult to explain, for virulent diphtheria bacilli are abundant in the nose and nasal secretion. Diphtheroid organs can occasionally be isolated from well people and those not known to have been in contact with diphtheria cases. The Klebs-Loffler bacillus can be isolated from the throats of nearly 7 per cent, of the presumably healthy population1 ; in the throats of contacts the percentage rises to~3"3 or more. Murray and the writer2 found diphtheria-like bacilli in 58 out of 385 children (15 per ceut.) admitted into the Victoria Hospital, Chelsea. Ford Robertson believes that diphtheroid organisms — possibly the Klebs-Loffler bacillus itself — may play an important part in the production of general para- lysis of the insane. His views have not gained general acceptance, and Eyre [loc.cit.) found that the percentage incidence of all diphtheroid organisms and of the Klebs- Loffler bacillus in the throats of the insane was not greater than in well persons, and was unable to isolate the B. diphtherias post mortem from cases of general paralysis. Traces of antitoxin can be detected in the blood after an attack of diphtheria, usually at the end of the first week of convalescence : the antitoxin has probably little to do with the actual recovery from the disease (see 4 1 See Eyre, Brit. Med. Journ., 1905, vol. ii, p. 1104. : Brit. Med. Journ., 1901, vol. i, p. 1474. See also Graham-Smith, Journ. of Hygiene, vol. iii, 1903, p. 216. Pathogenicity of the Diphtheria Bacillus 287 p 917) A small__amo""h of antitoxin, has also been n^l^lly found in well peoj^e and iiiuntreated horses. ItTSbeen suggested that nTsueh cases there has been a latent infection with the B. diphtheria, but on Ehvlich s side-chain hypothesis it seems more likely that in such cases there happens to be an excess of the receptors which constitute antitoxin naturally free m the blood. Guinea-pigs are the animals generally employed for experimental workon diphtheroid organisms. In order 'tb^ompare the~effe~cts and virulence of various bacilli it is customary to make the inoculation with a measured volume of a forty-eight hours' broth culture. From O'l c.c. to 2 c.c. of such a culture, according to the virulence, inoculated subcutaneously, is usually required to kill a 250-grm. guinea-pig within three days. At the seat of inoculation hemorrhagic oedema forms, haemorrhages occur in the serous membranes, and especially in the adrenals, while the renal epithelium and the liver-cells undergo cloudy degeneration. Inoculated into the trachea of the guinea-pig, rabbit, and chicken, pseudo-membranes form, and the same occurs with the superficially injured conjunctiva and vagina. It is stated by some that the diphtheria bacillus does not develop on a normal mucous membrane — this must first be injured, and the staphylococcus and streptococcus, so often associated with the diphtheria bacillus in the human subject, may play a part in pre- paring the way for infection by damaging the cells and tissues. Rabbits usually live somewhat longer than the guinea-pig after inoculation and paralysis frequently develops if life is prolonged, simulating the post-diph- theritic paralysis of man. The question of the occurrence of the Klebs-Loffler bacillus in the lower animals is of considerable impor- tance with regard to the spread of the disease and the 288 Manual of Bacteriology conveyance of infection. The so-called diphtheritic - ^affections of pigeons, poultry, and calves (referred to more in detail below, p. 313) are as a rule diseases quite distinct from human diphtheria, and are~not communicable to man. A number of observers assert, however, that cats may suffer from the disease, which in these animals runs a chronic course, and is associated with bronchitis, lobular pneumonia, nephritis, and wasting. JGein1 points ont thai, not only aro catejiable to the disease in houses where diphtheria has occurred, but that a similar infectious disease exists naturally"" among cats, and symptoms similar to this natural disease may be produced by inoculating healthy cats with the Klebs-Loffler bacillus. The diphtheria bacillus has also been isolated from the horse.2 Several epidemics of diphtheria have been traced to an infected milk supply. In some instances the infection has undoubtedly been derived from contamination from a human source, but in others this mode of infection has not been demonstrated, and it has been suggested ■ ' Do that certain eruptive conditions on the teats and udder of the cow may be caused by the Klebs-Loffler bacillus and the milk become infected therefrom. Klein3 made experiments with a view of determining this point. He inoculated healthy cows in the shoulder with a bouillon culture of the diphtheria bacillus. This caused fever and local swelling, and in about a week a papular and vesicular eruption appeared on the udders and teats. The B. diphtherias was isolated from the contents of the ^vesicles and also from the milk on the fifth day, but not subsequently. The cows diedjn. two to four weeks, and the B. diphtherias was obtained from the local 1 Rep. Med. Officer. Loc. Gov. Board for 1889, p. 162. 2 Cobbett, Centr.f. Bakt., xxviii, No. 19, p. 631. 3 Rep. Med. Officer Loc. Gov. Board for 1889 and 1890. Diphtheria Toxins 289 lesions. Abbott1 obtained somewhat different results, but Klein2 points out that these experiments were not performed under exactly the same conditions as his own. Klein, Eyre, Dean and Marshall3 have isolated the diphtheria bacillus from milk. It is to be noted that diphtheria-like, but non-pathogenic, bacilli are often to be found in milk and cheese (see section on "Milk"). Toa;in$. — Diphtheria toxin has not been obtained in a state of purity and its exact chemical nature is unknown. Loffler first investigated the chemical products formed by the diphtheria bacillus, and by precipitating bouillon cultures with alcohol obtained a white toxic substance which he classed among the enzymes. Roux and Yersin precipitated the toxin from filtered broth cultures by means of absolute alcohol, and also by the addition of calcium chloride. They found that 0*4 mgrm. was sufficient to kill eight guinea- pigs or two rabbits, and considered it to be an enzyme. From the blood and spleen of cases of diphtheria Sydney Martin4 isolated albumoses (chiefly deutero- albumose) and an organic acid, but no basic body. Injected subcutaneously the albumose produces much oedema and irregularity of temperature ; in larger doses depression of temperature with paralysis and coma. Small multiple doses, not sufficient to destroy life, may give rise to some fever, and in two or three days to paralysis of the hind legs in rabbits, with general weakness and loss of weight. Post-mortem, thejierves are found to have undergone degeneration — breaking 1 Journ. Path, and Bad., vol. ii, 1894, p. 3.1. 5 Ibid., p. 428. 3 Journ. of Hygiene, vol. vii, 1907, p. 32 (Eefs.). * Brit. Med. Journ., 1892, vol. i, p. 641. 19 290 Manual of Bacteriology up and disappearance of the myelin and interruption of the axis cylinder, while the heart is fatty. The organic acid is also a nerve poison, but is not so toxic as the albumose. From diphtheritic membrane, extracted with a 10 per cent, salt solution, only traces of albumose and organic acid were obtained, but the extract was highly toxic, producing fever and paralysis. Sidney Martin suggests that a substance of the nature of a ferment may be present, and that the ferment in the membrane on absorption may perhaps form the albumose in the body. From cultures of the diphtheria bacillus in alkali-albumin, albumose and organic acid, with similar actions to those isolated from the body, were obtained. Brieger and Frankel (1890) were unable to find any basic substance in cultures, and concluded that the toxic substance was a protein body, which they designated a " tox-albumin." It was destroyed by a temperature of 60° C. but not by one of 50° C, even in the presence of an excess of hydrochloric acid, and hence is probably not an enzyme. The tox-albumin is non-dialysable, is precipitated by saturation with ammo- nium sulphate but not with magnesium sulphate, and hence is neither a peptone nor a globulin, contains a large amount of sulphur, and gives the biuret and Millon's tests. A curious property of this substance is that small quantities (2'5 mgrm. per kilogramme of the body-weight) do not produce their effects until the lapse of weeks. Brieger and Boer in a later research .prepared the diphtheria tox-albumin by precipitating a bouillon culture with a 1 per cent, solution of zinc sulphate or chloride. TlTe precipitate of the zinc double salt was washed with slightly alkaline water and decomposed with a stream of carbonic acid gas. The purified tox-albumin gives the xanthoproteic, biuret, Diphtheria Antitoxin 291 and Adamkiewicz's reactions, and the red coloration on heating with Millon's reagent. According to Ehrlich the toxin broth is a complex mixture of toxic constituents belonging to the proteins, but this is denied by Madsen and Arrhenius (see p. 1 72). Its poisonous property gradually diminishes on keeping, and is destroyed by boiling in five minutes, at lower temperatures more slowly, and also by light. Diphtheria antitoxin. — By the injection of sub-lethal and increasing doses of the toxin into an animal an anti- toxin is generated. For the preparation of a potent antitoxin for therapeutic use the first essential is a highly toxic toxin, and for obtaining this a diphtheria i bacillus of" high virulence is required, and. but few strains possess the necessary virulence. The virulent bacillus is grown in an alkaline broth (rendered alkaline to the extent of about 5" 7 c.c. of normal caustic soda solution per litre beyond the neutral point to litmus) in Erlenmeyer flasks containing half to one litre for eight to twelve days at 37° C. Various small details have to be attended to in order to obtain toxin of maximum toxicity ; it is important that growth should occur upon the surface of the broth. The use of meat some days old has been advocated, or of acid beef-broth in which B. coli has been grown for twenty-four hours, in order to eliminate the glucose (p. 27). L. Martin makes use of " peptone " prepared by the auto-digestion of a pig's stomach with dilute hydrochloric acid. The cultures,, are then filtered through a Berkefeld or Pasteur- _' tflTamberland .filter to remove the bacilli^ The filtra.tjT* is 'gemi^free and very toxic, and a little carbolic acid may be added to preserve it. In New York 10 per cent, of a 5 per cent, solution of carbolic acid is added to the culture, the bacilli are allowed to deposit by standing for forty-eight hours, and the culture is filtered /t' ^297' Manual of Bacteriology ^ > through paper j in this way filtration through a filter- « candle is dispensed with. Less than 001 c.c. of the _toxin should kill a 250-grm. gumea-pig in three to iour days. Selected Jiotges which have been tested with mall em and tuberculin, and kept under observation for some time to ensure that they are healthy, are then inoculated with this filtrate, commencing with a dose of O'Ol to O'l c.c. according to the toxicity of the toxin, or 20 c.c. of the toxin together with 10.000 units of antitoxin may be given for the first three doses. Individual horses vary very much in their susceptibility to the toxin, so that care has to be exercised with the first injections. The injections are given subcutaneously over the shoulder, and produced a local swelling and some rise of temperature and general disturbance, lasting two or three days. When this has passed away the inoculation is repeated, a larger dose being admini- stered provided the reaction due to the former one was not too severe. The treatment is continued for five to six months, the dose of toxin administered being gradually increased until it may attain 500 c.cjir more._ Cartwright- Wood 'found that by growing virulent diphtheria bacilli for three or four weeks in ordinary peptone broth, with the addition of 10 or 20 percent, of blood-serum or plasma, subjecting the culture to a temperature of 65° C. for an hour and filtering before injection, much larger initial doses can be given and some degi-ee of immunisation attained, and subsequently the ordinary broth cultures may be injected in large _doses. Individual horses vary muriijnjhejr^p to yield antitoxin: on the whole those that are moderately sensitive tof the toxin seem to produce most antitoxin ; a horse to be of value should after three months' treatment yield an antitoxic serum containing not less than 300 units per c.c. The required potency having Standardisation of Antitoxin 293 been attained, as shown by the test described below, the horse is bled with aseptic precautions, the blood is , allowed to cpagiilate, and the serum is drawn off and fii]ed_ into sterile bottles each containing a dose of the antitoxic Jfo^^fl seruuT A~small amount of antiseptic, such as tri- kres£L_is generally added as a precautionary measure IcT prevent the multiplication of any stray germs that may have gained access during the various manipula- tions. Standardisation of antitoxin— The potency of diph- theria antitoxin is always described in "units " and is ^estimated by ascertaining the quantity of 'antitoxin "required just to neutralise a certain amount of a standardised toxin when both are injected into a 250- grm. guinea-pigT Formerly, by Koux's method, the minimal letlnilj&QSe of the toxin is nj^t_a^c^rtai]ied, and 41w then the number of grammes of guinea-pig which 1 c.c. T of antitoxin will protect against this minimal lethal dose is determined. If 0-01 c.c. of antitoxin protects a 300- gnu. guinea-pig against the minimal lethal dose, 1 c.c. will protect 300x 100 = 30,000 grm. of guinea- pig, and the immunising value of the antitoxin would be described as 30,000. This method is open to the fallacy that \j only a portion of the lethal dose be neutralised the guinea-pig may survive, and a fictitious value be given for the potency of the antitoxin, fk 1 • Behring_later adopted ten minimal lethal doses as thel^*€^tLiAMJL test dose of toxin, and he termed ten times the amount of ^ « ' , antitoxin which protects a guinea-pig against the ten > t minimal lethal doses a unit (the Behring unit, which Irl/*^f tlierefore = 100 minimal lethal doses of toxin), from which the Ehrlich unit, now universally adopted, is / ^££4^ derived. Though this method eliminates to a large « *„ /^b extent the objections to the lioux method, Ehrlich found that by it the same antitoxin tested with different 294 Manual of Bacteriology toxin broths yielded different values. This he explained by assuming that diphtheria toxin broth contains not only toxin but also other substances which combine with antitoxin. These substances, though non-toxic, or comparatively so, vary in amount in different toxin broths, and variable results, therefore, may be ob- tained by the simple method of testing. These substances, having an affinity for antitoxin, are toxoids and toxone. There are several varieties of toxoids, viz. (1) those having a greater affinity for antitoxin than toxin itself, fn-oloxoids ; (2) those having the same affinity, mjntoxoids ; (3) and those having a less affinity, ept- tovojdJ? Toxoids are probably derivatives of toxin ; they increase in quantity infold toxin broth which has been kept, and which at the same time decreases in toxicity. The toxones also combine with antitoxin, having a less affinity for it than toxin, are primary secretory products of the diphtheria bacillus, and while not acutely lethal, induce induration, necrosis and paralysis. TJue_.toxoid*- are comparatively scanty in a fresh toxin broth and are negligible, but it is otherwise with the toxone, which is always present in appreciable quantity. Owing .to the fact that toxone has less affinity for antitoxin than toxin has, if an exactly neutral mixture of toxin broth and antitoxin be prepared, considerably moi'e than the minimal lethal dose of the toxin bi-oth must be added to render the mixture acutely toxic, because the first portion of the added toxin simply displaces the toxone from its combination with the antitoxin, and is neutralised by the antitoxin so set free. Thus, suppose a certain amount of a toxin broth contains 90 units of toxin and 10 units of toxone, and to this amount 100 units of antitoxin are added so as 1 See pp. 172-175 for other views on the constitution of diphtheria toxin. Standardisation of Antitoxin 295 to form a physiologically neutral mixture, the com- bination which occurs is shown by the following " equation " : 90 toxin-antitoxin + 10 toxone-antitoxin = Ln (i. e. neutrality). If an amount of the toxin '. broth be nowaaaeH; corresponding to 11 units of toxin, the effect will be as though only one unit of toxin has been added, as is shown by the following "equation " : 90 toxin-antitoxin + 10 toxone-antitoxin + 11 toxin = 100 toxin-antitoxin + 10 toxone (free) + 1 toxin (free) = L + {i.e. just acutely lethal). Thus although the equivalent of eleven minimal lethal doses of toxin has been added to the physiologically neutral mixture of toxin broth and antitoxin, only one minimal lethal dose of toxin remains free and active, because ten toxin units displace the ten toxone units from the toxone- antitoxin complex and are neutralised by the antitoxin thus set free. Ehrlicli, therefore, devised a method of standardisation which eliminates irregularities clue to the variable proportions of toxone and toxin in the toxin broth by adopting n.ntitoxm and not toxin tas the standard!? TTi order to standardise an antitoxin, a virulent toxin broth is employed and its minimal lethal ' dose is approximately ascertained — i. e. that amount which is just sufficient to kill , a 250-grm. guinea-pig on the fourth or fifth day. A solution of accurately standardised antitoxin, which can be obtained from the Serurnspriifung Institut, Frankfort-on-Maine, is then prepared, containing one "unit" of the antitoxin in 1 j?.c, and thejtoxin is standardised with this by mixing with one unit various quantities above and below one hundred minimal lethal doses. It is required to ascertain the amount of the toxin broth, which, when mixed with one unit of antitoxin, just suffices to kill a 2o0-grin. guinea-pig on the fourth or fifth day after the injection of the mixture ; this amount of toxin is 2W Manual of Bacteriology known asJl^L^dose^ The L+ dose may be defined that amount of a given diphtheria toxin WTi'^jgh is not completely neutralist by nnp « » »f ^jTmhrnj antitoxin to the extent that exactly one simple lethal dose_of toxin remains unneutralised ; it corresponds usually to jO^l^Q minimal lethal dose's. For example, suppose 0-003 c.cT of the toxin was found to be the minimal lethal dose, with separate "units "of standard antitoxin, 0'2, OS, 0'4 and 0-5 c.c. respectively of the toxin might be mixed, and each mixture injected into a guinea-pig ; probably the guinea-pigs receiving the " unit" of antitoxin plus 0'2 and 0"3 c.c. of toxin would remain alive, while the animal receiving the 0-4 c.c of toxin would die in twenty-four to forty-eight hours. The death in the last case is too rapid ; more than a simple lethal dose has remained unneutralised, and therefore the L+ dose of toxin lies between 0'3 and 0*4 c.c, and further experiments would have to be performed with amounts of toxin between these limits in order to ascertain the exact dose. Death of the guinea-pig on the fourth or fifth day has been chosen because it has been found that if the dose of toxin be diminished ever so little below that producing this result, death does not ensue under nine or ten days. That is to say, an acute intoxication is fatal at the Jatest on the fourth nr fifth day, a fatal result after then being due to a chronic intoxication. The amount of toxin which is exactly neutralised by one " unit " of the standard antitoxin is known as the LQ dose. By exact neutralisation is meant absence of any reaction, general or local, at the seat of inoculation, in the inoculated guinea-pig. If toxin broth were a single substance, containing only toxin, then L+— LQ = D, the simple lethal dose, would be equal to the minimal lethal dose. But because of the presence of toxone, which Standardisation of Antitoxin 297 also has an affinity for antitoxin, D, the difference between the LQ and the L+ doses, is usually a multiple (8-12) of the minimal lethal dose. From these considerations we are now in a position to define n-nit. of antitoxin : aJ^uiuVMsthat amount of antiiaxi n which will neutralise about 100 minimal lethal doses for the guinea-pig- of diphtheria Joxim From certain considerations Ehrlich considers that the unit would ftva.nhly neutralise 200 minimal lethal doses of a theoretical toxin, containing only toxin and neither toxoid nor toxone, but, inasmuch as such a toxin is unknown practically, the unit corresponds usually to 105-120 minimal lethal doses of a toxin broth, the "extremes which Ehrlich has found being 16 and 136 lethal doses. Having standardised a specimen of toxin by means of standard antitoxin, this standardised toxin is in its turn used to standardise the antitoxic serum Avhich has been prepared for therapeutic use. The toxin is preserved by the addition of toluol, and is kept in a cool, dark place ; it needs to be re-standardised Iii standardising antitoxin, the L+ dose of the stan- dar di sed.J.oxJ n is mixed with varying amounts of the antitoxin, the mixtures are injected, into guinea-pigs, and the amount of the antitoxic serum which neutral- ises the L+ dose of toxin is thus ascertained. If, for example, it were found that 0-05, 0*04 and 0'03 c.c. of the antitoxic serum neutralised the L+ dose of toxin, but that the guinea-pig receiving 0*025 c.c. suffered from some local necrosis, wasted, and died in a few days, and the animal receiving 0'02 c.c. died in two or three days, 0*03 c.c. of this antitoxin would be about equivalent to one unit of standard antitoxin, and the antitoxic serum therefore contains 33 units per c.c. For all the experiments the conditions must be kept as 298 Manual of Bacteriology constant as possible, guinea-pigs weighing 250 gnu. or thereabouts employed, and to eliminate irregularities a number of animals must be used. The _ antitoxic constituent of diphtheria antitoxin is globulin in nature, or is intimately associated with the glcdniTin content of the serum. Thus Atkinson found that if the serum is precipitated by saturation with magnesium sulphate, the whole of the antitoxin is carried down with the precipitate, and also that the globulin content of the blood-serum of antitoxin horses is increased. II is results were confirmed by Ledingham.1 • There can now be no doubt as to the value of the antitoxin treatment: of diphtheria. Since the introduction of antitoxin treatment, which was commenced about the middle of 1894, there has been a steady decline in the case mortality from diphtheria, especiall y in London, where probably the majority of the cases are injected with antitoxin. From 1891 to 1894 the ease mortality from diphtheria in the hospitals of the Metropolitan Asylums Board averaged about 30 per cent., in 1895 it was 22"8 per cent., and afterwards steadily fell, until during the last eight years it has ranged between 8'3 and 10 per cent . Not less than 2000 units should be injected for a dose, and early treatment is of paramount importance. As soon as there is a reasonable probability that the case is one of diphtheria the antitoxin should be used, and treatment should not be delayed for the result of the bacteriological examination. The statistics show that in cases treated on the first day of the disease the case mortality is 3-0, on the second day it is 6"5, on the third day 10-6, on the fourth day 12-9, and on the fifth day and afterwards 14-8 per cent. In bad cases, and in those coming under treatment at a late stage of the disease, the dose may be increased to 10,000 20,000, or even 30,000 units with advantage, and to bring the patient under the influence of the antitoxin as rapidly as 1 Journ. of Hygiene, vol. vii, 1907, p. 65. Antitoxin Treatment 299 possible the first dose may be administered intravenously. The dose may have to be repeated once or twice in mild cases, in bad cases perhaps every six or twelve hours until several doses have been given, the guide being the general condition of the patient and the rapidity of the separation of the membrane. In addition to antitoxin, the recumbent nustuj-e and general and local treatment should be pursued as usual. In* cases of mixed infection, in which the diphtheria bacilli are associated with streptococci or staphylococci, diphtheria antitoxin mav prove of less value, as it has no influence on the streptococcic or staphylococcic infection, and injections of anti-streptococcic serum may be given in addition. Diphtheritic paralysis seems to be rather more frequent after the use of antitoxin than in the cases not treated with it, probably because a greater number of cases survive. The antitoxin has also been employed as a prophylactic in schools or other places where susceptible individuals are congregated together, and where cases of diphtheria have occurred, with excellent results. The procedure in such circumstances should consist of a bacteriological examination of the throats of all the inmates in the institution, isolation of those in whom the B. diphtheria} is found, and the injection of everyone, or at least of all children, with a prophylactic dose, repeated if considered desirable, ten days later.1 For this purpose a dose of about 500 units should be given. The immunity so produced does not last for more than three weeks. The objection to the use of antitoxin for prophylaxis is that should the patient subsequently develop diphtheria, treatment with antitoxin may induce serious symptoms due to super- sensitation or anaphylaxis. The writer beleives that all the advantages of antitoxin without its disadvantages may be obtained by the use of a vaccine, consisting of diphtheria endotoxin. Some clinicians assert that antitoxin exerts its effect when 1 On the prophylactic use of antitoxin see Norton, Lancet, 1907, vol. ii, p. 85. 1 300 Manual of Bacteriology administered by the mouth or the rectum. Hewlett was un- able to detect any absorption of tetanus antitoxin from the stomach or rectum, nor Sternberg of diphtheria antitoxin from the rectum, of rabbits. Pseudo = diphtheria and Diphtheria=like Bacilli. Diphtheria-like bacilli are not uncommon in wounds and in pathological exudates, etc., and in connection with diphtheria an important question must be dis- cussed, viz. the occurrence and nature of the so-called pseudo-diphtheria bacilli. The term was originally used by Loffler, and by the rule of priority should be reserved for the organism described by him under this name. The pseudo-diphtheria bacillus of all authors is an organism occurring in the throat in various anginal conditions, scai'let fever, etc., and occasionally in the tln*oats and noses of well persons, and is non- pathogenic to guinea-pigs. Park and Beebe met with it in twenty-seven out of 330 healthy throats examined by them. Boux and Yersin, Abbott and Frankel de- scribe it as morphologically resembling the Klebs- Loffler bacillus, while Loffler, von Hofmann, Koplick, Park and Beebe, Peters, and Hewlett and Miss Knight,1 consider that an organism differing somewhat from the Klebs-Loffler bacillus should alone be termed the pseudo-diphtheria bacillus ; to avoid confusion it is best to designate it the Hofrnann bacillus. Moiyhology. — Typically, the Hofmann bacillus is a shortish rod tapering towards the ends, which are rounded, the average length being from P5 n to 2,u, and it occurs in pairs, resembling two suppositories placed base to base. It is non-motile, does not form scores, is arranged in a parallel grouping like the 1 Trans. Brit. Inst, of 1'rev. Med., vol. i, 1897. The Hofmann Bacillus 301 Klebs-Loffler bacillus (due to the same mode of divi- sion), and involution forms are, as a rule, not met with (Plate VII a) It is Gram-positive, and stains deeply and regularly with Loffler's methylene blue, segmenta- tion and polar staining usually being absent With «fam, no inky granules are perceptible, as is > It ITu^sewjjiLthP^iphthPvia bacillus. f/ I - Cultural o-eactions.-'rhe Hofmann bacillus is almost \ a strict aerobe j there is no growth anaerobically m hydrogen. On Bernm,,agar,_an&^iain it forms cveB^ OyJUcA^H coloured colonies or growths, barely distinguishable I TroTrTfluSse of the Klebs-Loffler bacillus. On ordinary / potato it hardly grows at all, what growth there is being quite invisible. On alkaline potato,* however, it forms distinct cream-coloured colonies, usually visible by the second day. In stab-cultures in gelatin and C^yf Q ^W^P-qgai- Tin_g».« ™ formed, and the growth is con- 6 fined to the upper part of the stab. In broth it forms sometimes a granular deposit, sometimes a general turbidity. On neutral litmus glucose-agar a blue colour is developed, indicating the production of alkalinity. Cultivated in peptone water an indole-like reaction with sulphuric acid alone can be obtained after a variable time, three to four weeks, while the diph- theria bacillus gives it in about a week ; with a nitrite £p K+Qp and sulphuric acid the indole-like reaction, can be $AA~*rv~*~*. obtained with both the pseudo- and diphtheria bacilli in about a week. "The substance giving this indole-like 1m>IU4>«_&1 reaction is not indole, but_skatole-carboxylic acid." A_ broth culture reduces a weak solution of methylene f ^ * >>c blue. The bacillus does not curdle milk or liquefy ]AMj^i \) , * ■ S > 1 Ordinary potato rendered alkaline with a 10 per cent, solution of sodium carbonate before sterilisation. 2 Hewlett, Trans. Path. Soc. Lond., vol. li, 1900, p. 187 ; vol.lii, 1901, p. 113. 302 Manual of Bacteriology gelatin, can be cultivated at from 22° to 37° C. and is non-pathogenic to guinea-pigs in doses of 5 c.c. or more of a forty-eight hours' broth culture. Some of the differences between the Hofmann bacillus and the Klebs-Loffler bacillus are shown in the table on the next page. Mandelbaum and Heine- mann1 state that if a glycerin-agar plate be smeared with human blood and inoculated, the diphtheria bacillus produces colonies surrounded by a yellow zone, while the colonies of the Hofmann and xerosis bacilli do not change the red colour of the blood. In addition, the Hofmann bacillus does not ferment any sugar, etc. (see Table, p. 306). The histories of several cases investigated by Miss Knight and Hewlett seemed to show that the Hofmann bacillus is associated with mild anginal conditions, which are free from complications, end in recovery, and are not followed by sequelae. In many of the cases the anginal condition was associated with distinct patches of membrane, and in two symptoms were present suggestive of the toxaemia which is met with in diphtheria. In . a long series of expei'iments Hewlett and Miss Knight believed that some fwidftnre_jvas obtained of the conversion of the Hofmnun into the Klebs-Loffler bacillus and vire- t-prstn.^. Moreover, the Hofmann bacillus seemed in many instances to replace the Klebs- Loffler bacillus in the throat during convalescence, and it is possible in a large series of cultures to obtain con- necting links between the Klebs-Loffler bacillus on the one hand and the Hofmann bacillus on the other. Cobbett,2 however, suggests that these facts are capable of another explanation, viz. that during the acute 1 Centr.f. Bakt. (Orig.), liii, 1910, p. 356. : Journ. of Hygiene, vol. i, 1901. The Hofmann Bacillus 303 Differences between the Klebs-Loffler and the Hofmann Bacillus. Morphology Staining with Loffler's hlue Neisser's stain . Alkaline potaTb Neutral litmus agar Litmus milk Stab - cultures in glucose agar and gelatin Anaerobic cultures in hydrogen Indole-like reaction, (peptone- water cultures, with sul- phuric acid alone) Fermentation reac- tions Rods 1-5 to 2/i in length, tending to be slightly thicker at the centre than at the ends. Is "plumper," shorter, and less variable than the Klebs- Loffler bacillus Involution forms rare Stains more deeply and regularly than the Klebs-Loffler bacillus. Polar staining rare Negative Distinct cream - coloured colonies or growth visible in two days Alkaline reaction Alkaline reaction Growth only at upper part of stab No Only after three weeks5 growth. ( Due to skatole- carboxylic acid) See table Eods averaging 3 fi to 4 fi in length. Slen- der and (excluding involution forms) of more uniform diameter than the pseudo. Consider- able variation in size. Involution forms ixsually present. Staining generally more or less ir- regular, and polar staining common. Positive. *— Grows well, hut growth is almost invisible. Acid reaction Acid reaction Growth length of stab along whole Grows well. After one week's growth. (Due to skatole - carboxylic acid) . on page 306. stage, diphtheria bacilli being readily found, the Hof- mann bacillus is likely to be overlooked, whereas at a later stage a more careful search may be necessary to detect the diphtheria bacillus, and in the course of 304 Manual of Bacteriology that search the Hofmann bacillus is therefore more frequently seen. Miss Knight and Hewlett came to the conclusion that in some cases, at least, the Fofmaiin bacillns_^s_a. modified Klfths-T,nffjpr bacillus, and the view taken of its relation to the Klebs-Loffler bacillus was, that it is a very attenuated Klebs-Loffler bacillus, i. e. one far removed from virulence. It would therefore seem wise to treat anginal cases in which the pseudo-diphtheria bacillus is found as possibly infective, though it would probably be inexpedient to admit to a general diph- theria ward (unless a prophylactic dose of antitoxin or of an endotoxic vaccine be given), nor would antitoxin be needed in the majority. Most authorities have been unable to convert the pseudo-bacillus into a virulent Klebs-Loffler bacillus, or vice versa, and many are of opinion that it has probably nothing to do with diphtheria (Park and Beebe, Petei-s, Washbourn, Cobbett, Clark). A few fatal cases have been recorded (e. y. by Stanley Kent) in which a careful search has failed to reveal any but Hofmann bacilli. Boycott1 found that the seasonal prevalence of the Klebs-Loffler and Hofmann bacilli does not correspond, the former prevailing during September, October, and November ; the latter is more frequent from May to August. Priestley records an outbreak of what he terms " pseudo-diphtheria," in which the Hofmann bacillus seemed to be the causative organism, and expresses the opinion that this bacillus is not related to the Klebs- Loffler bacillus.2 Salter 3 claimed to have found that the Hofmann 1 Journ. of Hygiene, 1905, vol. v, p. 223. 3 Public Health, July, 1903. :l Trans. Jenner Inst. Prev. Med., vol. ii, p. 113. (Bibliog.) The Hofmann Bacillus 305 bacillus is virulent to many small birds (goldfinch, chaf- finch, canary, etc.), and that by successive passages it becomes converted morphologically into a Klebs-Loffler form with feeble virulence for the guinea-pig. He also found the filtered broth cultui'e of the Hofmann bacillus, though harmless to guinea-pigs, to be toxic to small birds, and that it contains a non-toxic substance (toxoid) which has the power of combining with, and neutralising, diphtheria antitoxin. Salter concluded, therefore, that diphtheritic organisms are to be met with of every grade of virulence, the weakest, known as Hofmann's or the pseudo-diphtheria bacillus, representing the most attenuated form of the Klebs-Loffler bacillus. The writer/ Cobbett,2 Petrie,3 Williams,4 and Clark5 have, however, quite failed to confirm Salter's results. To sum up: the Klebs-Loffler-like avirulent bacilli met with in the thoat, the pseudo-diphtheria bacilli of Roux and Yersin, are probably modified and avirulent diphtheria bacilli. As regards the Hofmann bacillus, the general trend_oj_opiiiion at present is to consider it as quite distinct from the Klebs-Loffler bacillulT ' Another view is to regard it as in reality including several species, of which one may be a modified Klebs- Loffler bacillus, the others having no relation with this organism. The Klebs-Loffler-like avirulent bacilli might, therefore, be regarded as true diphtheria bacilli thghtly removed from virulence, the Hofmann bacillus if derived from the Klebs-Loffler, as a diphtheria bacillus far removed from virulence. In determining the fermentation reactions of the diphtheria- Brit. Med. Joum., Sup., July 9th, 1904. Joum. of State Med., vol. xi, p. 609. Joum. of Hyijiene, vol. v, p. 134. Journ. Med. Research, 1902, p. 83. Journ. Infect. Diseases, vol. vii, 1910, p. 335. 20 1 306 Manual of Bacteriology like bacilli, the organisms should first he grown in broth until they become acclimatised to this medium, or should be grown in a medium which suits them, e.g. broth with the addition of serum or of ascitic fluid. Hiss's serum-water medium is satisfactory— serum 1 part, water 3 parts, with 1 per cent, of carbohydrate or other substance, tinged with litmus and sterilised in the steamer on three consecutive days. Graham-Smith1 gives the following table of fermentation tests : Organism. B. diphtherise, virulent and avirulent Hofmann bacillus* Xerosis bacillus* . B. coryzm* Diphtheria-like bacilli : From the ear* . From the urethra* From the throat* From the fowl* . (* Avirulent to the guinea-pig) Hiss's medium (10 days' growth). c A ~TT C _A_ C A 0 A C A 3 02 c A 03 0 0 0 0 0 0 0 0 0 0 c c A 0 1 0 0 0 c A 0 A C A o I 0 A A A C A 0 c A C A i 0 A C A 0 0 0 0 0 c A 0 0 0 C A 0 c A 0 0 0 0 0 0 0 0 0 - coagulation » - = no coagulation ; A = acid; 0 = no reaction. Slight variations were occasionally noted: for example four out of twenty otpttheria bacilli gave no acid with lactose and the amount of acid production and of coagulation was somewhat variable. Clinical Diagnosis. (A) In man and animals .—I. In some cases the diph- theria bacillus can be identified in the membrane or discharge, and the diagnosis established thereby. • Journ. of Hygiene, vol. vi, 1906, p. 286. Diagnosis of Diphtheria 307 Films are. made with the exudation, or with a fragment of the membrane teased up as finely as possible on a slide, a droplet of water being added if necessary. One of these films should l>e stained with Loffler's methylene blue, another by H-ra.in's method. The bacilli will be found lying parallel to one another in larger or smaller groups, together with involution forms. Films stained with Neisser's or Pugh's stain (see below) may also be of assistance. Another method is"~To stain the films for five seconds in dilute carbol- methylene blue (seven drops to 10 c.c. water), rinsing and drying, and counter- staining in dilute carbon-fuschin (ten drops to 10 c.c. water) for one minute, rinsing and drying (Higley). II. Frequently the membrane is so crowded with different forms of organisms that it is extremely difficult to recognise the diphtheria bacilli with any degree of certainty. Eecourse must then be had to cultivation. For this purpose sloping blood-serum tubes, or tubes of serum-agar, must be employed; simple agar is unsuitable. A piece of membrane or a swabbing from the throat is rubbed over the surface of one or two serum tubes, care being taken not to break up the medium. The tubes are incubated at 37° C. for eighteen to twenty hours, and are then examined microscopically whether there is any visible growth or not. If there be no visible growth a scraping is taken by means of a sterilised platinum needle from the whole surface and a film is prepared. If there is a visible growth the film should be prepared from the most likely colonies, or, if the growth be confluent, from the upper half inch or so. A microscopical examination must always be made, for some colonies — certain staphylococci and torulse, for example — simulate those of the diphtheria bacillus very closely. The films may be stained with Loffler's methylene blue for five to ten minutes, or by Pugh's method, then washed and dried. If the films are made on a slide, after staining, washing, and drying, a drop of cedar oil may be put on the stained patch, which is then examined directly without a cover-glass. If, however, there is very little growth, it is better to make a cover-glass specimen, 308 Manual of Bacteriology as the position of the material is so much more easily located. The preparations are examined with a ,Vm- oil immersion lens magnifying not less than 800-1000 diameters, and the Klebs-Lofner bacillus is identified from the description ;j,iven in the text . Prausnitz considers that if negative results are obtained after eighteen to twenty-four hours' incubation the tubes should be incubated for a further twenty to twenty-four hours and re-examined, and undoubtedly occasionally a positive result may be obtained by tins longer incubation. Loffler's methylene blue gives much more characteristic preparations than Gram's method. Although eighteen to twenty hours is recommended for incubating the cultures, a microscopical examination will sometimes reveal the bacilli at a much earlier period. The writer has found them in as short a time as six hours, but if bacilli are then not found the tubes must be incubated for the longer period. Neisser's method of staining is as follows : ~ (a) One gramme of methylene blue (GrrilbTer's) is dissolve.! in 20 c.c. of 96 per cent, alcohol, which is then mixed with 950 c.c. of distilled water and 50 c.c. of glacial acetic acid. (b) Two grammes of Bismarck brown are dissolved in one litre of boiling distilled water and the solution is filtered. The preparations are stained in (a) for one to three seconds, rinsed in water, and stained in (h) for three to five seconds, washed in water, dried, and mounted. The bacilli are stained brown, and contain two, rarely three, inky-blue dots. This is a valuable confirmatory stain for the diphtheria bacillus, but staining for a longer time than that recommended by Neisser is advisable, viz. half a minute in the blue and one minute in the brown. Tanner treats with Gram's iodine solution for half a minute after the blue. The staiuing solutions seem to keep well but occasionally fad to act, so should be controlled on an undoubted diphtheria culture. Pugh's stain is also a very good one. It is a mixture Diagnosis of Diphtheria 309 containing 1 gnu. of toluidine blue dissolved in 20 c.e. of absolute alcohol and added to 1000 c.c. of distilled water and 20 c.c. of glacial acetic acid. The mixture is applied for two minutes. The protoplasm of the bacilli is stained a pale blue and the polar bodies are deeply stained and stand out in marked contrast ; by artificial light they appear a reddish purple. In the majority of cases, after a little experience, the Klebs-Loffler bacillus will be readily recognised if present, Occasionally, however, bacilli may be present which resemble the Klebs-Loffler very closely, and of which it is difficult to be certain. In such a case the following points should be noted in attempting to arrive at a decision : 1. The character of the growth on the medium. 2. The depth of staining with Loffler's blue, and the presence or absence of segmentation or polar staining : The Klebs-Loffler bacillus usually stains somewhat deeply, while the bacilli resembling it stain but feebly. 3. The presence or absence of involution forms, clubbing, etc.- 4. The presence or absence of thread forms : The Klebs-^ Loffler bacillus does not form threads.1 5. The presence or absence of spores : The Klebs-Loffler bacillus does nojjlorni spoi-es. 6. futility in a hanging drop : The Klebs-Loffler bacillus is non-motile. Q"ram's method of staining : The Klebs-Loffler bacillus stains^wep.. 8. The grouping of the organism : The parallel grouping of the Klebs-Lofflerbacillus is~lsonTewhat characteristic. The bacilli when lying side by side do not seem quite to touch, while the bacilli which resemble the Klebs-Loffler and show a parallel grouping frequently lie much closer together than the Klebs-Loffler bacillus ever docs. 9. The reaction with Neisser s or Pugh's stain (the culture 1 Klein and others have described thread and branched forms in cultures of the Klebs-Loffler bacillus in certain circumstances, but the.se are not likely to be observed under the conditions mentioned. 310 Manual of Bacteriology must be a young serum one) : The pseudo-bacillus and other bacilli do nut give the diphtheritic reaction (polar staining). 10. The filial test of virulence may be applied. For this purpose the organism must be isolated in pure culture by plate cultivations. Two guinea-pigs, of 250 300 grm. weight, are each inoculated with 2 c.c. of a forty-eight hours' broth culture, one receiving at the same time 1 c.c. of diphtheria, antitoxin.. If the guinea-pig inoculated with culture only dies, while the one receiving culture and antitoxin lives, this is complete proof that the organism is the diphtheria bacillus; if both live no inference can be made except that the organism is non-virulent ; if both die, it shows that the organism is virulent, but that it is not neutralised by antitoxin, and therefore is not the diphtheria bacillus. In cases in which bacilli persist, the test of virulence is frequently applied. If the organism proves to be non- virulent, presumably the patient is non-infective. Such a presumption, in the writer's opinion, however, is not necessarily true. 11. Agglutination tests are unsatisfactory and not of service. It occasionally happens that a conclusion cannot be arrived at without an extended investigation. If serum tubes are not available an egg may be used. It is boiled hard, the shell cTiipped away from une end with a lvrnjle sterilised by heating, and the inoculation made on the exposed white ; the egg is then placed, inoculated end down, in a wine glass, of such a size that it rests on the rim and doPs! not, tivuc.h the bottom- A few drops of water may with advantage be put at the bottom of the glass to keep the egg- white moist, The preparation is kept in a warm place for twenty-four to forty-eight hours and then examined. Antitoxin itself may be used as a culture medium, provided if contains no antiseptic (this is now rarely the case.) A test- tube is sterilised by heating, or with boiling water or steam from a kettle, antitoxin to the depth of about an inch is poured in, and is coagulated by holding the tube very obliquely in boiling water or steam. After coagulation and PLATE VII. b. Vincent's angina. Smear from exudation showing- fusifor bacilli (dark; and spirilla (light), x 2000. face page 310. > I Vincent's Angina 311 cooling the medium is inoculated. If no incubator is available, the culture may be kept in a warm place, or in au inside pocket Many laboratories will now undertake the examination ot material. Culture outfits are supplied by some, consisting of a sterilised tube containing a sterilised swab. Failing this, a piece of membrane may be forwarded in a tube or bottle which has been sterilised by heating, or with boiling water or steam. If there be no membrane, a swab can be readily extemporised by wrapping a little wool round the end of a piece of wire, knitting needle, hair-pin, penholder, or splinter of wood. The wool may be sterilised by moistening with water and then holding in a flame. Membrane or secretion may also be forwarded on pledgets of wool, pieces of lint or calico, and even on paper, but these are not so suitable. (B) In milk. — See section on " Milk." Vincent's Angina. An infective malady characterised by sore throat, fetor, dysphagia, and ulceration and membrane simulating diph- theria. The diphtheria bacillus, however, is not present, and the affection is caused by an apparent association of a bacillus and a spirochaete. The bacillus (B . j'usiformis) measures 6-8 /a to 10-12 jjL in length, has ■pointed ends and is usually some- what bent, not straight, often appears feebly motile, and does not stain by Gram. It can be cultivated anaerobically on the ordinary media to which human blood- serum, ascitic or hydrocele fluid has been added. The spirochaete is long and sinuous and very motile, but cannot be cultivated, and is stated to be developed from the fusiform bacillus. Smears may be stained with methylene blue or dilute carbol-fuchsin, and the appearance of the associated organisms is so charac- teristic that a diagnosis is easily effected (Plate VII., b). Fusiform bacilli have been met with in various necrotic processes, e. g. noma (see Chap. XX). The Xerosis Bacillus. The xerosis bacillus was isolated by Neisser fi-oni cases of xerosis conjunctivae, and is met with in follicular con- 312 Manual of Bacteriology junctivitis. Lawson and also Grriffith isolated it from nearly 50 per cent, of all normal conjunctival sacs. In morphology ^audstaining reactions it resembles the Klebs-Loffler bacillus jery closely. It differs from the Klebs-Loffler bacillus in the following particulars: (1) Usually, but not always, in the primary cultivations from the eye on blood-serum, colonies do not appear under about thirty hours, while those of the Klebs-Loffler bacillus are visible in sixteen to twenty hours. This does not apply to the secondary cultivations, in which the colonies appear as soon as those of the Klebs-Loffler bacillus. (2) Upon agar it will seldom or never grow in primary culture, and in secondary cultures it forms only a thin, translucent, dru film. (3) Upon gelatin it will never grow in primary culture and seldom in secondary culture. (4) It does not give rise to acid production in milk or glucose broth. (5) It is non-pathogenic to guinea-pigs. (6) The "Neisser stain is 7ip.ga.tiYP!. The fermentation re- actions will be found in the table on p. 306. In all probability the organism is not causative of xerosis conjunctivas. To isolate the organism, blood-serum tubes are inoculated with a looped platinum needle from cases of follicular conjunctivitis or xerosis, and incubated at 37° C. for forty to forty-eight hours. Half the tubes will usually show a growth. Preparations may be stained with Loftier' s blue and by Gram's method. Bacillus coryzas (segmentosus). An organism first described by Cautley, of frequent occur- rence in the nasal secretion in cases of " influenza " cold. It bears a striking resemblance morphologically to the B. diphtheria when stained with methylene blue, and is Gram- positive, but does not show granules either with Loftier blue or with Neisser's stain. On agar it grows more slowly than B. diphthenne, and in glucose broth and litmus milk acid production is slow and feeble. It is non-pathogenic to guinea- pigs. The fermentation reactions will be found in the table on p. 306. Pigeon Diphtheria 313 Other Diphtheria = Iike Bacilli. As already mentioned, diphtheria-like bacilli are not in- frequent in wounds, pathological discharges and secretions. Some of them may be positive with Neisser's stain. They are always non-virulent. The fermentation reactions of some of these organisms will be found in the table on p. 306. Bacillus diphtherias columbarum. Pigeon diphtheria is an infectious disease of pigeons, characterised by the formation of diphtheritic-like membranes on the tongue, fauces, and corners of the mouth ; occurs in extensive epizootics from time to time. Loftier isolated a bacillus to which he gave this name. It is short, with rounded ends, non-motile, does not form spores, and does not stain by Gram's method. On gelatin it forms a whitish growth without liquefaction, on agar a creamy growth, and on potato a thin grey film. Milk is not curdled and is unchanged in reaction. It is pathogenic for the mouse and pigeon, but only slightly so for the fowl and guinea-pig. It is possible to prepare a vaccine, and an anti-serum for the disease.1 Eecent research has, however, suggested that the disease may be due to a filter-passer.2 Diphtheritic roup of poultry is a different disease, and is stated to be due to a protozoan parasite.8 Macfadyen and the writer1 found Klebs-Loffler-like organisms to be' present in the mouths and throats of healthy pigeons and fowls. These organisms resembled the true Klebs-Lofner bacillus in their cultural reactions, but were quite non-virulent to guinea-pigs (see table, p. 306). The so-called diphtheria of calves is produced by an anaerobic streptothrix. 1 See Ann. de I'Inst. Pasteur, xv, 1901, p. 952. 2 Dean and Marshall, Journ. of Path, and Bart., vol. xiii, 1908, p. 2U. 3 See also Gordon Sharp, Lancet, 1900, vol. ii, p. IS. '> Trans. Path. Hoc. Land., vol. li, 1900, p. 13, and Brit. Med. Journ., 1900, vol. i, p. 994. Manual of Bacteriology CHAPTER IX. " AC ID- FAST " BACILLI. TUBERCULOSIS LEPROSY THE SM E G M A BACILLUS. GLANDERS. •"Acid-fast" Bacilli. An important characteristic of the tubercle, leprosy, and smegma bacilli is the property they possess when stained with fuchsin of retaining the red colour after treatment with a strong mineral acid (25 per cent, sulphuric or 30 per cent, nitric). They are therefore termed " acid-fast." Most other organisms are rapidly decolorised even by 1 or 2 per cent, sulphuric acid, but it must be recognised that several apparently saprophytic bacilli are also " acid-fast." The retention of the fuchsin colour in spite of treatment with the acid seems to be due to the presence of substances of a fatty or waxy nature within the organisms with which the fuchsin either combines or is protected from the action of the acid. Moreover, by cultivating many saprophytic bacilli in media containing butter, Pienstock and Gottstein con- verted them into " acid-fast " forms. " Acid-fast " bacilli are also present in butter (Petri, Rabinowitsch, Rubner), on certain Graminaceas (the "Timothy-grass bacillus" of Moeller), and in dung (the "Mist bacillus"). It has been suggested that these Tuberculosis 31j saprophytic acid-fast bacilli may be derived from the tubercle bacillus, but Panisset's work gives no con- firmation of this. The Strejototricheaj occasionally exhibit " acid-fast " properties. Dean has found acid-fast leprosy-like bacilli in rats (see p. 355). All the acid-fast bacilH seem to be Gram-positive. 1 — " Tuberculosis. Tuberculosis is, unfortunately, only too common in the human subject, and most of the domestic animals and wild animals in a state of captivity may be attacked by it. The conception of tuberculosis was originally a purely anatomical one, the name being given to a condition in which the organs were studded with little yellowish points or nodules, which were termed tubei'cles. Laennec was the first to indicate the characters of these nodules or tubercles, and traced with considerable accuracy their development from minute lesions, the miliary tubercles, up to the large cheesy masses which may be met with in the glands and lungs. Microscopically, the structure of a young and typical tubercle is characteristic. At the centre one or more giant-cells are found — large protoplasmic masses, each containing ten to twenty nuclei arranged round the periphery (Plate IX.. 6); They are of the nature of Plasmodia, similar to the masses of fused cells which surround a foreign body in the lower animals (Adami). Around the giant-cells are well-defined epithelial-like cells with large and distinct nuclei, which are known as epithelioid, or more properly endothelioid, cells. A zone of. smaller cells with scanty protoplasm and small nuclei 316 Manual of Bacteriology surrounds the endothelioid cells; they are known ;is lymphoid cells from their likeness to the cells of lymphoid tissue. This is the structure of a typical tubercle, but one or other of the components may be wanting-, and none can be said to be absolutely charac- teristic of the tubercle. The nodule possesses no blood-vessels, and as its size increases by growth at the periphery the central parts undergo degenerative changes, and may become either structureless or hyaline, or be converted into a soft yellowish material somewhat like cheese and termed caseous. More or less extensive inflammatory reaction ensues in the tissues surrounding the tubercle, and the cellular elements so produced often become spindle-shaped and ultimately fibrous, so that the tuberculous nodule becomes enclosed by a capsule of fibrous tissue which may contract and convert it into a fibrous nodule. Alter caseation has occurred calcification may ensue — that is, lime-salts are deposited and the nodule is converted into a calcareous mass. So far back as 1865 Yillemin showed that inocula- tion of rabbits with human caseous material was followed by a development of nodules similar in all respects to the miliary tubercles in man. Cohnheim, Burdon Sanderson, and Wilson Fox confirmed this observation, but they also showed that the development of tubercles apparently followed the introduction, not only of tuberculous material, but also of setons, pieces of putrid muscle, and gutta-percha. It was pointed out, however, that in all probability these results were due to accidental contamination or inoculation with tuberculous matter, and, by adopting suitable precau- tions m order to prevent such sources of error, it was conclusively shown that non-tuberculous matter is unable to set up tuberculosis. Tuberculosis is there- The Tubercle Bacillus 317 fore ihoculable, and is an infective disease, and as such must be due to a specific infective agent, to the discovery of which observers then directed their atten- tion. In 1862 Koch announced .that he had discovered a special bacillus, the tubercle bacillus, in tuberculous tissues, which could be isolated and cultivated, and which reproduced the disease on inoculation. The Tubercle Bacillus. Morphology. — The tubercle bacillus (B. tuberculosis) is a slender rod with rounded ends, often slightly curved, and averaging 2-3 jli in length, though the length varies in the tissues from 1*25 /x to 6"5 it; in cultures it tends to be short, on serum being about 1 /n. In stained preparations one or more unstained intervals are often seen in the rods (Plate VIII., a) ; these have been considered by some obseiwers to be spores, but there are many objections to this view. Spores are usually single and not multiple, and are regular spherical or ovoid bodies, whereas the unstained spaces in the tubercle rods are irregular. Moreover, in the same specimen of sputum a varying amount of " bead- ing," as it is termed, may be brought out by different staining methods (Plate VJIL, b) ; in a preparation stained by Grain's method it is usually more pro- nounced than in one stained with carbol-fuchsin. In class work also it will be found that one student's specimen will show beading much more markedly than another's. These considerations render it probable that the beading is partly due to segmentation of the protoplasm, and partly, perhaps, is an artifact clue to the staining process, and is not a spore formation. The tubercle bacillus, however, probably does form spores, though this is a debated point. Some observers have 318 Manual of Bacteriology described clear, regular, unstained spaces in bacilli from old cultivations, and consider these to be true spores. k Jh> 0 . A The tubercle bacillus is a non-motile, strictly para- f***^^^1 sitio organism (it has been descinbetl as being- both motile and flagellated). It usually occurs singly, occasionally linked in twos or threes so as to form short chains, and under certain conditions, especially in old cultures, filamentous forms develop, and Foulerton1 and others include it among the Streptotricheee. The bacillus is agglutinated by the blood-sei'um of a tuber- culous animal (see p. 347). Staininc/ reactions. — The tubercle bacillus stains in- differently with the ordinary watery solutions of dyes, prolonged treatment with, or warming, the solution being required. It stains well by Gram's method. It also stains well and -deeply with carbol-fuchsin, particu- L*4A larly on warming, and when so stained is markedly (\J2t*^ V resistant to the decolorising action of 25-30 per cent. 9b 0 * Oi ' mineral acid > tnat is to sa^5 ifc is stron8-1y "acid-fast," H^AL^ ^ and this property is made use of for demonstrating its ^ J^. presence in tissues, etc., and for diagnostic purposes. MyfAX/f Koch states that the peculiar staining reaction of the tubercle bacillus is due to a coating of two fatty acids, which take up the stain, and are not decolorised by the mineral acid. De Schweinitz and Dorset {loc. cit. p. 324) have found the fatty substances to be princi- pally a glyceride of palmitic acid, together with small amounts of lauric acid and of two other undetermined acids. Bulloch and Macleod {loo. cit, p. 325) found that the fat is not acid-fast, and by saponification yields oleic, isocetinic, and myristinic acids. The acid- fast substance, according to these observers, is an alcohol. i "Milroy Lectures," Lancet, 1910, vol. i, p. 551, et seq. PLATE VIII. To face page 318. The Tubercle Bacillus 319 Cultural characters. -The tubercle bacillus is aerobic and facultatively anaerobic, ar.d thrives best at a tem- perature of 37° C. or thereabouts, and development even then is slow, six weeks at least being required for an appreciable growth. The simplest method of isolat- ing the bacillus from the tissues is to make use of Roux's potato tube (Fig. 9), the bulb being filled with 5 per cent, glycerin. The potato is inoculated with an emulsion of the tuberculous material, and in- cubated at 37° C. In six or eight weeks cultures will be obtained in, perhaps, 50 per cent, of the tubes. Twort1 has successfully isolated the bacillus from sputum by direct cultures in an ericolin medium. Other media that can be employed for cultivating the organism from the tissues are glycerinated serum (preferably dog's), and .glycerin brain agar. The latter is prepared by making a 3 per cent, nutrient agar of + 20 reaction, adding an equal volume of pounded ox-brain, and sufficient glycerin to make 5 per cent, in the mixture, and steri- lising. After culture on these media for some generations, the tubercle bacillus will develop on 5 per cent, glycerin agar (reaction + 15 or 20), and in 5 per cent, glycerin broth (veal is best) ; it will also grow, though very slowly, on glycerin gelatine at 22° 0. _Gelatin and blood-serum are not liquefied. On glycei-in agar the Pig. 37. — Tubercle bacillus. Glycerin - agar culture three months old. R20 Manual of Bacteriology growth forms j^jlrv^rinklerl rmd wrinkled, cream. coloured or brownish-yellow film, which has been well described as resembJinglhe" patches of lichen met with on trees (Fig. 37). The growth, however, varies con- siderably, both in colour and in the amount of wrink- ling, though retaining more or less the characteristics just mentioned. In broth it forms soft cream-coloured, flaky masses, which increase slowly both in size and number, the broth remaining perfectly bright and clear. Sometimes a dry crinkled film forms on the surface of the broth, and may spread all over it, and tends to Fig. 38. — Flask for growing tuberculin. creep up the sides of the vessel. This film formation seems to be essential for the preparation of a satisfac- tory old tuberculin, but it is necessary in order to start it that some of the inoculated particles should float and form nuclei from which the film spreads. The virulent organism from the primary cultivations is difficult to grow on anything but glycerinated potato or serum, or brain agar. Tuberculins. — Extracts of, and suspensions of tritu- rated, tubercle bacilli are employed in treatment and in the diagnosis of tuberculosis. The preparations are known as tuberculins. Old tuberculin. — This is prepared by growing the tubercle bacillus in glycerin veal broth in a shallow layer Old Tuberculin 321 in flat flasks (Fig. 38), so that there is a free supply of oxygen. After some weeks an abundant growth with copious film formation develops ; the latter seems to be essential, but it does not appear to matter whether the bacilli be virulent or non-virulent, or whether they be of human or of mammalian origin. The cultures, bacilli and all, are heated at 115° C. in the autoclave for half an hour, then concentrated over" a watgr^bath to about one tenth of their volume, and finally are filtered through jjorous porcelain ; the resulting fluid is thick, owing to the concentration of the glycerin by the evaporation, is of a dark amber colour, and possesses a eurious chai-acteristic smell. The large proportion of glycerin preserves the fluid, which keeps indefinitely in a cool dark place. This old tuberculin possesses remarkable properties. Relatively large amounts (OT— 0*5 c.c.) may be injected into a healthv animal or individual without effect, but in a tuberculous one a minute dose, 0"001 c.c, gives rise to a marked reaction — elevation of temperature with constitutional disturbance more or less severe, and swelling and tumefaction of tuberculous lesions (glands, ulcers, etc.), and this reaction is made use of for diagnostic purposes (see p. 348). By cautiously in- CTeasing the amount a toleration is gradually induced, so that considerable doses cause little or no disturbance. Injections of tuberculin tend to produce marked changgs_ in the tuberculous^ parts, leading to necrosis and ex- foliation, with subsequent healthy reaction and repair. Tin's is especially seen in cases of lupus ; by continued injections a, marvellous improvement results, so much so that a cure is apparently effected ; but, unfortunately, when the treatment is discontinued the scar usual iv breaks down and the disease returns. Nevertheless, a few cases have remained permanently healed. 21 322 Manual of Bacteriology For treatment, the dose to commence w_ith should not be morg_ than_-DiQQlrrQ:jQU2 c.c^ dilutions being made with 0"5 per cent, carbolic solution, and the dose is repeated when all reaction has passed away and is gradually increased. Tuberculin R, or tuberculin BE, is now generally employed (see below). Healthy guinea-pigs bear considerable injections of_ tuberculin without harm ; but if they be_jtuherimlons, if the disease is advanced (eight to ten weeks after inoculation), doses of 0*01 c.c. produce death; if less advanced (four to five weeks after inoculation) a larger dose, 0-2 to 0-3 c.c, is required; but 0'5 c.c. always j)rpj£es fatal. The post-mortem appearances are con- gestion of the lymphatics and viscera, and dark red spots, from mere points to the size of a hemp-seed, on the liver and spleen. These are due to enormous engorgement of the capillaries ki the immediate neigh- bourhood of tuberculous deposits, actual extravasations of blood being rarely found. The hamiorrhagic-like spots on the liver are almost pathognomonic of death from tuberculin. Absolute alcohol precipitates the_active principle of tuberculin in the form of a whitish"flocculent precipitate #r_~which chemically consists of proteoses. This precipitate, ^ ft AAj^SlWe-aisstive&) is made use of intiie_ojoMhalmic reaction (p- 349). Tuberculin applied to the scarified skin also gives a cutaneous reaction in tuberculosis (p. 348). Tuberculin R, or TR, new tuberculin, is prepared from young and "virulent cultures of the tubercle bacillus. ^ rJuJitfoie growth is collected, dried in vacuo and tnhirated, by machinery.1 Of the triturated material, 1 grm. tfV^M is treated with 100 c.c. of distilled water^nd^ntri^ ^jTS > fugalised. TLp^ipprnatant liquid is rejected, and the ^1^^ i Deutsch. med. Wochenschr., 1897, April 1st (translations or abstracts yk£$f" » * in most of the medical journals of about this date). Tuberculins 323 residue is collected, dried, again triturated and centri- fugalised. The supernatant liquid is carefully pipetted off and kept , while the residue is again submitted to t he same treatment, and the jjrocess is repeated until no solid residue is left. The fhiids are then mixed, the solid content is estimated gravimetrically, some gjycerin__^ isadded, and the liquid is diluted to the correct volume, so as to contain 2 mgrm. of solid matter per cubic ceutinTetre_Xnot 10 mgrm. as formeidy stated), and for use is diluted with 20 per cent, sterile glycerin solution. Tuberculin E, according to Koch, possesses distinct immunising properties, and causes neither reaction nor suppuration. For treatment of tuberculosis in man the initial dose is equivalent to not more than to-oVtto — TTrixoo" ~ g-^Vo mgrm. of solid matter, according to the nature of the case. The doses are given subcutaneously at inter- vals of ten to fourteen days, and the treatment may be controlled m the earlier stages by opsonic determina- tions. According to Latham, tuberculin may also be given by thn month Cases of cutaneous or localised tuberculosis, and those in which the opsonic index to tubercle is moderately reduced, react best. In phthisis and visceral tuberculosis no striking results have been obtained. ^Tuberculin, bacillary emulsion QTFTj^Js an emulsion j/h^fi • oFthe powdered bodies of tubercle bacilli in 50 per cent. ' -» aqueous glycerin. The mixture is allowed to sedimerrt until all heavy particles have deposited, the milky supernatant fluid is pipetted off, and standardised so as to contain 5 mgrm. of solid matter per c.c. The dosage is similar to that of tuberculin R. J^^j Bejjrjnp- has prepared another tuberculin, tulase or ~~yL_cv TC, by treating tubercle bacilli with chloral, which he / P ' Sfcates hgtjgL-markea curative action, and is better (tj^^j 324 Manual of Bacteriology ^administered by the mouth than by subcutaneous inoculation. By giving- tnlase to cows, the milk is said to acquire immunising and curative properties which are transmitted to those consuming it. Other tuberculins are also on the market, and any tuberculin may be prepared with a human or with a bovine strain of bacillus. Chemical 'products. — The., tubercle bacillus produces no extra-cellular toxin. Crookshank and Herroun obtained from glycerin broth cultures of the tubercle bacillus a proteose and an alkaloidal body. The pro- teose was also obtained from " perlsucht." Both the alkaloid and the proteose (from both sources) pi-oduced a rise of temperature in tuberculous guinea-pigs, while in healthy animals the former caused a slight, and the latter a marked, fall in temperature. De Schweinitz and Dorset1 described chemical pro- ducts isolated from the tubercle bacillus grown in a special glycerin-asparagin mixture. From the bacilli themselves an acid body was isolated, probably teraconic acid, an unsaturated acid of the fatty series. A certain amount of the same body was also obtained from the special culture medium, but only a trace from glycerin broth, in which the bacilli had been cultivated, in the latter case not because it was not formed, but because of the difficulty of isolation. This acid seemed to produce on injection depression of temperature and necrosis of the tissues locally, possessed some immu- nising power, and may be the substance producing caseation in the tuberculous nodules. The bacilli ex- tracted with hot water yielded an albuminoid, which -g^-^tu^^^ they regard as the fever-producing substance. ^ Med. Journ. N. T,, 1897, July 24th, p. 195. Also Fifteenth Annual Rep. Bureau of Animal Industry, U.S.A., 1898. Action of Heat 325 Bulloch and Macleod1 state that the, acid-fast sub- stance of the tubercle bacillus is an alcohol. Hot xylol \vill remove this substance troin the tubercle bacillus, and ether or 5 per cent, caustic soda that from the smegma bacillus j the organisms after this treatment are no longer "acid-fast." Maragliano states that toxic bodies are present in the blood and urine of tuberculous individuals. Cellu- lose also seems to be present in small amount in the bacilli (it has also been found in tuberculous nodules). Tubercle bacilli, living or dead, are with great difficulty absorbed when in any quantity. The" dead bacilli when injected nnder the skin invariably cause suppuration, and several months later it is still possible to detect in the pus numerous bacilli which stain well ; introduced into the circulation of rabbits they give rise to nodules in the lungs similar to the tuberculous nodules produced by living bacilli (Koch). Action of heat and antiseptic* on the tubercle bacillus. — The thermal death-point of the bacillus has been the subject of some controversy. Sternberg found that tuberculous sputum exposed for ten minutes to a temperature of U0°, 80°, and 66° C. Jailed to infect guinea-pigs in inoculation, while another specimen of the same sputum heated for ten minutes to a tempera- ture of 50° C. produced tuberculosis in a guinea-pig, so that from these experiments the thermal death-point lies between 50° and 66° C. Yersin in 1888, by culture methods, failed to obtain any growth from bacilli which had been heated to 70° C. for ten minutes, while those heated to 55° C. and 60 C. gave growths in glycerin broth in ten days and twenty-two days respectively. Macfadyen and the writer, in the course of some experiments on the 1 Journ. of Hygiene, vol. iv, 1904, p. 1. 326 Manual of Bacteriology sterilisation of milk, found that milk to which powdered dried sputum had been added was rendered innocuous by a momentary heating to 67°— 68° C. These experi- ments indicate that a temperature of 65° C. and over is probably rapidly fatal to the tubercle bacillus, so that milk which has been pasteurised (/. e. heated to 68°-70° C. for twenty to thirty minutes) may be regarded as quite safe. Experiments by the Royal Commission on Tuberculosis with virulent tuberculous milk gave somewhat irregular results ; in one instance heating to 65° C. for two and a half minutes rendered the milk innocuous, in another instance after five minutes at 70° C. it was slightly virulent, but twelve minutes at the same temperature rendered it inert (see also section on "Milk"). Foulerton found that emulsified tuberculous material fiom tuberculous guinea-pigs did not lose its power of infecting unless heated to 70° C. or over for ten minutes. The tubercle bacillus offers considerable resistance to the action of antiseptics and germicides. Yersin found that it was killed by 5 per cent, carbolic acid in thirty seconds, by 1 per cent, in one minute, by absolute alcohol in five minutes, and by mercuric chloride, 1-1000, in ten minutes. Crookshank found that tuberculous sputum mixed with an equal volume of 5 per cent, carbolic was rendered innocuous in a few minutes, and this without any special precautions as to breaking up the masses. For disinfecting sputum mercuric chloride is unsuitable. (See also Chap. XXI.) Pathogends, etc.— Man is, unfortunately, only too frequently attacked with tuberculosis, the manifestations of which tend to differ somewhat at different age periods. Thus, in the very young, general miliary tuberculosis, tuberculous meningitis, and tuberculous disease of the peritoneum, intestine, and mesenteric PLATE IX. i-cell in tubercle containing tubercle bacilli, x 1000. To face paye ijltj. ^ukit^ /Wyj r Distribution of Bacilli 327 glands (tabes mesenterioa) are the commonest ; in older children, up to the age of puberty, the lymphatic glands, especially in the neck, joints and bones, and the skin (lupus) are mostly attacked ; young adults suffer from disease of the lung (consumption, phthisisTT and older people from chronic disease of the lung and tuberculous disease of the urinary organs and testes, and of the suprarenal capsules ^Addison's disease). Scrofula and struma were terms formerly much employed ; both denote a swollen neck, and were applied to cases suffering from chronic tuberculous inflammation with enlargement of lymphatic glands, especially of the cervical glands, with which other conditions, such as inflammation of the ear, throat and eye, and implication of bones and joints, are frequently associated. The distribution of the bacillus in the tissues varies considerably. In young and active tubercles the bacilli are more plentiful and more easily demonstrated than in older and more chronic ones. They tend to be more numerous in some animals than in others — in the ox and horse than in man, for example. In man the bacillus is difficult to demonstrate (by staining) in enlarged and caseating glands, in pus, in synovial membranes, and in lupus. In some animals, especially the ox and horse, bacilli can usually be readily demonstrated, and may be present in large numbers, and frequently have the typical distribution, viz. within and at the periphery of the giant-cells, though they are by no means confined to this locality (Plate IX b). It has been asserted, particularly by Rosenberger and Forsyth, that tubercle bacilli can be detected in the blood m the majority of cases of pulmonary tubercu- losis. Hewat and Sutherland/ however, made twenty- 1 Brit. Med. Journ., 1909, vol. ii, p. 1119 (References). Manual of Bacteriology two blood examinations on twenty patients in-all stages of the disease and in only one detected two acid-fasl bacilli. Schroeder and Cotton tested the blood of forty-two cattle in all stages of tuberculosis by inocula- tion into guinea-pigs with negative results. Tuberculosis in animals. — The majority of the domestic animals are subject to tuberculosis. It is most common in the ox, pig, and horse, much less so in the sheep and goat, cat and dog. Wild animals, both mammals and birds, in a state of captivity are also specially prone to be attacked, and a large number of the deaths in Zoological Gardens, particularly among the apes, are due to this disease. In carp, tubercle-like nodules are occasionally met with in which a bacillus resembling the tubercle bacillus in morphology and staining reactions is present. It grows, however, much more freely than the true tubercle bacillus, and though inoculable into fish and frogs, is non-inoculable into warm-blooded animals. But it yields a tuberculin which reacts .with mammalian tuberculosis, and by feeding carp on the mammalian tubercle bacillus this can apparently be transformed into the piscian variety. Bird or avian tuberculosis undoubtedly differs in many respects from mammalian tuberculosis. The tuberculous new formations may be very large, but do not show nearly such a disposition to caseation or suppuration as the human lesions. Epithelioid cells form the major part of the growth, and giant-cells are very infrequent. One remarkable feature is the enormous numbers of bacilli which may be present in the tissues ; in places they may be so numerous and closely packed as to form distinct masses or nodules. The bacilli of avian have the same staining reaction as those of mamma- lian tuberculosis, but on cultivation and inoculation Avian Tuberculosis 329 various differences between the two races become evident . The mammalian bacilli flourish best at about 37 C, and growth ceases at 41° C, whereas the avian bacilli thrive luxuriantly at 43° G , and the growth of the latter 'on glycerin agar is much moister and more wrinkled, and often more pigmented, than that of the former. Fowls and dogs are with difficulty infected with human bacilli, but dogs are susceptible to infection with avian bacilli. By cultivation on boric-acid agar and on ea'°"s, etc., the mammalian bacilli are stated to assume the characters of the avian._ Avian tuberculosis is of practical importance not only as attacking poultry, but also in human pathology, as several cases have been recorded in which the bacilli cultivated from human cases seemed to be of the avian type, and were therefore probably derived from an avian source of infection. Two types of tuberculosis also occur in the horse— one in which the lesions are chiefly abdominal, in the other the lungs and bronchial glands are most affected. Nocard states that generally the bacillus obtained from the pulmonary variety is of the ordinary mammalian type, while that of the abdominal one belongs to .the avian. Relation of human and 'mammalian tuberculosis. — It has long been noticed that there are certain differences between the bacilli of human and of bovine tuberculosis, the latter tending to be shorter and thicker and less readily cultivated than the former ; also, while human tuberculous material injected into a rabbit generally produces small discrete lesions in the organs which tend to retrogress, bovine material induces a progressive disease with large caseating masses.1 These 1 The bacilli derived from tuberculosis of the sheep, pig and horse are also of the bovine type. :;:!() Manual of Bacteriology distinctions were regarded as being duo to varia- tions in the bacilli as a result of growing upon a different soil and not to any fundamental difference between the two strains of bacilli. In 1901, however, Koch stated1 that young cattle and swine cannot be infected with human tuberculous material, and ho therefore concluded that human and mammalian tubercle bacilli are essentially different. As a result of his experiments he made the statement that "though the important question whether man is susceptible to bovine tuberculosis at all is not yet absolutely decided. if such a snscejTtihjlity really exists, the infection of human beings is hut very rare occurrence^" . This view met with considerable opposition, and a second Eoyal Commission was appointed to investigate the question, and the following summarises the results obtained up to the present, from which it will be gathered that while there is no justification for assuming that man is infected from human sources alone, infection from human sources is probably vastly more frequent than from any other. Thirty different viruses isolated from cases of tuberculosis occurring spontaneously in bovines have been studied, and the results of introducing them into a number of different animals by feeding and inoculation are recorded. In calves, inoculation usually results in generalised progressive tuberculosis, but the effect is somewhat dependent on the dose, i. e. the number of bacilli, administered. Thus whereas 50 mgrm . of culture always induced a fatal generalised progressive tuberculosis, in two instances much smaller doses— 0'01-0-02 mgrm. produced only limited retrogressive tuberculosis. Feed- ing, on the other hand, usually produced lesions limited to the neighbourhood of the digestive tract, which 1 See Brit. Med. Journ., 1901, vol. ii, p. 189. Bovine Tuberculosis 331 generally retrogress and become calcareous. The bovine bacillus, when introduced into rhesus monkeys or chimpanzees, either by inoculation (even in so small a dose as 0-001 mgrm.) or by feeding, induces rapid generalised tuberculosis, and, considering the close relation that exists between the anthropoid apes and man, these results are of the highest importance. In pigs, generalised progressive tuberculosis is readily set up both by feeding with, and by the inoculation of, bovine bacilli. Goats, dogs, and cats are relatively less sus- ceptible, but more or less tuberculous infection can similarly be produced in them. On this part of the investigation the Commissioners remark that the bacillus of bovine tuberculosis is not so constituted as to act on bovine tissues only, and the fact that it can readily infect the anthropoid apes, and, indeed, seems to produce this result more readily than in the bovine body itself, has an importance so obvious that it need not be dwelt on. The viruses isolated from sixty cases of the disease in man were also studied, and the results obtained show that they may be divided into two groups, subsequently referred to as Group I. and Group II. The bacilli of Group I. comprised fourteen viruses, one obtained from sputum, three from tubercu- lous cervical glands, and ten from mesenteric glands of primary abdominal tuberculosis in children. The results produced by introducing these viruses into animals are identical with those produced by the bovine bacillus. The bacilli of Group II. comprised forty viruses obtained from various forms of human tuberculosis— cervical glands, mesenteric glands (8), lungs and bronchial glands (10), joint and bone disease (9), testis, kidney, etc.— grow more luxuriantly in culture than those of Group I., and inoculated into calves and rabbits do not produce the generalised and 332 Manual of Bacteriology fata] disease caused by the bovine bacillus, hut in rhesus monkeys and in the chimpanzee set up a general tuberculosis. Certain human viruses, differing in cer- ium respects from those of Groups I. and II., were also met with and are classed as Group lib, hut an opinion on their significance is reserved for a future report. The Commissioners conclude that the tubercle bacillus in its nutritive and reproductive powers resembles other simple organisms, and that the essen- tial difference between one strain and another depends on variations in these factors, and they classify those bacilli that grow with difficulty on artificial media as dysgonic, and those that grow readily on media as eugonic. The bearings of the results obtained are thus sum- marised : " There can be no doubt that in a certain number of cases the tuberculosis occurring in the human subject, especially in children, is the direct result of the jntrcH. duction into the human body of the bacillus of bovine "tuberculosis^ and that in the majority of these eases The disease is introduced through cow's milk. Our results clearly point to the nece"sslty of measures more stringent than those at present enforced being taken to prevent the sale or the consumption of tuberculous milk." As regards the histological appearances of the tuber- culous process in different animals, Dr. Eastwood states that there is an underlying unity of the morbid processes produced experimentally by infection with every variety of bovine and human tubercle bacillus. Eber,1 in an extended investigation, succeeded in infecting calves from three cases of human pulmonary tuberculosis. The bacilli isolated from the human i Cadr.f. Bald., AM. I (Orig.) lix.. 1911, p. 193. Channels of Infection 333 material were of the human type, but after passage through the calf became transformed into the bovine type. He affirms, therefore, the essential identity of the human and bovine types of tubercle bacilli. With regard to the channel of infection in human tuberculosis" opinions differ. Koch insisted that inha- I fcrfj^ lation of air-borne bacilli derived from dried human sputum is the principal source of infection; Von Behring, on the other hand, expressed the opinion that tuberculous milk fed to children is the main source of infection both of children and of adults; in The latteTcase he suggests that bacilli are ingested in childhood and lie dormant for years before becoming active. Calmette similarly believes that in the young, Qx£iUACtt infection by the digestive tract, especially by tuber- M» culous milk, is the more frequent, and attaches little* "ov no importance to dry dust containing tubercle bacilli as a source of infection. Eavenel considers that the alimentary tract, particularly in children, is a frequent portal of entry for the tubercle bacillus, which he believes is able to pass through an intact mucous membrane. Of sixty cases of human tuber- culosis investigated by the Royal Commission on Tuberculosis, twenty-eight possessed clinical histories indicating that in them the bacillus was introduced by the alimentary canal. Fliigge, on the other hand, states that his experi- ments show that tuberculosis can be communicated to animals by inhalation, and that the dose of bacilli required to infect by the respiratory tract is far less than that required to infect by the alimentary canal. The mode of infection in man doubtless varies, and he believes that children may be infected by the digestive tract, by tuberculous food, particularly milk, but the Manual of Bacteriology most extensive source of infection is the number of droplets of tuberculous expectoration coughed up by consumptives; these float in the air and serve as sources of infection to others. Ribbert and Schrotter, also, from the evidence of autopsies, considered inhala- tion as the chief mode of infection in man. Bulloch.1 from a careful survey of the literature, concludes that pulmonary tuberculosis is invariably caused by bacilli of the human type, and, therefore, is presumably due to inhalation of human bacilli. McFadyean,2 also, from a critical survey of the experimental evidence, concludes that (1) inhalation of tubercle bacilli suspended in the air is a very certain method of infecting- susceptible animals; (2) experi- mental infection by the digestive tract is comparatively difficult to realise; (3) inhalation is probably the commonest natural method of infection, both in man and in animals ; (4) infection by the digestive tract can be inferred only when the lesions are confined to the abdomen. He finally states that " the whole of the experimental evidence on which the theory of the intestinal origin of pulmonary tuberculosis in man was built up has been swept away." Thus there has been a reversion to Koch's original view, and, inasmuch as the death-i-ate per K'OO living from all forms of tuberculosis is about T64, that from phthisis is lvl4, so that by far the greater part of the mortality from tuberculosis must be ascribed to infection from human sources. While this book was in the Press, the final report of the Royal Commission was issued. The Commissioners conclude that a considerable amount of huroa.n tuberculosis is caused by bacilli of the bovine type, and that tuberculosis may be 1 "Horace Dobell Lecture," 1910. 2 Joum. Boy. Inst. Public Health, vol. xviii, 1910, p. 705. Commission on Tuberculosis 335 communicated to man from infected cow's milk, and from tuberculous meat, either begforjjork. ^So far, therefore, from any relaxation of the existing supervision of milk-production and meat-preparation being possible, the Commissioners_press upon the government the ftnforcement'of~food regulations, "planned to afford better "security against the infection of human beings through the medium of articles of diet derive.] from tuberculous animals." More particularly they urge such action " in order to avert or minimise the present danger arising from the consumption of infected milk." Of vouno- children who died of wasting disease of the intes- tine, the bovine bacillus was present in nearly half the cases. Further, TTarge proportion of cases of tuberculous cervical o-lands in both children and adults was due to the same bacillus. The wording of the report is: "Whatever, therefore, may be the animal source of tuberculosis in adolescents and in adult man, there can be no doubt that a considerable proportion of the tuberculosis affecting children is of bovine origin, more particularly that which affects primarily the abdominal organs and the cervical glands. And further, there can be no doubt that primary abdominal tuberculosis, as well as tuberculosis of the cervical glands, is commonly due to ingestion of tuberculous infective material. The evidence which we have accumulated goes to demonstrate that a considerable amount of the tuberculosis of childhood is to be ascribed to infection with bacilli of the bovine type transmitted to children in meals consisting largely of the milk of the cow. " We are convinced that measures for securing the prevention of ingestion of living bovine tubercle bacilli with niilkjvould greatly reduce the number of cases of abdominal and cervical "gland tuberculosis in children, and that sucli measures should include the exclusion from the food supply of the milk of the recognisably tuberculous cow, irrespective of the site of the disease, whether in the udder or in the internal organs." 336 Manual of Bacteriology The occurrence of tuberculosis in the domestic animals raises points of practical importance, especially the occurrence of infection from the consumption of meat and milk from diseased animals. In the ox the tuberculous lesions are most frequently met with in the lymphatic glands arid serous membranes, particularly the pleura, and in the lungs and liver, while the fat and muscular tissues, which constitute the major part of " meat," are very rarely affected. On the pleura the growths take the form of nodular masses, which from their arrangement are popularly termed " grapes " or " angle berries." There can be no doubt that the carcase of an animal extensively aifected with tuberculosis, especially if wasting has occurred, should be condemned as unfit for food, and likewise all parts in which there are tuberculous deposits. But it becomes an important question for the community, financially as well as from a hygienic point of view, as to the method of procedure with the meat from a beast comparatively slightly affected with tuberculosis— an enlarged gland or two, and a few nodules on the pleura. No doubt the ideal method in such a case is the con- demnation and destruction of the whole carca.se, be the amount of tubercle ever so little; but from financial considerations this procedure is hardly practicable on account of the large amount that would have to be paid in compensation. Experiment has demonstrated that the tubercle bacilli are practically confined to the tuber- culous areas and are extremely rarely met with in the muscular tissue, and these portions, therefore, it might seem, could be eaten with impunity, especially as they would be cooked before consumption. As regards swine, however, it is generally held that tuberculosis anywhere condemns the whole carcase. The report of the first Royal Commission on Tuber- Tuberculous Food 337 culosis, however, indicated two dangers. Firstly, in cutting up a carcase the butcher will most likely use tlie sainekn^^^j'oughout,, and in this way may infect tlie meat with tuberculous matter by smearing with the knife. Secondly, cooking cannot be depended upon to destroy the bacilli unless the joints are under OlbTiii weight : when the weight is above this the 0 Jb. in weight; when the weight is above this the temperature in the interior may not rise sufficiently high. Evidently one of the first measures tobe taken is the abolition of private slaughterhouses and the establish- ment of municipal abattoirs where the meat would have to be passed by competent inspectors. In this way all badly affected carcases would be condemned, and those only slightly affected could be separately dealt with and special precautions taken to eliminate tuberculous pieces, etc. Tuberculous, milk also raises many important points. Probably some J 0-15 per cent, of al^ samples are infective to guinea-pigs, but t."hi« A^T^t. ^^fiiy indicate that this proportion would be dangerous to man, for the material is introduced into the guinea-pigs by inoculation after concentration by centrifuging (see also section on "Milk"). Tubercle bacilli are present 111 milk jK>t on1v whft" foe udder is tuberculous, but also when the cows are suffering from tuberculosis elsewhere winch is clinically recognisable. Thus, when the lungs are affected, bacilli are disseminated from the air-passages and also by the faaces. It is noteworthy that the incidence of abdominal tuberculosis in youno- children occurs just when cow's milk in the staple article of their diet. At the same time this incidence does not seem to fall on those who consume most milk Much might be done by the registration of all dairy premises, the use of selected cows, the elimination of all tuberculous animals, and by enforcing the inspection 22 338 Manual of Bacteriology of dairy cattle by competent inspectors at intervals ol not longer than a fortnight, making the notification til' any disease of the udder compjiksoryj_ancl the sale of milk from such a diseased udderjllegajj^nder a heavy "penalty (Roy. Com. on Tuberculosis). In the absence of inspection and the use of selected cows, treatment of milk intended for the food of infants and young children by pasteurisation, or sterilisation has been recommended, but has disadvantages (see section on " Milk ") . The ideal method, and one which commends itself at first sight as being the most satisfactory, is the elimin ati onby_jd3ghter_ojLal^^ ^e.nlnns. _This wasadopted"lnthe State of Massachusetts ; under an order of the Board of Cattle Commissioners all beasts in the State were tested with tuberculin, and every animal that reacted was slaughtered, and strict quaran- tine combined with the tuberculin test imposed on all imported cattle. Even in this small state such a plan was found to be unworkable, the expense being so_ heavy. A middle course seems to be the only practi- *^ab!e one, viz. all manifestly tuberculous animals, especially where wasting or tuberculous udder is present, to be slaughtered; other animals to be tested with tuber- culin, and those which react to be separated from the healthy and to be disposed of as soon as convenient, and in the meanwhile kept as much as possible in pasture. Measures should be adopted by local authorities and others to prevent the spread of tuberculosis, and, thanks to the attention directed to the subject, tuberculosis is diminishing It can hardly be doubted that the disease, or at least phthisis, should be made notifiable, though there are many difficulties in carrying this out. Patients should be warned of the danger of disseminating therr expectoration, andj^uld usepocket^jtopontaining antiseptic, or handkerchiefs (such as the Japanese an Measures against Tuberculosis :;:;'.! paper ones) which can be destroyed. ^Eooms which have been inhabited by tuberculous patients should b"e~ disinfected, for which purpose Delepine recommended spraying with a 1—100 solution of chloride of lime. Although the occurrence of direct infection can rarely be proved, the possibility of this cannot be ignored. Not only should the dissemination of infection be prevented, but the resistance of the individual should be raised by providing a healthy environment and by inculcating the importance of fresh air. Serum therapeutics and vaccine. — Maragliano's serum is pr-epared by injecting cows with watery tuber- culin and with a bacillary pulp made by grinding up tubercle bacilli, emulsifying in water and filtering- through a porcelain filter. Subcutaneous injections of the two preparations are given in increasing doses, commencing with 5 c.c. of each, until 20 c.c. is reached, the frequency of the injection being determined by the temperature reaction and general symptoms produced. Marmorek's serumjs prepared by growing tubercle bacilli in a medium consisting of a leucotoxic calf-serum (prepared by injecting calves with leucocytes) and~ glycerine liver bouillon.1 The filtered culture is injected into horses, and their serum, after several injections, becomes antitoxic to a slight degree. It cannot be said that even encouraging results have been obtained with these or other sera. For vaccine treatment, tuberculins E and BE are usually employed (p. 322). Latham has found that tuberculin given per us produces its characteristic effects. _ Immunity.— Attempts have been made from time to time to produce immunity against the B. tuberculosis, particularly in cattle. Thus McFadyean2 found that 1 Bull de I'lnst. Pasteur, i, 1903, p. 851. Trans. Path. Soc. Lond., vol. liii, 1902, p. 20. 340 Manual of Bacteriology heifers which had previously been subjected to repeated doses of tuberculin (old) in some cases resisted infection with virulent bacilli. Behring1 also employed human tubercle bacilli for the vaccination of cattle with satis- factory results. .His tulase likewise confers immunity when given either by the month or by the stomach. Theobald Smith2 also concludes that vaccination of calves with the human type of bacillus is harmless, and that the procedure leads to a relatively high resistance to fatal doses of the bovine bacillus. Clinical Examination. I The " complement-fixation " test was first used in tuber- -cTuWVW^s^^ The method has been further elaborated by Euaerjr.3 He makes use of a, standard emulsion of tubercle bacilli in salt solution, contammg about 4 per cent, by volume of solid bacillary substance. This is sterilised bv intermittent sterilisation and keeps for four to six weeks. Bacilli from various sources vary somewhat, so that, the emulsion should be standardised so as to give an absorption-time with normal sera of about 20 minutes U the complement of normal serum should be just completely absorbed in about 20 minutes. A water-bath kept at a constant temperature of 38° C. is used to warm al the con- stituents and mixtures. One part of the serum to be tested is mixed with four parts of the bacillary emulsion m a small ube (e.g. a Durham's tube) in the water-batb the time of Ltlg being accurately noted. After 2h minutes' incubation 4 volumes of the mixture are removed by means of a Ipillary pipette with teat (Fig. 35, p. 225), into which d» a single volume of sensitised corpuscles (i.e. a hamuli, ystem, P- 191) ^ taken up and the whole is expel ed into small tube already standing in the ^water-bath. The pieces is repeated after 5, 10, 15 and 20 minutes, and longei i Brit. Med. Jonrn., 1 906, vol. h, p. 5/ 1 . » Jonrn. Med. Research, vol. xviii, 1908, p. 451. a Lancet, 1911, vol. i, p. 485. Diagnosis of Tuberculosis 341 necessary. By the occurrence or absence of haemolysis in the various tubes, the time taken for the absorption of comple- ment is ascertained, the complement used being- that con- tained in the serum itself, which therefore should be fresh. A control with normal serum should always be performed at the same time. With normal serum complete absorption should take place, in about 20 minutes ; with tuberculous sera it is often complete in 2| minutes. If, then, absorption of complement is complete in much less than the time necessary for absorption with a normal serum, presumably the serum is derived from a tuberculous individual. (But see Emery's paper for limitations.) IT. The examination of sputum, etc., for the tubercle bacillus is a routine procedure of the greatest value in forming a d iagnosis. Fortunately, owing to the peculiar staining reaction of the tubercle bacillus, discovered by Koch, the method is comparatively simple. 1. Sputum. — Film specimens are prepared by smearing with a platinum needle a little of the sputum on a slide so as to form a thin film covering two thirds of the surface, or by placing a particle of the sputum on one slide, applying another slide, pi-essing together, and then drawing apart so that a thin film is left on each slide. The. thick portion of the sputum should be used, the thin mucoid portion being rejected. If there are any small yellow caseous particles present these should be chosen, and sufficient material should be used so as to form a distinct but not too thick film ; a little experience will soon decide the right amount. Preparations may also be made by smearing the sputum on a cover-glass or between two cover-glasses instead of using slides. Which- ever plan is adopted, the film is dried and fixed in the usual manner (generally by heat), and then stained by one of the following methods : (a) Zielil-Neehen method. — Film specimens on slides are most conveniently stained by flooding with filtered, undiluted carbol-fuchsin and warming for 2 to 5 minutes on a piece of asbestos cardboard supported on a tripod, or on a, heated penny (p. 114), or slides or cover-glasses flooded with the stain :U2 Manual of Bacteriology may be held in the forceps and carefully warmed over a flame, or the preparations may ho immersed in a watch-glass or dish of the stain, covered, and placed in the warm incubator for half an hour. In no case must the stain he allowed to boil; it should only be warmed sufficiently to steam (50°- 60° C), and with slides or cover-glasses as evaporation takes place more stain (always filtered), or better, -5 per cent, carbolic, should be added. After staining, the preparations are rinsed in water and are then decolorise! by treating with 25 per cent, sulphuric or 30 per cent, nitric acid. The preparation may be flooded with the acid, but a better method is to have a pot (fig. 20, p. 113) containing the acid in which it is immersed. In the acid the colour changes after a few seconds to a yellowish brown, the preparation is then rinsed in water, and some of the pink colour returns. The treatment with acid and with water alternately is repeated until the prepara- tion is nearly colourless when rinsed in water. With sputum this is usually the case after three or four rinses in the acid, but it varies with the thickness of the film and with the number of tubercle bacilli present ; when these are absent, the film often decolorises more readily than when there are many. The presence of blood renders the decolorisation difficult. After decolorising and washing, the preparations are stained for one minute in Loffler's methylene blue, washed in water, and mounted in water, or, better, dried and mounted in Canada-balsam or cedar oil. When the preparation is made on the slide, after washing and drying, it can he examined directly without a cover-glass with the oil-immersion after applying a drop of cedar oil, unless a permanent specimen is desired, in which case it should be mounted in Canada-balsam. The tubercle bacilli appear as delicate red rods, often beaded or segmented, on a blue background composed of cells, mucus, and putrefactive orother bacteria. Occasionally here and there a little red colour may be present in addition to the tubercle, bacilli. Hair and keratinised material generally, such as horny epithelium, and red blood-corpuscles, retain the red colour after the foregoing treatment, and the spores of bacteria- arc also liable to retain the red somewhat persisted ly. Diagnosis of Tuberculosis 343 These exceptions are not, however, likely to prove a source of error, for the tubercle bacilli should be recognised not only by their red colour, but also by their characteristic size, shape, and 0- eneral appearance. It is conceivable that acid-fast bacilli not tubercle might be present in sputum, but such an event is a verv unlikely one. For the microscopical examination, a 1- inch with <*ood illumination is sufficient when the tubercle bacilli are present in any number. When they are scanty it is necessary to use a x\-inch oil-immersion, and this is the better lens in anv case. (See Plate IX., 6, and Plate X., a.) If tubercle bacilli are not found, other specimens should be prepared and examined. It is only by repeated examina- tions onjsgr*"* "fusions that the negative evidence, the nbsenc-e of tubercle bacilli, becomes of any value. " Thetubercle bacillus is occasionally not acid-fast"1 ; probably the bacilli insuchcases are degenerate, and, like all degenerate * YJ^a/ bacteria, failTostain well. Dole"! has examined a number otO<^.fY methods that have recently been introduced for staining and tOLjl differentiating the tubercle bacillus, but does not find that any * presents a marked advantage over those usually employed. In material whin.h has been preserved a longtime, e. g. sputum with carbolic, or tissue in spirit, the bacilli may be much less acid-fast than in fresh material. Various methods have been recommended for the solution of the sputum and the examination of the sediment of the bacilli. In onejnethod 5 c.c. of sputum are mixed with 50 c.c. of normal _KOH solution ; the mixture is warmed in a water-bath to 60°-65° C. until the sputum is dissolved (about 3 hours) ; 50 c.c. of cold water are next added, the whole is well shaken, and again warmed for £ hour. Petroleum ether, 2 c.c, is next added, the whole is well shaken, and is then kept at 60° C. until the ether has separated. The bacilli will be concentrated in the fluffy, IggV^v at the -junction of the ether and water ; this is pipetted off and films are made with£Y Q - it and stained. Antiformin (a mixture of sodium ^IV^-^^tL/^ chlorite and sodium hydrate) has -also been recommended. / Into a boiling-tube or small flask of 50 c.c. capacity, 5 c.c. of 1 gee J^tHcrl, L908, vol. i, p. 1222, 344 Manual of Bacteriology the sputum are introduced. To this are added 25 e.c. of antiformin solution (10-20 percent, aqueous solution) diluted with 10-20 c.c. of water according to the density of the sputum. The mixture is well shaken until homogeneous (about 15 minutes), then centrifuged, the deposit is washed three times with salt solution by centrifuging, and films are made with the washed deposit and stained by the Ziehl- Neelsen method. If it is inconvenient to examine the sputum for a day or two a little 1-20 carbolic should be added. This preserves the sputum and the tubercle bacilli retain their staining power for some time. If the tubercle bacillus cannot be detected microscopically after repeated examinations, and a certain diagnosis is important, the inoculation method may be employed. A couple of guinea-pigs are inoculated subcutaneously in the thigh or abdomen with 0 5 to 1 c.c. of the sputum. If tubercle bacilli are present the animals will show signs of tuberculosis in three to six weeks (see below, "Urine"). (b) Gobbet's method. — Prepare and slain the specimens in carbol-fuchsin as in method („). Then wash and treat with the following solution for two to three minutes; wash, dry, and mount : Alcohol ...... 50 parts Water . . . • • • 30 „ Nitric acid 20 „ Saturate with methylene blue and filter. The solution both decolorises and counter-stains. 2. Tissues^— The histological appearance of the t ubercle is usually sufficient for diagnostic purposes without the demonstration of the tubercle bacilli, which in many instances may be difficult in human material, as the bacilli may be very scanty, or practically impossible to find, e g. in lupus. Secti< ms should be prepared either by the freezing or the paraffin method, stained with hematoxylin, and counter-stained with eosin, or orange-rubin, or with the Ehrlich-Biondi mixture. In order to demonstrate the tubercle bacillus in fresh tissue smears may be made and stained like sputum, or Diagnosis of Tuberculosis 345 soot ions prepared and stained in warm carbol-fuchsin for about ten minutes. For frozen sections the stain may be contained in a watch-glass or small glass capsule, and is warmed until it steams, but not boiled, on a piece of asbestos cardboard or a sand-bath. Paraffin sections should be fixed to the slides with glycerin albumin, and may be stained by Hooding with the carbol-fnchsin and warming on asbestos cardb< >ard, or a heated penny, for ten minutes. After staining, the sections are washed in water and are then decolorised in 25 per cent, sulphuric acid. This is a longer process than with sputum, and the sections after being in the acid for a few seconds are washed in water and then returned to the acid, and this alternate rinsing in acid and in water is repeated until they are nearly colourless when placed in water. It is not necessary to remove the colour absolutely ; a faint pink remaining does not matter. After rinsing in fresh water to remove all the acid, the sections are counter- stained in Loffler's methylene blue for two minutes, rinsed in methylated spirit, passed through absolute alcohol somewhat rapidly to avoid removing too much of the blue, cleared in cedar oil or xylol, and mounted in balsam. The sections may also be counter- stained with hematoxylin or Bismarck brown. Instead of using the strong acid solution for decolorising, an acid alcohol solution may be used with advantage. G-ram's method may also be used, but is, of course, not distinctive for the tubercle bacillus. The following is a good method for staining tubercle in sections (Ki'ihne, modified by Deh'pine1) : ( a ) The tissues should be fixed in corrosive sublimate, acidu- lated or not, hardened in alcohol, embedded in paraffin, and the sections fixed on slides with glycerin albumin. (6) The sections are stained with haematin solution for ten to twenty seconds to obtain a pure nuclear stain (not too deep), then washed thoroughly in water. (c) They arc then stained with carbol fuchsin, kept at a temperature of about 47° C. for twenty to thirty minutes. ' See Med. Chronicle, vol. v, 1896, p. 17. 34(5 Manual of Bacteriology The slides are during that time kept in a moist chamber to prevent the stain from drying on the specimen. (d) The stain is rinsed off with water, and the sections are treated with 2 per cent, watery solution of hydrochloride of aniliu for a few seconds. • (e) The sections are then decolorised in 75 per cent, alcohol till they are apparently free from stain ; this will take from fifteen to thirty minutes. (/) They are then counter-stained with a solution of orange (one part of saturated watery solution of orange to twenty to forty parts of 50 per cent, alcohol), dehydrated, cleared, and mounted. Where a positive diagnosis is important, a small piece of the tissue may be inserted under the shin of the thigh or abdomen of a guinea-pig. If tuberculous, the animal will show signs of tuberculosis in two to three weeks (see below, "Urine"). Cover-glass specimens of pure cultivations of the tubercle bacillus maybe stained in warm carbol-fuchsin for two to five minutes, rinsed in the sulphuric or nitric acid solution, washed, dried , and mounted. They can also be stained by G-ram's method, which usually brings out the beaded appear- ance very markedly . Differentiation from the leprosy bacillus will be found at p. 356, and from the smegma bacillus and other acid-fast organisms at p. 357. 3. Urine. — The tubercle bacillus is often very difficult to demonstrate m urine. The urine must be allowed to stand in a conical glass for twenty -four hours or centrifugalised, and film specimens are prepared with the sediment and treated by one of the methods for sputum given above. Several speci- mens should be made and must be very carefully examined. It is important to exclude the smegma bacillus, and the urine is preferably drawn off by a catheter. Staining may be carried out by Housell's method, by which the smegma bacillus is decolorised, viz. after staining in warm carbol-fuchsin the specimen is washed and dried. It is then immersed in acid alcohol (alcohol + 3 per cent, hydrochloric) for ten minutes, washed in water, counter- stained for a few seconds in a saturated alcoholic solution of methylene blue, washed, dried, Diagnosis of Tuberculosis 347 and mounted (see also p. 357). An electrolytic method for the concentration of the tubercle bacilli has been devised by Kuss.* If a diagnosis is of importance inoculation should be presorted to. _ Two guinea-pigs are inoculated subcutaneously in the thigh or abdomen with 0-5 to 1 c.c. of the deposit from the sedimented or centrifugalised urine, or one may be inoculated subcutaneously, the other intra-peritoneally. If tubercle bacilli are present the animals may show signs of tuberculosis as early as two to three weeks after inoculation. Delepine3 recommends the inoculations to be made on the inner aspect of the leg about the level of the knee. The order of infection after inoculation is as follows : the popliteal, superficial and deep inguinal, and sub-lumbar glands, the retro-hepatic, mediastinal and bronchial, deep cervical, and subscapular glands, the spleen, liver, and lungs. The inocu- lated animals are killed in two to three weeks, dissected, and the lesions examined microscopically. Others inoculate two guinea-pigs, one subcutaneously in the abdomen, the other intra-peritoneally. Negative results are nearly as valuable as positive ones. In f&ces, if definite yellow caseous particles can be found, these should be picked out, and films made and stained. Anti- formin may also be used. About a cubic | in. of feces is mixed with 20 c.c. of |15 per cent, aqueous antiformin in a conical glass, will agitated and broken up, and an equal volume of the dilute antiformin is then added. The mixture is allowed to stand for an hour, and films are prepared from the white curdy layer which forms, stained, and examined. 4. Milk— See section on milk (Chapter XXI). III. Aqphdination reaction— The method of agglutination was proposed by Arloing and Courmont for the diagnosis of tuberculosis, but is difficult to carry out and is jmt, much employed. A special method has to be employed to obtain nomogeneous cultures of the tubercle bacillus or a powder of 1 Proc. Roy. Soc. Land., b. 1909. - Brit. Med. Journ., 1893, vol. ii, p. 664. The results only apply to ordinary forms of tuberculosis, and not to certain modified forms such as lupus and the avian variety. 348 Manual of Bacteriology pulverised or ground-up bacilli may be used : this powder may be purchased. The reaction may be carried out either microscopically or macroscopioally ; for the latter small sterile test-tubes may be employed. For each test three dilutions of the serum are made, a 1 in 5, a 1 in 10, and a 1 in 20, and the tubes filled with these dilutions are allowed to stand in an inclined position (45°) for five to ten hours. In man the serum of normal individuals may agglutinate up to a dilution of 1 in 5, Avhile in animals this is variable — imperceptible in the guinea-pig, rabbit, and calf ; feeble in the goat ; in the adult ox up to 1 in 5, but in the dog it may be up to 1 in 10 or even 1 in 20. A positive serum reaction in a suspected subject is a sign of great value in establishing the diagnosis ; a negative serum reaction is of less value. 'IV. The ftjismt.ic method. — The general mode of carrying this out is described at pp. 224-230, the tubercle bacilli being suspended in 1*5 per cent, salt solution. V. Tuberculin reactions. — The old tuberculin is used for diagnostic purposes ; it is not perhaps very safe. A dose of 0-002 c.c. is injected subcutaneously, and the temperature taken Eour-hourly during the succeeding thirty- six hours. A rise of 2°-3° F. or more ensues a few hours after injection in tuberculous subjects. If no reaction occurs another dose of 0-00f) c.c. may be given after the lapse of some days. This method has now almost completely been superseded by the cutaneous or by the ophthalmo reaction. The cutaneous tuberculin reaction.— Von Pirquet1 discovered that when tuberculin is introduced into the superficial layers of the skin of tuberculous individuals, as in vaccination, a reaction occurs consisting of the formation of a papule with redness, slight swelling and exudation, and sometimes small vesicles. This reaction is usually at its height twenty-four to forty-eight hours after inoculation. In healthy individuals no reaction follows the inoculation. The method is to scarify a small spot on the forearm through a drop of a dilution of the old tuberculin, and protect the patch with a simple dry 1 Wien, vied. Work., July 6th, 1907, Diagnosis of Tuberculosis 349 dressing. Moro lias modified the method by applying the tuberculin to the skin in the form of ointment. T1,p. nphthdmn-tubercidin reaction.— C&lmette transfer** 1 the site of inoculation from the skin to the conjuuctiva. He makes use of material prepared by precipitating the old tuber- ^ culin with alcohol, of which a 1-100 solution is prepared m ULU^fc distilled water. One drop of this is instilled into the inner 1 half of the conjunctiva of one eye. In tuberculous individuals a reaction follows, usually in six to sixteen hours after medication, consisting of a conjunctivitis, ranging in intensity from a local redness to a redness extending over the whole eye and having the appearance of an acute conjunctivitis. The reaction soon passes off, generally without leaving ill effect. On the whole the reaction appears to be fairly constant in tuberculous individuals, but absence of reaction is not certain proof that the case is not tuberculous.1 VI. Tuberculin for veterinary use— The dose of the various preparations in the market varies according to their strength ; it corresponds to 0T c.c. or 0*2 c.c. of Koch's original tuberculin. (a) The dose is injected subcutaneously in the neck. (6) The temperature should be taken immediately previous to inoculation, and, if possible, morning and evening for two or three days previous to inoculation. (c) The temperature should be taken at the twentieth hour after injection, or, if it can be done, at frequent intervals from the twelfth to the twentieth hour. (d) The reaction consists of a rise of temperature of T5° to 6° F. above the average normal, occurring eight to twelve hours after injection, and lasting twelve to fourteen hours, accompanied by some systemic disturbance. (e) A healthy animal is unaffected by the injection, and if an animal be very extensively affected with tuberculosis the reaction may not be given, or may be masked by the febrile condition present. An ophthalmoreaction may also be employed in cattle. 1 See articles in Brit. Med. Journ. and Lancet, 1907, vol. ii, and 1908, vol. i. :;;,(> Manual of Bacteriology Johne's Disease. Johne's disease,1 a bovine enteritis, is due to an acid-fasi bacillus closely resembling the tubercle bacillus in morphology. It occurs in scrapings of the affected mucous membrane of the bowel, and also in sections of the intestinal wall. The Johne bacillus is not inoculable into the guinea-pig or rabbit, and does not grow on any of the ordinary laboratory media. Twort states that it can be cultivated on the medium employed by him for growing the leprosy bacillus (p. 354). Pseudotuberculosis. The term " pseudo- tuberculosis " (which is not a good one, and should be discarded) has been applied to a number of different conditions which have as a common character the presence of tubercle-like nodules, but which are not caused by the tubercle bacillus. Such are produced by certain parasitic worms, by Blastomycetes, Sbreptothrix and Aspergillus, Protozoa, and by several bacteria. Pfeiffer's Bacillus pseudo-tulerculosis produces nodu- lar deposits in the organ, accompanied by wasting, very like true tuberculosis. The disease, however, runs a more rapid course, death ensuing in the guinea-pigs two to three weeks after inoculation. Guinea-pigs, rabbits, mice and monkeys can be readily infected. The nodules consist of masses of round cells which undergo necrosis and caseation. The bacillus in the tissues is not readily stained, carbol-methylene blue being the best solution, as it is not acid-fast, nor does it stain by Gram's method. Morphologically it is a small rod 1—2 /u in length, usually non-motile, although, according to Klein, it possesses a single flagellum or two flagella at one end. On gelatin it forms a whitish 1 See MacFadyean, Journ. Comp. Path, and Therap., vol. xx, 1907, p. 48. Leprosy 351 growth without liquefaction, like that of the colon bacillus, but confined to the needle-track. It produces alkali, forms no gas, and does not curdle milk. J3roth remains clear, with a whitish stringy flocculent deposit. The bacillus grows readily and rapidly. MaeConkey has found that the fermentation reactions of this organism and of the plague bacillus are practi- cally identical (see "Plague/' p. 416), and sterilised' cultures of either will protect against the other. Ovine caseous lymphadenitis, a disease of sheep simulating tuberculosis, is due to a short plump bacillus with rounded ends which stains well by Gram's method, and grows best on blood-serum, on which it forms greyish colonies.1 Leprosy. ]Lep_rosy, the elephantiasis G-rascorum or true elephantiasis, is a disease which has existed from the earliest times, and was recognised by the ancients. It was undoubtedly somewhat prevalent in the British Isles from the twelfth to the fifteenth centuries, as the many leper houses and enactments against lepers testify. At the same time, no doubt a number of other skin diseases, syphilides, psoriasis, lupus, etc., were at that early period of medical diagnosis confounded with it. In the present day leprosy, although extinct in the British Isles, may be said to have a world-wide distri- bution, for it is met with in Iceland and Scandinavia, Russia and the Mediterranean coasts ; in Persia, India, China, Siberia, and Japan ; in Africa from north to south, m the American continent in- many districts, and also in the Pacific Islands. Three varieties of leprosy are described- the tuberculated or nodular, the anees- thetic, and the mixed. 1 Sixteenth Ann. Rep. Bureau of Animal Indust. U.S.A., p. C3S. Manual of Bacteriology The mode of spread is probably by personal contact (though possibly insects play some part), and throughout ancient and medissval times leprosy was considered bo be a contagious and communicable disease, as witness the stringent regulations in the Mosaic and other laws for the segregation of lepers. J. Hutchinson supposes that fish in the diet, particularly if stale, decomposed, or badly cured, in some way is a causative factor; but he is practically alone in this view. A bacillus, the Bacillus lepra', is abundant in the tissues, and was discovered by Hansen in 1879. In form it resembles the tubercle bacillus, hut is slightly more slender; it probably does not form spores, though in stained preparations the same irregularity in staining — namely, the occurrence of unstained inter- vals, the so-called " beading " — is met w ith as in the tubercle bacillus, and is assumed by some to be due to the presence of spores. 'The organism as obtained from the tissues is non-motile, stains readily with the ordinary anilin dyes, and by dram's method, which brings out the beaded appearance very well, and is markedly acid- fast, thus closely resembling the tubercle bacillus, and the methods used to demonstrate it are the same as for the latter organism. The Bacillus lepras is round in enormous numbers, usually crowded together in bundles or masses, in the leprous nodules in the skin (Plate X., a), liver, spleen, and testicles, and in the affected nerves in the anass- thetic form ; it has also been found in the blood, but only in the febrile paroxysms which set in when the disease is approaching a fatal termination. The exact situation of the leprosy bacilli in the tissues has been a matter of controversy. By some it has been held that they are contained within certain round cells, the so-called leprous cells, and this may be the case, but to PLATE X a. Leprosy. Section of akin x 1500. To face />) In animals the agglutination reaction is stated by Moore and Taylor1 to give accurate results. In man this test might give an inconclusive result (see aide). (7) In the tiss_!ias_the glanders bacillus is difficult td demonstrate. Sections may be stained for half an hour with carbol niethylene-blue, treated with 4 per cent, acetic for a few seconds, washed, and rapidly dehydrated with alcohol, cleared and mounted. McFadyean recommends, after treating with the acetic and washing, flooding with a saturated solution of tannic acid in water for fifteen minutes, washing, counter- staining in a 1 per cent, aqueous solution of acid fuchsin for fifteen to thirty seconds, washing, dehydrating, and clearing in cedar oil. Twort's method may also be employed (see section on Amoeba coli, "Clinical Diagnosis"). 1 Joum. of Infect. Diseases, Sup. No. 3, May, 1907, p. 85. 24 370 Manual of Bacteriology ^ 0 CHAPTER X. TYPHOID l-'KVKIl — L'ARA-TYl'HOID MVJCK — BACJLLUS KNTKUI- TID1S AND THE GtAIiTNEJA GKOUf — SWINE E.BVEK — BACILLUS DYS K N T K Bl -K BACILLUS COL 1 . The organisms considered in this chapter form a natural group or family, and pass as it were by gradations in cultural characters from the typhoid bacillus to the colon bacillus. Loftier classes them together in a family, the Typhacese, which is divided into sub- families by the reactions of the organisms included in it on certain culture media. These culture media are: ( 1 ) the_/ uphold solution, an aqueous solution containing 2 per cent. peptone, 1 per cent, nutrose, 1 per cent. ^rape_ "su^ar, 5~per~ceut. lactose, and T5 c.c. per cent, normal potash ; T27~the para-typhoid solution, having the same composition with (he exception that the glucose is omitted. To 100 c.c. of the solution, in each case. 1 c.c. of a 0'2 per cent, solution of chemically pure malachite- green crystals (Hoechst, No. 120) is added. Loffler's classification is as follows: Family. — Typhacese. 8%1-family (1)— Typhese. This group does not ferment the typhoid solution, but either leaves it unchanged or precipitates it, The para-typhoid solution is either not fermented, is unchanged, or becomes milky. The following organisms are contained in it: (a) B. typhosus, actively motile, precipitates the typhoid solution, has no action on the para-typhoid solution. (6) B. dysenteric (Flexner), motile,1 does not precipit ate the typhoid solution, and the para-typhoid solution is unchanged, (c) B. dysenteric (Shiga-Kruse), non- ' it is usually staled to lie non-motile. Typhoid Fever 371 motile, has no action on either solution, but precipitates the typhoid solution if the green be omitted. (. coil and other organisms (see also p. 405). Chatterjee2 finds that agar on which the typhoid bacillus has been grown contains substances which inhibit further 1 Occasionally a feeble indole reaction may be obtained by careful testing. - Trans. Fourteenth Iniernat, Cong, of Hygiene (Berlin, 1907), Bd. iv, p. 34. 374 Manual of Bacteriology development of the organism if if be inoculated on to an agar culture which has been scraped so as to remove all growth. Pathogenicity. — In cases of typhoid fever in man the Bacillus typhosus is widely distributed in the body, in the various tissues, and in the blood, from which it may be obtained by cultivations made from at least 0"5 c.c. (see "Clinical Diagnosis," p. 889). The bacillus is con- stantly present in the blood from the commencement of the disease, though not in large numbers, and cultures from the blood in competent hands result in the recovery of the organism in approximately 100 per cent, of the cases ; in the later stages of the disease it is less frequently recovered.1 In addition to being present in the Peyer's patches, mesenteric glands, and spleen, the B. typhosus has been found in the rose-spots of the eruption, in the sweat, in the sputum and lungs in the pulmonary complications, and in the urine. In the urine it is so frequently present that special disinfection should be practised, more particularly during con- valescence, and in some eases it may be SO abundant as to produce a turbidity (typhoid bacilluria) and cystitis. It is also pyogenic, and occurs (usually in pure culture) in concurrent or post-typhoidal complications, e.g. empyema, abscesses, osteomyelitis, suppurating ovarian cysts, etc. Clumps of bacilli in the gall-bladder have been suggested as the nuclei of gall-stones, and the bacilli may be so numerous in the gall-bladder and bile- ducts as to cause cholecystitis and cholangitis. The organism is by no means easy to isolate from the stools, simple plate cultivations usually fail, and the best medium to employ is the Conradi-Drigalski or malachite- green agar (see "Water"). Injected intraperitoneal! y into mice and guinea-pigs, l Coleman and Buxton, Avicr. Jovm. Med. Sri.. June, 190". Bacillus Typhosus 375 the B. typhosus usually produces death, and the same result follows from intra-venous injections m rabbits, but the pathogenic effects so obtained are not specific. By continuous cultivation it loses its pathogenic proper- ties Given by the mouth no result follows, and the same is the experience of most observers who have fed animals on typhoid stools; a disease process analogous to typhoid fever in man has rarely been induced experimentally. Remlinger1 states that by feeding young rabbits on vegetables, cabbage, etc., soaked m water, to which had been added some culture of the typhoid bacillus, he has succeeded in inducing a con- dition resembling typhoid fever in man. The charts which accompany the paper show a typical rise of temperature, a period of pyrexia with morning remis- sion, followed by a typical fall of temperature. The animals suffered from diarrhoea, and their blood gave the agglutination reaction. Post mortem, the intestine was congested and filled with yellow diarrhceic matter, the Peyer's patches were swollen and in some places commencing to ulcerate. The spleen was increased to two or three times its normal size, and cultures of the typhoid bacillus were obtained from it. Metchnikoff2 has infected the chimpanzee par os with typhoid faeces. The proof of the causal relation of the Bacillus typhosus to enteric fever is based on the following facts. It is met with in the tissues in cases of enteric fever, can lie obtained from the spleen during lii'e by puncturing with a hollow needle, and may be isolated from the urine and blood during the course of the disease, and is not met with in other diseases. The writer has had under his care three eases, and knows of several others, in which the disease was almost 1 Ann. •<> with eases which recover, and cases in which agglutination is feeble or absent tend to be severe. Toxins. — From cultures of the typhoid bacillus Brieger isolated a base which he termed typhotoxin, and which is isomeric with gadinine. In animals it produced salivation, profuse diarrhoea, paralysis, and death. Brieger and Kninkel isolated from cultures a toxic protein body. Fen wick and Bokenham2 extracted from spleens of typhoid fever patients a proteose, an alkaloid, and a fatty residue. The proteose produced fever, anorexia,, and loss of weight in guinea-pigs and rabbits, bnt the alkaloid and fatty matter were without effect. The toxins of the typhoid bacillus, however, seem to be largely iiitra-celbilar, and filtered broth cultures m are u^sjnjllxjilinost non-toxic^ Sidney Martin1 by cul- tivating in a protein medium was able sometimes to obtain a toxic filtrate, a few c.c. of which produced lowered temperature, diarrhoea and death. Macfadyen and Rowland,1 by disintegrating large quantities of typhoid bacilli, filtering, and so obtaining the intra- cellular constituents in the Hltrate, found that small doses of the latter produced a transient rise of tempera- ture in guinea-pigs and a loss of weight which was soon recovered from. Animals so treated were pro- 1 Malvoz, Ann. da Vlnst. Pasteur, xi, 1897, p. 582. * Brit. Med. Journ., 1895, vol. i, p. sol. :l Ihid., 1S9H, vol. ii, pp. 11 unci 7!i. •< Ccntr.f. Bald., xxx, p. 753. Survival of Bacillus Typhosus 379 tected against a certain lethal dose of typhoid bacilli, and their blood exhibited agglutinative and hacterjfllylig. properties towards the typhoid bacillus. Macfadyen later obtained the intra-cell ular juice of typhoid bacilli by disintegration after freezing with liquid air, and found it to be very toxic to guinea-pigs by infra-peritoneal, and to rabbits by iutra-venous inoculation. The writer found that cultures of the Bacillus typhosus&o. not give the " diazo " reaction.^ Survival of the typhoid bacillus in the body. — Bacilli may persist in the spleen for weeks, in the gall- bladder for years, and in suppurative lesions for six years or more. Foster and Kayser obtained pure cultures from the gall-bladders of seven out of eight cases, and in 2 per cent, of the cases this Ci chole- cystitis typhosa" becomes a chronic process, and typhoid bacilli may be discharged into the bowel for long- periods. Dean2 found this to be the case in a patient who had had enteric fever twenty-nine years previously. Such " typhoid carriers," have been the subject of much investigation recently;1 A. and J. Ledingham record three instances met with in an asylum in which mysterious cases of typhoid had occurred- — 31 cases during fourteen years. Davies and Walker Hall4 relate similar outbreaks, the carrier in this case being a woman who had suffered from enteric fever in 1901, milk serving as the vehicle of transmission, and a number of other instances have been recorded. Three fourths of the cases are women (and three fourths of the cases of gall-stones occur in women), and usually 1 Pror. Rot/. Soc. Lond., B. Ixxi, 1902, p. 77. 3 Brit. Med. Journ., J! 108, vol. i, p. 562. ;; See Ledingham, Sep. Med. Off. Lor. Gov. Board for 1909-10 ( Bibliog). 1 Proe. Roy. Soc. Med., vol. i, 1908, Epidemiolog. Sect., p. 175. 1 880 Manual of Bacteriology the serum of the carriers gives a marked agglutination reaction, and their stools frequently contain such large numbers of typhoid bacilli that these largely replace: the natural bacterial iiora of the intestine and may often be recovered from the stools by simple plating. Obviously the typhoid carrier is a source of serious risk to the community, and mysterious outbreaks of enteric lever, ascribed by some in the past to a " de novo " origin of the specific organism, become explicable. The typhoid bacillus may occur in the contents of ovarian cysts, usually causing suppuration, and may survive for months— twelve in a case recorded by Taylor1 — after the attack of typhoid. Survival of the typhoid bacillus outside the body. — The Bacillus typhosus has been isolated in a few instances from water supplies which have become infected, and have given rise to epidemics, as in the case of the Lincoln epidemic in 1905.2 This is the exception, however, and the isolation of the typhoid bacillus from an infected water is a very difficult rnatteV on account of the fact that the bacillus may have"died ont-bfifpro the investigation is commenced, that it is generally' in a small minonEy"Tnrd admixed with numbers of conform organisms, and that until recently no medium was available which inhibited the , crrnwth of f,hp rWi-form organisms without at the same *"tTme inhibiting the growth of the B. typhosus. By the use of malachite or brilliant gre^u-me^lia, thejast^ rl rljiffigWy seems to have beenovercome (see name< section on "Water"). In sterilised waters, including distilled water, the Bacillus typhosus maintains its vitality for upwards ol a month, 'and in some cases for much longer. The i Joum. Ohstet. and Gywecol. Brit. Empire, November, 1W7. ■ Rep. Med. Of Lac. Gov. Board for 1905-06. Survival of Bacillus Typhosus 381 survival is not necessarily longer in an organically polluted water than in a pure water. Infecting sterilised Thames water (from the Temple Embank- ment) and sterilised tup-water of the Chelsea Water- works with typhoid cultures, the writer found that, examining small quantities (1 c.c.) of the water, the bacillus appeared to die out in the former in two to three weeks, in the latter in four to five weeks. The survival of the typhoid bacillus in natural waters must be influenced by many circumstances- temperature, chemical composition, struggle for exis- tence with the natural bacterial flora, etc., of the water. Experiments by Russell and Fuller,1 in which the organism, suspended in collodion sacs, was subjected to the action of lake water, indicated that the maximum was eight to ten days, llouston^ using raw Thames, Lee, and New River waters artificially infected with varying quantities of ordinary laboratory typhoid cultures, and examining quantities of 100 c.c. of the water, found that in none of eighteen experiments was a negative result obtained in four weeks, and it was only after nine weeks that the typhoid bacillus could not be isolated from this quantity in all the experi- ments. But in subsequent experiments,3 in which typhoid bacilli, obtained directly from the urine of a carrier case by centrifugalising and without culturing, were added to the water, the number of bacilli was reduced by 99"99 per cent, after a week, and after ten days the organism could not be isolated from 100 c.c. of the infected water, indicating that the uncultured bacillus rapidly dies in a natural water, and that even a week's storage of water affords enormous protection 1 Jown. Infect. Diseases, Sap. No. 2, February, 1902, p. 40. 2 First Rep. on Research Work, Metropolitan Water Board, 11)08. ;1 Sixth Research Report, Metropolitan Water Board, 1911. 382 Manual of Bacteriology against water-borne typhoid. In aerated (C02) waters the B. typhosus does not survive a fortnight. The methods of isolation from water are given in Chapter XXI. The Bacillus typhosus may gain access to shell-fish,1 oysters, mussels, cockles, etc., particularly if obtained from sewage-polluted laying. Such polluted shell-fish may give rise to typhoid epidemics — as at Winchester and South amp tion in the case of oysters, and in the case of cockles, derived from the Thames Estuary and imperfectly cooked, to typhoid cases. Buchan found that out of 855 primary cases of typhoid fever occur- ring in households in Birmingham, 124, or 14"5 per cent., had a history of mussel eating, and in seventeen instances the histories were conclusive of mussel infection. Mussels, under certain conditions (which are not well understood), are liable to develop my tilotoxin, ele. (p. 39), which gives rise to gastro-enteritis. Shell-fish from sewage- polluted layings contain B. colt in varying numbers, but from uncont animated layings are free from this organism, which may therefore serve as an index of pollution (see "Examination of Shell-Eish," Chapter XXI). Contaminated shell-fish, removed to pure water, gradually cleanse themselves — probably after two to three weeks' sojourn. Klein obtained the typhoid bacillus from artificially infected oysters, kept in tanks of sea-water, after nine, sixteen, and even eighteen days from the commencement of the experiment, the oysters showing no abnormal condition. As regards the vitality of the Bacillus iyphosus in 1 On pathogenic organisms in shell-fish see Reports by Bulstrode to the Local Government Board, 1894 and 1911 ; Hep. Med. Off. Loc. Gov. Board for 1899-1900, p. 574; Houston, Fourth Report of the Sewage Commission, vol. iii, 1904; McWeeney, Loc. Gov. Board, Ireland, 1904; Buchan, Journ. of Eyffiene, vol. x, 1910, p. 569. Survival of Bacillus Typhosus 388 sewage we have little certain information ; probably it tends to die out within a few days. In sterilised sewage inoculated with it the B. typhosus hardly multiplies at all, and at the end of ten days has died out. Certain organisms in sewage seemed to have deleterious action on the B. typhosus, hastening Us extinction, viz. the B. fluoresces liquefaciens and B. fluorescens stercoral;*. Russell and Fuller, subject- ing the bacillus to the direct action of sewage, found the survival to range from three to five days. Jnjlry gai^e" p.ni'fix. according to Dempster,1 the Bacillus typhosus does not live longer than eightou days (Firth and Horroclcs recovered it up to twenty- five days), and in peat it dies within twenty-four hours. In moist soil, however, the bacillus still survived on the forty-second clay. In an artificially dried soil it was not found alive after the seventh day. Sidney Martin found that r^no^t^teriUse£^o\]_kejr)t at temperatures from 3° to 37° C, the B. typhosus maintains its vitality for upwards of fifteen months^ but that in unsterilised soil it rapidly dies/ Mair3 concludes that the typhoid bacillus can survive in natural soil in large numbers for about twenty days, and is still present in a living condition alter seventy to eighty days, but that there is no evidence that it is capable of multiplying and leading a saprophytic existence in ordinary soil. He suggests that Martin's result (the rapid extinction of the bacillus in un- sterilised soil) may be explained by the use of broth cultures for infection, the broth added causing a multiplication of the saprophytes. Firth and Horrocks1 1 Med.-Chir. Trans., vol. lxxvii, 1894, p. 263. 3 Raps. Med. Off. Loc. Gov. Board f&t L896-1901. ;l Journ. of Hygiene, vol. viii, L908, p. 37. 4 Brit. Med. Journ., 1902, vol. ii, p. 936. ;!s | Manual of Bacteriology similarly conclude bhat the typhoid bacillus displays no tendency to increase in numbers, nor to grow upwards or downwards in soil, though it may be washed by water through a thickness of 18 inches. Neil her virgin nor sewage-polluted soils differed much in these respects. Vitality of B. typhosus in dust, fomites, etc. — Firth and Horrocks found the 13. typhosus to be alive in soil dry enough to form dust for as Long as twenty-five days, and consider that infective material can be readily transmitted from dried soil and sand by means oF winds and air-currents. Doubtless much depends on the degree of dryness of the substratum. From khaki drill and serge inoculated with cultures the bacillus was recoverable for from ten to twelve weeks, and For From ten to seventeen days from the same materials fouled with enteric faeces. Semple and G-reig,1 with cloth and blanket infected with typhoid urine, tailed to obtain the bacillus after seventeen days. This, however, was in India, and the survival of the typhoid bacillus on Fomites probably greatly depends on the degree of drying of the material. A striking instance of the conveyance of infection by Fomites was that of the blankets used in the South African war and brought to this country, which gave rise to many cases of typhoid fever. Firth and Horrock demonstrated that house-Hies can convey enteric infective material from specific excreta or other polluted material to objects on which they settle or feed, and the Commission which investigated the prevalence of enteric fever in the Spanish-American war ascribed to flies the principal part in the dis- semination of the disease (see also p. 411). There has always been considerable discussion on the 1 Sc. Mum. Guv. of India, No. 32, 1908. Air-infection and Typhoid 385 ' exact relation of " sewer- gas " to disease. It is gene- rally held that sewer-gas is at least a predisposing cause to enteric fever, diphtheria and tonsillitis. Some have considered that the specific organisms are present in the emanations from sewers, and this may occasionally be the case. Thus Horrocks,1 in some experiments performed at Gibraltar, by pouring sewage artificially infected with typhoid culture down dizains, showed that specific bacteria present in sewage may be ejected into the air of ventilation pipes, inspection chambers, drains and sewers by (a) the bm-sting of bubbles at the surface of the sewage, (b) the separation of dried par- ticles from the walls of pipes, chambers and sewers, and probably by (c) the ejection of minute droplets from flowing sewage. " Sewer-gas ;' may also lower vitality and increase susceptibility. Thus Alessi found that animals exposed to di'ain emanations are at first more susceptible to infection, but after a month or so acquire tolerance and are no more susceptible than animals kept under ordinary conditions. There is no evidence that sewer-men or those employed at sewage- works suffer from ill-health. jLctiun of heat, germicides, etc. — The B. typhosus in broth culture is killed by a temperature of 53°-54° C. in -60 C. in ten minutes. It is half an hnnr and of 56 readily destroyed by antiseptics? {See table, ChapTXXII. Semple and Greig (loc. cit.) found bright sunlight to be germicidal in from two to six hours. Wines and spirits have some germicidal action on the typhoid bacillus. Champagne destroys the bacillus in ten minutes, white wines in fifteen to twenty minutes, red wines in thirty minutes or thereabouts. If diluted with water the germicidal action takes much longer to accomplish, and the acidity, not the alcohol content, 1 Journ. Boy. San. Inst., May, 1907, p. 176. 25 386 Manual of Bacteriology seems to be the active factor.1 Spirits, such as whisky or brandy, if diluted with not more than one to two times the volume of water, kill in ten to twenty minutes. Anti-typhoid serum. — Attempts have been made to prepare an anti-typhoid serum by inoculating horses with, increasing doses of typhoid bacilli, first killed (by heat, chloroform, etc.) and then living, but such sera have proved quite useless. Macfadyen2 prepared an endotoxic serum by treating horses with the endotoxin obtained by triturating the bacilli in the presence of liquid air. The writer continued the work, and obtained a serum which gave promising results.0 Chantemesse,4 by cultivating a virulent strain of the typhoid bacillus in a special broth made with ox spleen, heating the culture to 55° C, centrifugalising and injecting horses with the fluid, obtains a serum which he claims has marked curative properties, the mortality being 43 per cent., as against 17 per cent, for those subjected to ordinary treatment. The patients receive very small doses of the serum — five or six drops — and the dose is repeated only two or three times. This dosage is quite different from that of an ordinary anti- toxic or antimicrobic serum, and Wright suggested that toxins (and not anti-bodies) in the serum may be the active agents. Chantemesse has accepted this view, and the treatment, therefore, seems to be a vaccine one. The disease has also been treated with a vaccine (consisting of a killed culture) with promising results 1 Sabrazes and Marcandier, Ann. de I'Inst. Pasteur, 191 l7. 2 Proc. Roy. Soc. Loncl.,B, vol. lxxi, 1903, pp. 76 and 351 ; Brit. Med. Journ., 1906, vol. i, p. 905. 3 See Hewlett, Goodall and Bruce, Proc Roy. Soc. Med^vol. n, 1907-08 (Medical Section), p. 245 et seq.-, and Hewlett's Serum Therapy, p. 220. . ,QfV7 4 Trans. Fourteenth Inter nat. Con 4 Manual of Bacteriology microscopic. Some confusion exists in the nomenclature of the disease. Swine fever is the British, and hog cholera the American, name. In addition, a disease of swine was formally described under the designation " swine plague " (" Schweine- seuche," Schiitz). This clinically much resembles swine fever, but pneumonia is a prominent lesion, and a non-motile, stumpy, bi-polar staining bacillus belonging to the group of the hemorrhagic septicemic bacilli is present (see under " Chicken Cholera "). This is now regarded as a secondary infection and the disease as being really swine fever. The B suicholern', is apparently identical with the B. ictero'ides of Sanarelli. (See also Chap. XIX.) Although the lesions are very similar, swine fever has nothing to do with typhoid fever of man, nor with ulcerative colitis. Other organisms belonging to the Gartner group are : 1. The Danysz bacillus, used as a virus for exterminating rats (the Danysz virus). 2. The B. ictero'ides of Sanarelli, supposed by him to be the cause of yellow fever, but apparently identical with the B. suicholera (see " Yellow Fever," Chap. XIX). 3. The B. typhi murium of Loftier, used as a virus for exterminating mice. 4. The B. psittacosis of Nocard, causing an infective disease of parrots and transmissible to man (bird-fanciers, etc.), in whom it produces a severe and often fatal broncho- pneumonia. 5. Summer diarrhoea. — Morgan1 concluded that the summer or epidemic diarrhoea of infants is not caused by the dysentery bacillus (see p. 401). In 50 per cent, of the cases he isolated a bacillus which appears to be most closely allied to the hog-cholera bacillus, differing from the latter by producing alkalinity in litmus milk (without previous acidity) and much indole, and by failing to produce acid and gas from mannitol, arabinose, maltose, and dextrin. Eyre and Minett* » Brit. Med. Journ., 1906, vol. i, pp. 908 and 1131 ; ibid., 1907, vol. i, p. 16. 2 Brit. Med. Journ., 1909, vol. i, p. 1227. r Para-typhoid Fever 395 examined the normal faces of sixty young children, and in four only isolated a bacillus allied to the Morgan bacillus. The method of isolation was by means of plates of bile-salt agar containing 1 per cent, of mannitol and coloured with neutral red. (See also Chap. XX.) Para=typhoid Fever.1 The name " para-colon " bacillus was given by Gilbert in 1895 to races of bacilli intermediate in type between the typhoid bacillus and the colon bacillus, and this designation was also applied by Widal and ISTobccourt to a bacillus isolated by them from an abscess in the neighbourhood of the thyroid. The name "para-typhoid" bacillus appears first to have been used by Archard and Bensaucle in 1896, and was reintroduced by Schottmuller in 1901, and would seem to be the preferable designation for those micro-organisms that produce typhoidal symptoms. Para-typhoid fever may be defined as a disease much resembling typhoid fever in its clinical aspect, which is, however, caused, not by the typhoid bacillus, but by organisms belonging to the para-typhoid sub-group of the Gartner group of bacilli. Para-typhoid infections sometimes occur in epidemics, may be spread by drinking-water and by " carriers," and occur in all parts of the world. Para-typhoid bacilli are also occasionally the pathogenic agents in cases of "food poisoning" with gastro-enteritis, particularly B. suipedifer (or aertryck) . The para-typhoid bacilli are morphologically like the typhoid bacillus and are actively motile, but they ferment glucose with the production both of acid and of gas. A number of races have been isolated differing 1 See Savage, Rep. Med. Off. Loc. Gov. Board for 1908-9, p. 316 ; Bainbridge and O'Brien, Journ. of Hygiene, vol. xi, 1911, p. 68 (Bibliog.). 396 Manual of Bacteriology From one another in their source, rate of fermentation of glucose, action on milk, action on neutral red, and agglutination reaction, and are distinguished by the names of those who isolated them. Two groups of the para-typhoid may be distinguished which have been termed A and u by Buxton. Group A produces less gas in glucose media than group b ; with group A milk remains permanently acid ; with group b it becomes alkaline after a transient acidity ; and though group A changes neutral red to yellow, the red colour tend to return after three weeks or so, while with group b the yellow colour is permanent. That is to say, in its reactions group A is allied more closely to the typhoid bacillus than group b. B. paratyphosu* a or « is rarely found, the vast majority of eases of para-typhoid fever being associated with the presence of the B or /3 type. The fermentation reactions of some of the para-typhoid bacilli are given in the table on p. 400. * As regards the agglutination reaction, the blood of the para-typhoid fever patient either does not agglutinate the typhoid bacillus or agglutinates it only in low dilution — e.g. 1 in 10 to 40, while it agglutinates the para-typhoid bacilli in far higher dilution— e.g. 1 in 100 or 200, or even higher; thus in Cashing'* case the patient's serum agglutinated the para-typhoid bacillus isolated from it up to ] in 8000. The diagnosis of para-typhoid fever would be based on (a) the agglutination reaction ; {b) the isolation of a para-typhoid bacillus by cultures from the blood (p. 389). Bacillus dysenteric. In one type of dysentery, the so-called epidemic or bacillary form (see " Dysentery," Chap. XX), a bacillus, Bacillary Dysentery 397 B. dysmterix, is the causative agent. The 5. dysenteric includes a group of closely allied organisms. The dyseritery bacillus was first isolated m 1897 by Shiga in Japan. Somewhat later Kru.se isolated an almost identical bacillus in Germany, and this type is known as the Shiga-Kruse type. Later, Flexner and Strong isolated another type of the dysentery bacillus, and during the last few years similar organisms, but differing from the Shiga-Kruse and Flexner types in some of their fermentation and other reactions, have been isolated ; these are sometimes termed " pseudo- dysentery " bacilli. The Shiga-Kruse and other types of dysentery bacilli have been isolated by Flexner and Strong in the Philippines, Park, Duval, Bassett, Martini, Hiss, Russell and others in the United States, Castellani in Ceylon, Rogers and others in India, Puffer and Willmore in Egypt (El Tor), and Eyre, McWeeney and others in the British Isles. Morphology. — The B. dysenteric are small slender bacilli much resembling the colon bacillus. They are non-motile, but Brownian movement is often active,1 Gram-negative, and non-sporing, and are readily destroyed by heat (58°-6(T C.J~ahd antiseptics. Cultural characters. — The dysentery bacilli are aerobic and facultatively anaerobic. On agar a thinnish creamy growth develops ; on gelatin a white growth nearly limited to the inoculation track, and without liquefac- tion. The colonies on a gelatin plate closely resemble those of the typhoid bacillus. On potato the growth is either thin, grey and slightly visible, or thicker and yellowish or brownish. The colour of neutral red media is unaltered. Litmus milk first becomes faintly acid, 1 Flagella have been described l>y some observers, but cannot usually be demonstrated, 398 Manual of Bacteriology then markedly alkaline; no clotting. Indole is generally not formed; occasionally a trace may bo detected. All strains ferment glucose with the forma- tion of acid only,^jip__gas_| none ferments lactose. Some strains (the Flexner type) ferment mannitol with the formation of acid only, no gas ; other strains (the Shiga-Kruse type) have no action on this alcohol. The principal fermentation and other reactions are given in the table on p. 400. These reactions are very variable with different stains, but differentiation may be accomplished by agglutination, saturation, and com- plement fixation, tests. Agglutination reaction.' — The agglutination reaction is given by the blood of patients suffering from the bacillary form of dysentery, but not by the amoebic form (unless a double infection be present, which occasionally is the case). The agglutination reaction is obtained in dilutions of 1 in 10 to 1 in 100, but may occur only with the particular strain causing the infection.1 Thus by the agglutination reaction variations between different strains of the B. di/senterige may be detected. Pathogenic action. — The organism seems limited to the bowel and its mucous membrane and does not gain access to the blood. No chai'acteristic lesions are produced in animals by administration of the dysentery bacillus per os. In man, cultures given by the mouth are stated to have induced a typical dysentery. Animals such as rabbits, guinea-pigs and mice are very sensitive to injections of living and killed cultures; in fact, it is very difficult to immunise animals against the organism. Amounts of 0*1— 0-2 mgrm. of an agar culture given intra-venously or intra-peritoneally are fatal to these animals. In man the organism is abundant in the bloody 1 See Hewlett, Trans. Path. Soc. Lond., vol. lv, 1904, p. 51. Bacillary Dysentery 399 mucoid discharge from the bowel, and at an early stage is easy to isolate by means of Conradi-Dngalsla agar plates, on which it forms small blue colonies ; at a later stage (after two to three days) the other organisms m the bowel multiply to such an extent that isolation may become very difficult. " Carriers " occur and help to spread the disease, which may be conveyed by infected water and food and by flies. Toxins— The filtrate of dysentery cultures (four to six weeks old) in a somewhat highly alkaline broth (broth just alkaline to litmus + 7 c.c. normal NaOH per litre) is markedly toxic, 0:1 c.c. being a fatal dose for a large rabbit.1 Anti-serum and vaccine. — The serum of horses immunised with the toxin, or with dead and then with living cultures, possesses marked antitoxic properties, and the use of this antitoxic serum has been successful in cases of acute bacillary dysentery. Shiga obtained a reduction in mortality from 22 to 7 per cent, by the use of .serum in a severe epidemic, and striking results were obtained by Ruffer and Willmore2 in Egypt. It is necessary, however, to employ a serum prepared with the particular strain of the disease. When the disease has become chronic the use of a vaccine, consisting of a culture sterilised by heat, is sometimes beneficial. Castellani also suggests the use of a vaccine for prophylactic purposes. Para-dysenlery bacilli. — In the dysenteries of Ceylon, Castellani3 has sometimes isolated dysentery bacilli nearly related to the Shiga-Kruse type, but showing differences from it in agglutination, persistence of acid 1 Todd, Journ. of Hygiene, vol. iv, 1904, p. 480 (Bibliog.). » Brit. Med. Journ., 1909, vol. ii, p. 862, and 1910, vol. ii, p. 1519. 3 Journ. of Hygiene, vol. iv, 1904, p. 495. 1 400 Manual of Bacteriology fcD .•a o — — o> Pi o E 9Q o pq pq Si B ■ 8 '53 Oh % B 8 a. pq Pq Pq Pq K) Oh pq pq ' 11 1 1 I J. 1 IM J| S .l.HMl>[Sll.l,| 1 1 1 1 1 1 1 1 i + + 1 + 1 ■ 9SI 1.111 (•) III O.BH •■}ll83.lotl su;> >G CM ua CM o IG IO o oq — + rt + _ EM rH ?! H n I^h + ■H + + + 1 J + + 1 1 1 + 1 ■3[Opul 1 + H- H- + + + 1 + uiiuiaf) jo aoiioHjenljiT; 1 1 1 1 1 1 1 1 i 1 1 1 + 1 n\ O -t; %< X b "■J < < •ui[nni II 1 • ill! : I" 1. 1 1 • 1 1 1 1 1 i -H -H 1 1 ■< + + + + 1 < + + + + + + + •«aopY + + + + 1 + + + 1 1 1 1 1 1 + + + + 1 1 + + + + + + + < + + + + 1 < + + + + + + + ■jBSna-airuQ 1 H- 1 1 1 1 1 i i + + 1 + + •asoioiiq 1 + 1 1 1 1 1 i i + + + + + •880UHJUH + + + + < < + ■< + + + + + •asomquiy 1 + + + 1 < < + + + + + •9S013H1«9 < + + + + < < + + + + + + + < + + + + < + + + + + + + •BSOtnAasi < + + + + + + + + + + •asoDtiiO < + + + + < < + + + + + + + B O W «» II •- »H ii • S on ll s - — CD J) bt be a a a XZ o ° o c C 01 ai • .6 S 2 s C on O B 0 g B 1 *g e a* = it DO o a & cS *s s ^ 5 ~ -- J. II II II < + -H The Colon Bacillus 401 reaction in, litmus milk, and virulence ; these he has termed " para-dysentery" bacilli. Asylums dysentery and summer diarrhoea of infants. — Both in America and in England some cases of summer diarrhoea of infants are found to be associated with the B. dysenterise (see above, p. 394). The asylums or institutional dysentery, or ulcerative colitis, is also due to this organism, and the blood of patients gives the agglutination reaction.1 In both instances the B. dysenteric present is of the Shiga-Kruse type. Bacillus coli. The Bacillus coli, or colon bacillus (B. coli com- munis), is an organism of considerable importance, both in connection with the Bacillus typhosus, in- pathological processes, and in water supplies as an indication of pollution. As its name implies it is a constant inhabi- tant of the intestinal tract in man and animals (except perhaps in certain arctic animals), and is one of the most widely distributed organisms in nature. While the term "colon bacillus" is applied to a fairly well- defined organism (the "typical B. coli"), there are a number of allied organisms differing from the type in one or more characters— e. g. motility, indole production, fermentation reactions, rate and extent of milk curdling, etc.— and these varieties are said to belong to the " colon group/' or are termed " coliform." The B. coli may be readily isolated by inoculating litmus factose bile-salFBeDtone-w^, ^ „TT^~ of a suspensLen-ef-fres-h- feces, growing for from twenty-four to forty-eight hours at 42° C, and plating- he culture on litmus lactose agar, on gelatin, or on jZonradi-DriralRln ^ or by direct qf ^ 1 Hewlett, Trans. Path. Soc. Lond., vol. It, 1904, p. 51. 26 L02 Manual of Bacteriology fgeces suspension on the last-named medium (see also " Water Morphology* — The B. coli is a short rod with rounded ends, 2 or 3 long and 0/4 to 0*6 fx broad, frequently linked in pairs or more. It is often so short that it is merely ovoid in shape ; and, on the other hand, longer individuals and involution forms occur 10 /x or more in length (Plate XIII., b). It is feebly motile, and possesses lateral flagella to the number of three or four on an average, which are shorter and straighter than those of the typhoid bacillus. It is sometimes met with in a diplococcoid form, which by cultivation in ascitic fluid may become fixed. Capsulated forms have been described. Spore-formation does not occur, but variolation may sometimes be observed. The organism is well stained by the ordinary anilin dyes, but is Gram-negative. Cultural characters.— The B. coli is , aerobic and^ Fa.f.nU,a,tivelv anaerobic, and grows readily on the "ordinary culture media from 20° to 37° G. In gelatin plates the colonies are visible in twenty-four to forty- eight hours. The deep colonies are spherical, granular, and of a pale brownish colour, darker at the centre than at the periphery. The superficial colonies are at first punctate, round and almost transparent, but sub- sequently spread on the surface and may attain a diameter of 3 mm., their margins become irregular, the surface is smooth, they are finely granular, opa escent in appearance, and are thicker at the centre than at the periphery (Fig. 41). On a gelatin streak a copious white/shining, smooth growth develops the margins of which are irregular and crenated (Plate XIII , c), and m old cultures the medium becomes opalescent, In a gelatin stab-culture a white growth develops along the line of inoculation with one or more gas-bubbles, lhe gelatin To face page 102. The Colon Bacillus LOS is not liquefied. On agar and on blood-seruin a thick, moist, shining, greyish layer forms. There is abundant formation of gas in a stab-culture in glucose-agar and in gelatin shake cultures (Fig. 42), provided the medium be made with meat; " lemco " gelatin, however, gene- rally fails to give gas. On acid potato it forms a sfcraw-yellow or brownish-yellow, moist, thick growth, but if the potato is not fresh and acid in reaction the Fig. il— Colonies of the colon bacillus, superficial and deep. growth may be colourless. Milk is a good culture medium, and is curdled in twenty-four to seventy-two hours. This curdling is principally due, not to an enzyme, but to the formation of a considerable amount of lactic acid, though a milk-curdling enzyme has been described by Savage1 as being formed under certain conditions. The gas which is produced in culture media under anaerobic conditions consists of hydrogen 1 Joum. Pathol, and Bad., November, 1904, 404 Manual of Bacteriology and carbon dioxide. Under aerobic conditions marsh gas is stated to be also formed. Tho ratio of H to C03 is about 2: 1 for dextrose and lactose. In broth it produces a general turbidity without film formation, and tho culture gives the indole reaction on the addi- tion of a nitrite in twenty-four to forty-eight hours. The fermentation reactions are given in the table, p. 400. It will be seen that the B. coli is an active fermenter of many carbohydrates, alcohols, and gluco- ITig. 1-2.— Colon bacillus. Gelatin shake culture showing gas production. sides,1 e. g. glucose, lactose, galactose, mannitol and dul- citol, but not of adonit. Cane-sugar may or may not be fermented"; ' sometimes "only acid is formed, sometimes both acid and gas are produced. To the variety pro- ducing both acid and gas from cane-sugar Durham gave the name B. coli commnnior. Prescott and Winslow consider that the term B. coli should be applied only to an organism that_does not attack ketonic sugars. Neutral red in glucose broth is ' See Twort, Proc. Boy. Soc Lond., b, vol. lxxviii, p. 329. The Colon Bacillus 405 changed to a fluorescent yellow, and Houston describes a typical B. coli as " flaginac," i. e. producing fluores- cence in neutral red glucose peptone-water (fl), acid and gas from glucose (ag), indole in peptone-water (in), and acid and curd in milk (ac). The colonies on Conradi-Drigalski agar are large and red (see "Water"). The B. coli does not give the Voges- Proskauer reaction (p. 410). The differentiation of the B. coli from the B. typhosus should present no difficulty if the morphology and motility of the organisms and their fermentation and / agglutination reactions be compared. Bacteriologists usually make use of the following tests for the differentiation of B. c,4i: (1) Morphology, (2) motility^ (3) Gram staining, (4) character of growth and colonies on gelatin , (5) non-liquefaction of gelatin, (6) action on milk, (7) indole formation, (8) fermenta^ tion of glucose, (9) fermentation of lactose and saccharose, (10) action on neutral rgd. MacConkey suggests that instead of tests Nos. 4, G, 7, 8, and 10, the following should be substituted : (a) fermentation of dulcitol, but not of adonit and inulin ; (b) the Voges- Proskauer reaction. Other media which have been recommended for the differentiation of B. coli from B. typhosus are the Proskauer- Capaldi media and Petruschky's litmus whey, but are not now much used. The Proskauer-Capaldi medium No. 1 is an asparagin- mannitol solution with certain salts ; medium No. 2 is a peptone-water-mannitol solution. Both solutions are care- fully neutralised and tinged with litmus. If these media be inoculated with B. typhosus and B. coli respectively and incubated at 37° C. for twenty-four hours, the following changes will be noted : Manual ot Bacteriology Medium No. 2. Growth with strongly acid reaction. Growth with neutral or faintly alkaline reaction. Petruschky's litmus whey is prepared as follows: Fresh millc is warmed and the casein precipitated by the addition of a minimal amount of hydrochloric acid. It is filtered, and the nitrate of clear whey is carefully neutralised with dilute caustic soda solution. The fluid is then steamed for two hours and filtered ; the filtrate should be clear, colourless, and neutral in reaction. Enough neutral litmus solution is then added to render it well coloured, and the mixture is distributed into test-tubes and sterilised. This medium is rendered slightly acid (represented by 6-10 c.c. N/10 caustic soda per cent.) by B. typhosus, very acid (40-50 c.c. ditto) by B. coli. The thermal death-point of the organism, according to Weisser and Sternberg, is 60° C. with an exposure of ten minutes. The B. coli will grow freely in a slightly acid medium, and in media containing as much as 0.15 per cent, of carbolic acid. In this respect it is a more resistant organism than the B. iyphotnis. Chemical products. — The acids produced are mainly Isevo-lactic acid with some dextro-lactic acid from glucose, Isevo-lactic acid only from mannitol ; also acetic, formic and succinic acids, and alcohol. Accord- ing to Harden, B. coli attacks glucose in a character- istic manner, each molecular proportion of sugar yielding half a molecular proportion of acetic acid and of alcohol, and one molecular proportion of lactic acid, together with a small amount of succinic acid, and gaseous carbonic acid and hydrogen.1 Nitrates are reduced to nitrites. 1 See also Eevis, Ccn/r.f. Bald. Abt.), xxvi. 1910, p. 161. Medium No. 1. B. typhosus No growth or change in reaction. B. coli . Growth with acid reaction. Pathogenicity of Colon Bacillus 407 No toxin, or a trace only, is formed in culture^ but the dead bacilli are toxic and pyogenic, and a toxin is obtained by autolysis of cultures or by triturating the bacilli with liquid air (Macfaclyen). Vaughan,1 by washing large quantities of colon and typhoid bacilli, extracting the bacterial cells first with alcohol, then with ether, and then digesting the ground residue with alcohol containing 2 per cent. NaOH, states that two constituents are obtained, one soluble in alcohol and toxic, the other insoluble in alcohol and non-toxic. The latter confers a certain degree of immunity on animals injected with it. Pathogenicity. — The pathogenic action and patho- genicity of the B. coli are very varied. Introduced into the circulation or into the peritoneal cavity in wninea-pigs or rabbits it usually causes death in from one to three days with a general septicemia. Some varieties are, however, non-virulent to animals. In man the colon bacillus is associated with a number of important pathological processes. It is usually the organism causing the peritonitis which is due to infection from the intestine, as in hernia with obstruction or perforation, in ulceration of the bowel and enteritis, in cancerous growths, and affections of the appendix, biliary canals, and gall-bladder. The exudation in these cases is often characteristic ; at first it is clear and greenish, it then becomes greenish- yellow, thin, semi-opaque and foul-smelling, and finally purulent. An important point is that the colon bacillus may pass through the intestinal wall where it )ias bfipn damaged, as by strangulation, but not yet perfoi"ated. The />. coli is a pyogenic organism, and has been ' Trans. Fourteenth Internat, Cong. Hygiene (Berlin, 1907), Bd. iv, p. 28. 408 Manual of Bacteriology met with in ischiorectal abscesses (probably the B. pyogenes fetidus of Passet). Possibly it causes in some instances the pneumonia and pleurisy occurrm" after peritonitis, for it has been obtained from the lung and pleura in these conditions, but it must be recognised that the B. coli is a common secondary or terminal infection. Puerperal fever is another con- dition sometimes caused by the B. coli, and cystitis and infections after urinary operations are also commonly due to it. In the Pictou cattle disease, characterised by extensive hepatic cirrhosis, Adami found a minute diplococcus or short bacillus. A similar form was afterwards isolated by him in hepatic cirrhosis in man. Miss Abbot,1 from a study of several such cases, came to the conclusion that this organism is a variety of the B. coli. It has been suggested that hepatic cirrhosis is produced by poisons or toxins, e. g. of the B. coli, and that alcoholism, the usual cause assigned, is but an exciting or secondary agent. Anti-serum and vaccine. — Attempts have been made to prepare an anti-serum for B. coli infections but they have met with little or no success. A vaccine prepared by sterilising cultures by heat and standardising has been used successfully in the treatment of chronic B. coli infections, e.g. cholangitis, cholecystitis, pyelitis, and cystitis. The B. coli vaccine is more toxic than most vaccines, and small doses must therefore be given (see p. 232). Clinical Examination. (1) The appearance and odour of the pus are often charac- teristic. Smears of the pus show small bacilli, which are decolorised by Gram's method. 1 Journ. Path, and Bad., vol. vi, 1900, No. 3, p. 315 (Bibliog.). Varieties of Bacillus Coli 409 (2) The organism may be isolated by plating on gelatin, agar, litmus lactose agar, Conradi-Drigalski agar, or by the use of neutral red or bile-salt media (see " Water "), The isolated organism must be tested as to its morphology motility, non-Gram staining, non-liquefaction of gelatin, indole production, curdling of milk, and fermentation of glucose, lactose, dulcitol, mannitol, etc. (3) An agglutination reaction may likewise be tried, but if negative is of little value, as there are so many varieties of the colon bacillus, and one variety may not be agglutinated by the specific serum obtained with another variety. A positive reaction must also be carefully controlled, as the colon bacillus is much more readily agglutinated by normal serum than is the typhoid bacillus. Varieties of Bacillus coli. Organisms are frequently met with in faeces, manure, sewage and polluted water which resemble the typical B. coli in many of their characters, but which differ from it in certain particulars. Thus the colonies on gelatin, instead of being smooth, may be wrinkled ; milk may be but slowly curdled (three to eight days) ; acid or gas production, or both, in sugars may be less marked than usual. These organisms are generally regarded as varieties of the B. coli, and are perhaps derived from typical B. coli. There is, however, little evidence that B. coli can be transformed iuto such varieties, or that these varieties can be reconverted into typical B. coli. Eevis (loc. cit.) has obtained evidence of alterations of fermentive power, and in the characters of the colonies of certain coliform organisms. Organisms that have been Regarded as Variants of B. coli. A number of organisms have been regarded as being- varieties of the B. coli (consult table of fermentation re- actions, p. 400). 410 Manual of Bacteriology (1) Bacillus cavicida (Brieger). — This resembles B. ooli in most of its characters, but was stated to he non-motile; MacConkey says it is motile. (2) Bacillus neapolitanus (Emmerich). — Isolated from the bowel in cases of cholera. It differs from B. coli by not being motile, and by fermenting cane sugar. (3) G-as-forming bacilli of Laser and Gartner.1 (4) Aerobic bacillus of malignant oedema (Klein). (5) Bacillus lactij aeroaeiies of Escherich. — Found in the intestine of nurslings. Much like B. coli, but is non-motile. It differs from B. coli by not fermenting dulcitol, byTermenting saccharose and adonit, and by giving the Voges-Proskauer reaction (see table, p. 400). According to Harden and Walpole,2 its action on glucose differs from that of -B. coli, more alcohol being produced and formed at the expense of that part of the molecule of the sugar which in the B. coli fermentation yields acetic and lactic acids. The Voges-Proskauer reaction is obtained by growing the organism in 2 per cent, glucose broth in a fermentation tube (Fig. 17, p. 85) for three days and adding some strong caustic potash solution ; on standing exposed to the air a pink colour develops. According to Harden and Walpole^ the reaction is probably due to acetylmethyl-carbmol, which in the presence of air and potash is oxidised into diacetyl, which then reacts with some constituent of the peptone in the medium, giving the pink colour. The B. J art is aiirogenes (which may be classed among the capsulated bacilli, see p. 271) is occasionally pathogenic, causing peritonitis.1 In these circumstances, it is capsulated, but the capsule is difficult to stain. (I!) B. cloacie (Jordan).— Met with in sewage. In general character* it much resembles B. coli, but produces more gas ' Ccntr. f. BaJct. (I1" AM.), xiii, 1893, p. 217, and xv, 1894, pp. 1 and 276. . 2 Journ. of Hygiene, vol. v, 1905, p. 488; Proc. Roy. Soc. Lond., n, vol. lxxvii, 3906, p.,399. » Proc. Roy. Soc. Loud., b, vol. lxxvii, 1906, p. 399. 4 See Churchman, Johns Hopkins Hasp. Bull., vol. xxii, 1911, p. 118. Fly-borne Infection 411 (75 perceiit.J from glucose and liquefies gelatin in four to five to thirty days. Like B. lactis aerogenes, saccharose is always fermented and the Voges-Proskauer reaction is positive, but neither dulcitol nor adonit is fermented. (See table, p. 400.) Flies as Carriers of Infection. Flies and other " insects " may convey infection (1) by infecting food, etc., (2) by direct inoculation, (3) by inocula- tion after a cycle of development — in which case the carrier is more or less specific ; e. g. anopheline mosquitoes in malaria. In the first method the organisms are generally bacteria, occasionally ova of worms ; in the second, bacteria or protozoa ; in the third, invariably protozoa, filaria, etc., i. e. animnl organisms. The ordinary domestic fly, the. blue-bottle and other similar flies (of which there are many) have no biting proboscis, but undoubtedly carry infection by infecting food, etc., directly by organisms upon various parts of their body, or by the .organisms passing through the digestive tract and infecting the food with the faeces. In this way, typhoid, bacillarv dysentery, B. enteritidis, summer diarrhoea, cholera, and possibly anthrax may be conveyed. The ordinary house-fly breeds in dung and garbage containing dung, and it has a possible range of flight of about a mile. The house-fly experimentally infected remains grossly infected for at least three days, and a smaller degree of infection persists for ten days or even longer.1 1 See Reports to the Loc. Gov. Board on Flies as Carriers of Infection, Nos. 1-4, 19K ) and 1911. 412 Manual of Bacteriology CHAPTER XI. BUBONIC PLAGUE CHICKEN CHOLERA MOUSE SEPTICEMIA. Bubonic Plague. Plague was epidemic throughout Europe during the Middle Ages j in England in the fourteenth century it appeared as the Black Death, and in the seventeenth century as the Great Plague of London, while numerous other lesser visitations have been recorded. For some years plague has been practically pandemia The disease seems always to have been endemic in certain centres, e. g. in Asia Minor, on the Persian Gulf, in Yunnan, in Uganda, etc. A characteristic of plague is the manner in which it appears and remains prevalent for a time in a district and then disappears, to reappear again after a considerable interval ; this has happened not only in Europe, but also in Persia, Syria, India, and China. Three main types of the disease are recognised, the buljomc in which the femoral (rarely the inguinal), axillary and other glands become enlarged (whence the disease derives its name), the septicemic, and the pneumonic. In India the disease has been mainly bubonic (70 per cent, of the cases). Occasionally the majority of the cases are pneumonic, as was the case in Accra, in China in 1910-11, and in the small out- break in Suffolk in 1910. Septicemic cases are the Plague 413 exception, but any form tends to become septicemic on the approach of death. At the commencement and at the end of an epidemic the disease may assume an extremely mild type, the so-called " pestis minor." Bacilli were first observed in this disease in the blood, buboes, and organs by Kitasato in 1394. In the same year (1894) Yersin investigated the outbreak of bubonic plague at Hong Kong, and described the bacillus met with in the buboes and its cultural and pathogenic properties very fully. This organism is known as the Bacillus pestis. Morphology. — The B. pestis belongs to the group of hemorrhagic septicemic bacilli (chicken cholera, rabbit and ferret septicemia, swine plague, etc., see p. 427), and is a markedly pleomorphic organism. In the animal body it occurs for the most part as a short, plump, non-sporing rod, measuring 2-3 by 1-2 fx, 111 Manual of Bacteriology but longer forms may be seen here and there measuring as much as 5 ,x (Fig. 43). .Polar_ staining is a marked feature (Plate XIV'., a and"?,). Occasionally swollen involution forms occur. The typical form of the organism, the bi-polar staining, short, stumpy bacillus, is met with in smears from the' buboes, in the sputum m the pneumonic form, and in the blood in the septi- cemic variety,but only in the earlier stages of the disease. Later the typical forms tend to disappear, their place being taken by a few large, rounded, ovoid, or pear- shaped involution forms. Under cultivation the bacilli in young cultures (twenty-four to forty-eight hours) are so short as to be almost coccoid or slightly ovoid ; on agar their size is about the same as that in the animal body, on gelatin they are somewhat smaller, but a few well-marked rods and even threads are always present. In older cultures, rod, thread and involution forms occur more numerously ; on agar containing 2—3 per cent, of salt the latter are swollen and yeast-like. In broth chains of slightly ovoid organisms occur resembling streptococci (Plate XV., a). The organism is non-sporing and non-motile, although Oordon described the presence of one or two hue spiral terminal flagella (others have not found flagella). Sometimes in hanging-drop cultivations a capsule is apparently present, but the writer has failed to verify this by staining methods. The B. pentin stains well with Lotfier's blue ami anilin-gentian violet, polar-staining being a marked feature, especially in smear preparations. It does not stain by Gram's method. With old laboratory strains polar staining may be completely absent, but in such cases may sometimes be obtained by first treating the preparations with alcohol or by the Gram PLATE \r\ a. Bacillus pestis. Smear preparation from a bubo, x 1000. b. Bacillus pestis. Smear preparation of sputum. x 1000. To face page Hi. 1 mam The Plague Bacillus 415 'i a I method, and subsequently staining with Loffler b blue or weak gentian violet. Sections are Lest stained with oarbol methylene or thionine blue. Cultural charactvrs.-The B. pedis is aerobic and facultatively anaerobic. On blood-serum it forms moist, smooth, shining, cream-coloured colonies or growths, slightly raised above the surrounding medium. The blood- serum is not liquefied. On agar the colonies are raised, round and cream-coloured, finely granular, denser at the centre than at the margins, which are regular. Size 0-25 to 0 5 mm. in two days at 37° C. On surface agar the B. pestix forms a thick, opaque, moist, smooth, cream- coloured growth, the margins of which are usually markedly crenated (Fig. 44) ; the growth is very sticky and tenacious. Haffkine states that when grown on dry agar (agar which has been kept in the warm incubator for two to three weeks) and viewed from behind the growth has an F*<*- 44. — Plague, . . i surface culture on appearance like that given _by the glycerine ao-ar -i. e. a dull, silvery forty-eight hours old. back of a mirror- appearance. On a salt agar (2'5— 3"5 per cent, of sodium chloride) Hankin describes the development of remarkable spherical or pear-shaped involution forms. On gelatin the colonies are whitish, filmy, finely granular with regular margins. Size, O'l to 0"25 mm. in five days at 22° C. On surface gelatin the organism forms a thin, white, 416 Manual of Bacteriology granular growth, with slightly irregular surface and margins, and nearly confined to the inoculation track, ine growth does not penetrate into the medium nor does it render it cloudy. The growth is very adherent. In a stab gelatin culture a delicate whitish, finely granular growth develops to the end of the stab, with little tendency to spread from the needle track. ' The gelatin is notOiojiefied. Both in agar and gelatin cultures fresh punctate growths sometimes develop in the original growth, simulating a contamination. No growth occurs on ordinary potato, and milk is not coagulated. In broth the growth is somewhat characteristic. For two or three day the broth remains perfectly clear, but a flocculent growth forms and gradually increases in amount on the bottom and sometimes upon the sides of the tube. After some days the broth may become a little cloudy. A delicate flocculent film develops if the tube be kept absolutely at rest. In broth to which a little butter-fat or ghee has been added little islands of growth appear on the surface, and from these flocculent tapering dependent growths form in about a week, pro- vided the tubes or flasks be kept absolutely at rest. the bulk of the broth remaining clear. This is the stalactite growth of Haffkine. and is very characteristic (B. pseudo-tuberculosis a 1st. "gives it). Broth cultures reduce a weak solution of methylene blue. With sulphuric acid alone a feeble indole reaction can be obtained with week-old broth cultures. With sulphuric acid and a nitrite a well-marked indole reaction can be obtained under the same conditions. The fermentation reactions of the B. pestis, which MacConkey lias pointed out are practically identical with those by the B. pseudo-tuberculosis, are as follows : Acid production, ~5ut no gas, in glucose, Isevulose, PLATE XV ofaae par/e Mil. * (J»./C(( C^U^u. U^u^ UA^tJl The Plague Bacillus 417 galactose, maltose, mannitol, and dextrin, no change in lactose, cane-sugar, and dulcitol. Action of antiseptics, etc. — The plague bacillus is readily destroyed by antiseptics ; a 1 : 1000 corrosive sublimate or J : J00 chloride of lime solution being efficient. An acid solution of corrosive sublimate is preferable, and for the practical disinfection of native houses a 1 : 250 solution of sulphuric acid may be employed. A temperature of 65° C. kills the organism in about fifteen minutes. Desiccation over sulphuric acid at 30° C. is also rapidly fatal. Vitality and virulence of cultures. — Cultures retain ^ their vitality for at least a month. As regards viru- lence, the organism varies much according to the source from which it is obtained. Under cultivation it gradually loses its virulence^ unless subcultured in tlie following manner : The cultures are made every week on surface agar, are placed in the blood-heat in- cubator for twenty-four hours, and are then removed and kept at room temperature. If inoculated into animals the virulence may be heightened for a particular species by successive passages, but in so doing is diminished for other species. ^_PgMm^^2£lion- — In addition to man, the follow- ing animals are liable to contract plague under natural conditions— the monkey, cat, rat, mouse, squirrel, ground squirrel, ferret, bandicoot, and marmot. The guinea-pig and rabbit are also susceptible to inocu- lation. The horse, cattle, sheep and goat are relatively insusceptible, though Simpson1 stated that calves and poultry may be infected by feeding, and suffer from a chronic form of the disease (this observation of Simpson's has not been confirmed by other workers). Birds are not easily susceptible, and vultures feeding 1 Report on the Plague in Hong Kong. 27 418 Manual of Bacteriology on the corpses of the plague-stricken do not seem to contract the disease. The mouse, rat, and guinea-pig are the animals chiefly used for experimental purposes in the laboratory j the first two are highly susceptible," a simple prick in the thigh with an infected needle being sufficient to induce the disease. A guinea-pig inoculated with plague material or with a pure cultivation usually dies in from two to seven days, the symptoms being sluggishness and loss of appetite, sometimes a discharge from the eyes, and towards the end staring coat and perhaps convulsive and paralytic attacks. The jmst-mortem appearances are extensive hemorrhagic oedema at the seat of im_ -plQ 45 —Spleen of guinea-pig inoculated with plague. (Nat. size.) oculation, enlargement and congestion of the spleen, and enlargement of, and hasmorrhages into, the inguinal and axillary lymphatic glaiujs^ If the animal live six or seven days, the glands may be as large as small nuts (see some admirable preparations in the College of Surgeons Museum). JThg_ spleen may be enormous, six times its natural size, and studded with small yellowish "nodules Resembling miliary tubercles, consisting of rTecrotic areas with masses of bacilli (Fig. 45) j the luno-s also may be more or less inflamed, and contain small and large necrotic foci. The bacilli are ex- tremely numerous at the seat of inoculation; m the glands, and in the spleen, less so in the peritoneal fluid liver, and blood; if the death of the animal is The Plague Bacillus 419 delayed bhe exudation in the bronchi may contain considerable numbers. Some bacilli may generally be found in the duodenum, trachea, and larynx. Mice usually die in from two to three days; and rats in from three to seven days after inoculation. In rats and mice the post-mortem appearances are similar to those in the guinea-pig. A very small dose of a pure culture may fail to kill an inoculated animal. Rabbits are much less susceptible to plague than guinea-pigs, and may be injected with considerable doses of living cultures without showing marked illness. Rats can be infected by jeeding on the corpses or carcases of men or animals dead from the disease. In man the bacilli are found in large numbers in the ^fluid in the buboes, either alone or mixed with strepto- cocci or micrococci, and in the sputum in the pneumonic form. They are not usually found in any number in the blood except in the septicemic variety, or shortly before death, and in stained preparations appear as short plump bacilli, often in pairs, with polar sj^in^o- and unstained centres (Plate XIV., a and 6). "TFthb organisms are found to be free and numerous in the buboes the prognosis tends to be grave, but if they are largely present within the phagocytic polymorphonuclear leucocytes the prognosis is better and the disease will probably remain localised. „ Iwms;— The plague bacillus forms but Tittle tny,'^ the minimal fatal dose of the most active filtered broth culture for a mouse being about O02 c.c. In order to prepare a vaccin^ or an anti-serum it „ja_Jieccssarv. therefore, to ejnjpbyjmfiltered cultures— CeC^ e miovnht* themselves. mmm : IJaofadyen obtained an endotoxin by triturating the bacilli frozen with liquid air. ' Vaccines and immunity.— Of the plague vaccines, HP* 420 Manual of Bacteriology that of Jdaj^kuie, the Haffkine prophylactic, is the best known, and lias been extensively employed. It consists essentially of a four to six weeks old bnl ter-l'at broth culture of the plau'in- liii.cillns. killed, by heating to 65 C. for an hour, with a small addition of juitiatiptic. As to the value of Haffkine's prophylactic a mass of figures is available. By its use both the incidence of, and the mortality from, plague are markedly diminished. Wilkinson collected the following data of the efficiency of the vaccine : Among the inoculated the case incidence was 1-8 and the case mortality 23-9 per cent.; among the uninoculated the figures were 7'7 and 60" 1 respec- tively. The immunising products seem to be mainly intra-cellular, but the broth itself is not without action. Other vaccines have also been devised. Lustig aud (Jaleotti prepared one by digesting the growth from agar cultures with 1 per cent, caustic soda solution, filtering through paper, and precipitating with very dilute acetic or hydrochloric acid, or by saturation with ammonium sulphate. The precipitate is dissolved in a 0-5 per cent, solution of sodium carbonate, and filtered through a Chamberland filter ; this forms the vaccine fluid. Calmette prepared a vaccine by emulsifying an agar_ growth In water,- well washing the organisms^ with sterile water to remove~adherent. toxin, ^emulsifying againjn_jsterile_ water, heating to 70° C. for an Tiour, and nnany-ctrvmg_j2L, vacuo. The dry substance can be kept for a considerable time "without change. For use 1-2 mgrm. are emulsified in 2-3 c.c. of sterile salt solution and injected. Yersin proposed vaccinating with living culture of feeble virulence, which has been done by Strong in Manilla. Though such a method might be used in a plague-stricken district, it is obviously one that can be used only with the greatest caution. Klein1 has prepared a prophylactic by drying the organs of a guinea-pig dead of plague for three days at 46° C, 1 Rep. Med. Off. Loc. Gov. Board for 1905-06. Plague Vaccines and Serum 423 rubbing the material to a powder, and further drying at 37° C. for three days. Of this dry powder 15-16 nigrm. protected a rat, and 25 nigrm. a monkey. With reference to experimental immunity and protection in plague, Klein1 found that a guinea-pig which had been three times injected with an amount of living culture iiisuffi- " cieiit to kill was 1 'Still capable of being infected ; that the blood of a guinea-pig which had twice passed th 1 0 U ^ ll dill attack of plague did not contain an appreciable amount of germicidal substances ; and that the immunisation of guinea- pigs by sterilised cultures is an extremely slow and difficult process. Calmette also found that the guinea-pig was extremely difficult to immunise. Calmette, from laboratory experiments, surmised that pro- tection with a vaccine is not attained for some days, and tli at in the interval susceptibility to infection is increased. These observations are not borne out in practice, for Bannerman2 found that so far from there being an increase in mortality among those who have been inoculated and who develop plague within ten days of inoculation the reverse is the case, and that in a small community where the population had been partly vaccinated and partly not vaccinated, the incidence of plague during the week following vaccination was less among the vaccinated than among the unvacci- nated, pointing to the rapid production of protection. ^Anti-i>laque serum. — This is prepared by growing the B. pestis on the surface of ap-ar in phite boTtles, \vashing__off and emulsifying the growth, and for the earlier injections the emulsion is heated to 65° C. foy one hour, and the commencing close is ^T part of a flask. The injections are given intra-venously at intervals of a week. At the end of three months the bactericidal power of the blood willlTave become' very markedT and living cultures are then injected 1 Rep. Med. Off. hoc. Gov. Board for 1896-97, A pp. B., p. 2. 2 Centralbl.f. Bakt. (i» Abt.), Bd. xxix, p. 873 (Bibliog.). 422 Manual of Bacteriology for a further period of about three mouths until a whole Bask-culture is given at a dose. An interval of a_Jortmght is allowed to elapse between the last dose and the^nh^bng of the animal. The serum is tested upon mice. The >_ai^i-pjague_^ which is mainly anti- microbic, is not very potent, and to be of service large amounts and early treatment are essential.1 Epidemiology. — The mode of infection in man has been a matter of controversy. The pneumonic form arises generally from aerial infection by the respiratory tract. It is extremely fatal and infectious, while the bubonic and septicemic varieties are hardly even con- tagious. Although a gastric and intestinal form of the disease has been described, and there is evidence to show that food or drink may be the vehicle of infection, this must be a rare mode of infection. Yersin claimed to have isolated the bacillus from the dust and earth of a native dwelling, and Hankin from the brackish water in a held. The observations of Hankin and others indicate, however, .that contagion is likely to occur only from immediate contact with man or animals, or their excretions, infected with plague, and not from a saprophytic form of the organism. Certain animals, especially the rat (Musjrattu* and Mas decn.manji&L, are important agents in spreading the ""disease. The association of sickness and of death among the rats with an epidemic of plague has been established by a number of observations, and in some instances the epizootic among the rats has been defi- nitely shown to precede the epidemic in man. The epidemics at Sydney are perhaps the most striking instances of rat-borne plague; discussing the first 1 See Hewlett's Serum Therapy, 1910. Transmission of Plague 423 one Tidswell says : "The one clear faotiiLfll was that human bjmigs were riot becoming infected from one another." In the fir^^pidemic the mode of introduction of the disease was never traced to any human source. During an epidemic the rats may be found in all stages of illness and the plague bacillus can be found in large numbers in their carcases. In the various epidemics at Sydney, cases of plague first occurred among the rats and mice, followed after an interval of days or weeks by human cases. Other animals may also occasionally be the means of dis- seminating the disease. The experiments of the Advisory Committee on Plague Investigation in India have conclusively shown the important part played by rats in the dissemination of the disease, though the origin of the primary infection in rats is doubtful. They may possibly become infected from the dust of earth era floors of the native houses soiled with excreta or discharges of plague patients, or from their clothing, poultices or dressings, but the readiest method is probably by feeding on the dead. Once the epi- zootic has started, further infection is simple ; rats Jight. and so may directly inoculate one another; the sick rats may soil grain or other food-stuffs, and the dead rats are eaten by their fellows. Moreover, para- sitic insects, especially fleas, undoubtedly may transmit the disease from one animal to another. Thus it is found that if guinea-pigs be placed in a plague- infected house, many of the animals contract plague • ^LQq^o but if the animals be placed in cages of wire-gauze, i^**** the mesh of which is small enough to prevent access of fleas1the animals do not contract plague. The transmission of the disease from rats to man is similarly due to transmission by fleas f except in the_ pneuinonic forms in which infection is direct from Manual of Bacteriology the sick to the healthy). The great majority of rat fleas are Xenopsylla cheopis, Oeratophylius fasciattis, Cer. awisus, Ctenojisylla masculi, and Gtmwphthahmiis agyrtes, of which the first is most prevalent in the tropics and subtropical regions, the second in cooler regions.1 The manner in which the periods in the year when human plague does not occur are bridged over is unknown. In such periods rats suffering from plague have been found, but these are regarded as having a retrogressive form of the disease leather than a chronic infection. The destruction of rats, either by trapping, poisoning, or asphyxiating, or by the use of the Danysz rat virus (see p. 394), is, therefore, one of the means to be adopted in fighting the disease. The extermination of rats seems quite impossible, but by rat destruction there is a likelihood of destroying infected animals and the subsequent development of a healthy race. On Plague, see Simpson, Treatise on Plague (Cambridge University Press); Klein, Bacteriology of Oriental Plague; "Reports on Plague Investigations in India," Journ. of Hi/giene (extra numbers), vols, vi-xi ; Rep. of the Indian Plague Commission ; numerous reports published by the Indian Government. Clinical Examination. If it cannot be examined immediately, plague material may be placed in a solution containing glycerine 20 c.c, distilled water 80 c.c, calcium carbonate 2 grin.. The bacilli retain their vitality and virulence in this for thirteen days ( Albrecht- Grhon method). (1) Withdraw a little of the fluid from the bubo by means of an antitoxin syringe. Make smears and stain with 1 See Chick and Martin, Joum. of Hygiene, vol. xi, 1911, p. 122 (Bibliog;.). Diagnosis of Plague 425 methylene or Ihiouine blue. Search for short plump bacilli often m pairs, with polar staining and unstained centres. They are not stained by GramVmefrhod. N.B. — There may be a mixture of organisms in the buboes, (2) Make agar plates, and broth cultures. Incubate the cultures at 25°-27° C, not at 37° C. From colonies on the agar plates the organism may be isolated and its cultural and pathogenic characters ascertained. The ajppg.vtpip.P! of the broth cultures, if characteristic, would bevery suggestive of plague, but if uniform turbidity develops this may be clue to contaminating organisms, e. g. micrococci. (3) Inoculate mice, rats, or guinea- pig^s subcutaneously with the fluid or with the culture. Some of the animals should be inoculated by the cutaneous method — rubbing a little of the material on the shaved abdomen, and also as in (4). Inocula- tion of rats serves to distinguish the B. pseudo-tuberculosis from the B. pestis. If the animals die, investigate for the Bacillus pestis by staining and culture methods. (4) In the pneumonic form, dilute the sputum with a little boiled water, inoculate several agar tubes, and incubate at 25°-27° C. Examine in two to three clays. Also daub the nostrils of a guinea-pig or rat with a brush or pledget of wool dipped in the diluted sputum, avoiding wounding the mucous membrane. Smears of the sputum may also be made, stained, and examined. Gram's method will distinguish the B. pestis from the Biplococcus pneumoniae ; the latter stains well by Gram. (5) Agglutination reaction.— The Indian Plague Commis- sioners state that in their opinion no practical value attaches to the method of serum diagnosis in plague, but a modified method is considered by Dunbar1 to be of considerable value. The method is carried out as follows : A small quantity of peptone solution, inoculated with the tissue-juice from the. suspected organ, is mixed with an equal quantity of plague-serum of such a strength that the dilution reduces it to 1 = 200 (approximately). A second dilution of 1 : 400 and a third of 1 : 800 are also prepared. 1 Centralbl.f. Bald., xli (Originals), 1906, p. 860. 426 Manual of Bacteriology As a control, an equal quantity of the inoculated peptone water is mixed with normal serum (rabbit or horse serum), the dilution being 1 : 100. In a few minutes a distinct difference is observable. The "control" shows with the oil-immersion lens a few isolated non-motile bacteria, while the plague-serum dilution 1 : 200 shows larger and smaller masses of agglutinated bacteria. After two hours' incubation the same result is obtained with the plague-serum dilution of 1 : 400. No agglutination, however, is observed after incubation for twenty-four hours of the dilution of 1 : 800. This agglutination reaction, in conjunction with other suspicious phenomena, justifies an official notification of suspected plague. In the examination of rats suspected to be suffering from plague infection, it is essential not only to take the naked-eye characters into account, but to make microscopical prepara- tions and cultures, and to test the cultures by animal inoculations. Care must be taken not to mistake hsemorrhagic septicem ic bacilli (see pp. 413, 427), and other organisms for the plague bacillus. The B. coli, B.proteus, and other organisms are recorded by Klein (loc. cit.) as simulating the B. peslis. Chicken Cholera. Chicken cholera is a disease of poultry characterised by profuse diarrhoea ; its course may be very rapid, and the bird found dead without having shown signs of illness. The organism is a very short rod, non-motile, so short that it is almost ovoid, 06 to 0'8 M in length, and 0"4 to 0"5 ^ in diameter. It stains by the ordinary anilin dyes, but not by Gram's method, and the staining tends to be polar, so that Pasteur, who first investigated the disease, described it as a diplococcus (Plate XV., b). The organism grows freely on the various culture media from 20° to 38° C, on agar forming a thick, moist, cream-coloured layer, on gelatin a shining, white, expansive growth without liquefaction. In broth a general turbidity forms, but growth on potato is indifferent. It produces acid, does not ferment glucose or lactose, is aerobic Chicken Cholera 427 and facultatively anaiirobic, does not form spores, and is killed by a temperature of 60° C. in fifteen minutes. If dried it dies iu a few days, but retains its vitality for a considerable time in damp earth or in water, and so infection is readily conveyed. Fowls die after subcutaneous, infra-muscular or intra-venous inoculation and by feeding, the organisms beiug found abundantly in the blood. Post mortem, the serous membranes may be inflamed and hsemor- rhagic, the liver large and soft, and the intestine shows hemorrhagic spots, aud is sometimes ulcerated and contains a mucoid fluid stained with blood. Other birds, pigeons, pheasants, sparrows, wild and domestic ducks are also susceptible to the disease, and rabbits and guinea-pigs can be successfully inoculated ; in the latter animal a local abscess sometimes forms instead of a general infection. By con- tinuous cultivation with free access of oxygen the virus becomes attenuated, and Pasteur was able thus to prepare a vaccine which protected fowls. The bacillus of chicken cholera belongs to the group of hemorrhagic septicemic bacilli (p. 413), and seems to be identical with Koch's bacillus of rabbit septicaemia, and with the bacillus of swine plague (see p. 394). These organisms tend to form a stalactite growth in butter broth. Organisms have been described by Klein in fowl enteritis, grouse disease, etc., differing somewhat from the bacillus of chicken cholera. Mouse Septicaemia. This disease may be conveniently described here. Koch first obtained a minute bacillus by injecting putrefying material subcutaneously into mice. It seems to be identical with the bacillus found in swine erysipelas. The organisms are met with in large numbers iu the blood and tissues of mice. They measure only 1 fi iu length, and occur in consider- able numbers in the leucocytes. The bacillus stains well by Gram's method, and is stated by some writers to be motile. l-s Manual of Bacteriology It grows readily, forming on agar extremely delicate, almost invisible colonies ; in stab gelatin cultures after some time a delicate cloudiness radiates from the central puncture. Prom an agar culture the bacilli are somewhat larger than those found in the animal body, and form filaments. It is pathogenic for swine, rabbits, and mice. Pneumonia 429 CHAPTER XII. PNEUMONIA. INFLUENZA, AND WHOOI'ING-COUGH. Pneumonia. Pneumonia is of two types, lobular, catarrhal, or broncho- pneumonia, and lobar or croupous pneumonia. The former may be primary, or may be secondary and arise in connection with many of the specific fevers, as in measles, whooping- cough, diphtheria, enteric fever, influenza, plague, etc. The broncho-pneumonia occurring in the course of other diseases may be due to the causative organism of the disease, or may be due to other organisms. Eyre1 examined 62 cases of broncho-pneumonia occurring in the course of other diseases and 102 cases in which the broncho-pneumonia was the primary lesion. Of these 164 cases, 52-4 per cent, yielded pure cultivations of some one or other of six bacteria — pneumococcus, Strep, longus, M. pyogenes var. audits, M. catarrhalis, B. pneumoniae, and B. influenza?, ; whilst 47-5 per cent, gave a mixed growth of one or more of these six in association with one or more of five other bacteria — M. tetra genus, B . jjeHyj&i&T B. pyocyaneus, B. typhosus, B. diph- tlierise. The B. coli also occurs in broncho-pneunionia. Acute croupous or lobar pneumonia in many of its characters resembles an acute specific infection, and while frequently a primary disease, may also occur secondarily in almost any condition. Friedlander in 1882-83 first described organisms in cases of pneumonia. 1 Jouru Path, and Bad., vol. xiv, 1910, p. 160. 430 Manual of Bacteriology In 1883-85 Talainon, Klein and Sternberg each described in pneumonic sputum an oval encapsuled organism, which induced pneumonia in animals ; it was termed by the former the Micrococcus lanceolatus, and by Sternberg the Micrococcus Pasteuri. This and Friedlander's organisms were at first believed to be identical, but Frankel and Weichselbaum subsequently showed that they are quite distinct, and that the former is the setiological agent of acute croupous pneumonia. The majority (95 per cenU of cases of acute croupous pneumonia are caused by the Biplococcus pneumonia, and Fried! finder's organism, now termed FriedliiiideTs pneumo- bacillus, or B. vneumnni;i\\s of aetiologieal significance in only a f small minority, if at all. The latter is, however, associated with certain pathological processes which will be referred to below. From pleuro-pneumonia of cattle, Nocard and Roux suc- ceeded in cultivating in broth in collodion sacs in the peri- toneal cavity of rabbits an organism just visible as minute granules with a magnification of 2000 diameters. Bordet1 states that it may be grown on the medium employed by him for the cultivation of the B. pertussis (p. 441), and' then appears as fine, straight, curved, undulating or even spirillar filaments not unlike spirochaetes. The Diplococcus (Streptococcus) pneumoniae.2 Synonyms, Fri'iukel's pneumococcus, Micrococcus Pasteuri (Sternberg), Micrococcus lanceolatus (Talamon), Micrococci!?; pyogenes tenuis (Rosenbach). Morphology. — The Diplococcus: -pneumoniae, in the sputum and tissues occurs as an oval or lance-shaped coccus united in pairs, occasionally in chains of three or four elements, and then often almost spherical, and is generally surrounded by a well-marked capsule (Plate XVI., a). In order to isolate the organism several tubes of glycerin agar, serum or serum-agar may be inoculated with rusty sputum and incubated for 1 Ann. de I'Inst. Pasteur, xxiv, 1910, March. 2 On the pathology of pneumococcus. infection sec Brit. Med. Journ., 1901, vol. ii, p. 7(50 ; Eyre, Lancet, 1908, vol. i, February 22nd. The Pneumococcus 431 forty-eight hours ; in some a pure culture may be obtained. A more certain method is to inject a drop or two of thej-usty sputum into The peritoneal cavity of a mouse or young rabbit. The animal will die in from twenty-four to thirty-six hours, and the organism will^ be found in considerable numbers in the lung- and blood, from which cultures may be obtained" 'it is. f "fl 4 A nmi-mnt-.ilft. stains with the ordinary anilin dyes and by y\Mn|Ot«v G-ram^s method . "1 Ju~Uaral characters. — The D. pneumonias is aerobic ' and almost facultatively anaerobic. On glycerin agar at •'j'V'"1 C. it forms minute, transparent, almost invisible qolonies like droplets of fluid ; on serum the growth has much the same characters, but is somewhat more abundant. It hardly grows on gelatin at the ordinary temperature, but in a 20 per cent, gelatin at 25° C. minute white colonies develop jothout liquefaction. In broth it produces a slight cloudiness ; it does not grow on potato but develops in milk, which is usually coagu- Jjlifid. ; neutral litmus glucose-agar becomes red during growth, indicating the production of acid. The fermentation reactions are given in the table on p. 248. Hiss's medium (p. 306) with inulin is fermented and coagulated; most other streptococci fail to ferment inulin. On the ordinary culture media it retains its vitality for a short time only, not more than about a week ; but if a little blood be smeared over the surface of the agar the vitality may be prolonged for a month or even longer. Washbourn recommended an agar rendered alkaline to the extent of 4 c.c. of normal caustic soda per litre, after neutralisation, rosolic acid being the indicator. This medium is smeared with blood, placed in the incubator for twenty-four hours to ascertain whether it be sterile, then inoculated, capped, and kept at 37° Q. IWs method for keeping Frankel's 432 Manual of Bacteriology pneumococcus alive and virulent is to receive the infected blood of an inoculated animal into a small glass tube 5 mm. in diameter and 20 cm. long, so thai the blood completely fills the tube, which is then sealed and kept away from the light at the ordinary tempera- ture. If inoculated on to ordinary gelatin, which is then kept in the blood heat (37° C.) incubator, the organism retains its vitality for a month or six weeks. Under cultivation the D. pneumoniae usually assumes the form of a short streptococcus (Plate XVI., b) (included by ^Gorchpn in his 8. hrevis class) and the capsule is lost, but is regained again on passage through a susceptible animal, or by growing in fluid serum. A good deal of variation occurs in the morpho- logy of the organism obtained from different sources Og^t/Clf and under cultivation. The thermal death-point of the i/^lCfix^ D. pneumoniae according to Sternberg is_ 52° C., the time of exposui*e being ten minutes, and it is readily destroyed by the ordinary germicides, by light, and by desiccation j but in dried sputum it may retain ^ its vitality and virulence unimpaired for weeks. " Pathogenic action. — The D. pneumonias is pathogenic for a number of animals, the most susceptible being miye. then in decreasing order, rabbits_, rats, guinea- pigs, and dogs. Pigeons and fowls are immune. f5eath follows after subcutaneous, intra- venous, intra- peritoneal, or intrathoracic injection of a virulent culture, or of rusty pneumonic sputum, into mice and rabbits in twenty-four to forty-eight hours. The virulence of the organism varies considerably ; under cultivation it may be completely lost, while by a series of passages through a susceptible animal it may be much increased. The less virulent it is the longer it tends to retain its vitality under cultivation. Except when injected into the lung or into the trachea, pneumonia does not result, PLATE XVI To face paj/e 132, 1 The Pneumococcus but tho disease runs the course of a septictemia with high temperature and dyspnoea, death being generally preceded by a subnormal temperature and often con- vulsions. The post-mortem appearances are much oedema and inflammatory infiltration at the seat of inoculation, haemorrhages in the serous membranes, enlargement and congestion of the spleen, and congestion of the lungs. The organisms occur in 1n,ro-» numbers in the blood, lungs, and spleen, usually in the form of oval diplococci with well-marked capsules (Plate XVI., a), but sometimes as short chains of streptococci. When injected into the lung or trachea a typical fibrinous or croupous pneumonia results. The D. pneumonia} is the cause of acute croupous pneumonia m man, and occurs in large numbers in the rusty sputum and hepatised lung, and in 20 per cent, of the cases can be isolated from the blood if 5-10 c.c. be cultured. The production of a typical pneumonic process experimentally and the presence of the diplo- coccus in a large proportion of cases of acute croupous pneumonia point to its specific relationship to the disease. With regard to the latter observation, Weichselbaum obtained it in 94 cases out of 129 examined, Wolf in 66 out of 70 cases, and Netter in 75 per cent, of the cases examined. In America the disease has of late been much on the increase, in Chicago the mortality having reached as high as 20 per 10,000 inhabitants. Acute croupous pneumonia sometimes occurs m epidemic form. The organism is frequently present in the saliva of healthy individuals, as shown by Netter, Sternberg and others, and the generally accepted idea of the relationship of « catching cold » to an attack of the disease is explicable on the theory that the action of cold lowers vitality, and renders the tissues vulner- 28 4o4 Manual of Bacteriology able to the attacks of the organism already in close proximity to them. Besides acute croupous pneumonia, more than half the cases of broncho-pneumonia, both primary, and secondary in the course of other diseases, are .due to the D. ■imp.vMonifB. which is also associated with a number of other important pathological conditions in man. It is a pyogenic organism, producing abscesses when inoculated into a relatively insusceptible aninia, such as a dog, and has been met with in abscesses, empyema, suppuration in the antrum, and purulent arthritis. It is also found in about half the cases of purulent meningitis, sometimes in cerebro-spinal nienin- "gTtis, in about~athird of the cases of otitis media and urfective endocarditis, sometimes in purulent j)ermar^ ditis, and occasionally in peritonitis. ' Tusinn— Auld separated a proteose and an organic^ acid from the blood and organs of infected animals, — aCici trom uie uiuuu *im «iB«"» — d>O^W^) ^ from cuitivations of the Dipldcoccus pneumonias m 'Ci2IXvU> alkali-albumin the same products were apparently £uaUru$X< obtained, the alkaline medium soon becoming per- , 0 manently acid. The proteose on subcutaneous or intra- venous injection produced some fever ; on intra-thoracic injection fever and dyspnoea, and post-mortem pleurisy and consolidation of the lung were found. The organic acid produced slight rise of temperature, but no other symptom. Macfadyen1 obtained an endotoxin by triturating cultures with liquid air. hdi-serum.— Immunity can be conferred on suscep- tible animals by treating them with attenuated calturea, and also by inoculation with increasing doses of faltered broth cultures of the virulent organism G. and 1 . Klelnperer used recent broth cultures heated to 60 C. for one or two hours. Washbourn used filtered i Brit. Med. Joum., 1906, vol. ii, p. 77<3 (Kefs.). Pneumococcic Serum and Vaccine 4S5 cultures in defibrinated blood, 20 c.c. o£ wliicli injected subcutaneously into a rabbit conferred immunity against virulent cultures, an immunity persisting for fifty or sixty days. The _blood-serum of such immunised animals will protect other animals when injected, and Klemperer, issaer, and Washbourn have prepared a pneumonic anti-serum. The latter, by first immunising a horse with filtered cultures, increased the immunity by injection with gradually increasing doses of living virulent cultures, until a very high degree of inimunitv is obtained. This anti-serum has been used in the treatment of pneumonia and other pneumococcic infec- tions, but the results have not been verv encourao-ino- I he protective serum seems to produce aggregation of the cocci when added to a culture of the diplococcus. Klemperer and Washbourn found that the serum of convalescent patients possesses some degree of protec- tive power. The serum, however, taken during the pyrexia! stage of the disease rather increases the sus- ceptibility of animals to pneumococcic infection. Vaccine. A vaccine prepared from cultures killed, by jieat^and standardised has been founcfof service in chronic pneumococcic infections, and has also been employed in acute croupous pneumonia.1 Friedlander's Pneumo = bacillus. This organism, already referred to above in the general discussion of pneumonia, and originally believed by 1 riedlander to be the cause of the disease, has been obtained by recent observers in only a small proportion of cases_of pneumonia. Morphology. -The B. pneumonia, is a very pleo- morphic organism, occurring in sputum or in the bW 1 WiUcox and Morgan, Brit. Med. Joum., 1909, vol. ii, p. 1050. 436 Manual of Bacteriology V of mi inoculated animal generally as a short rod w i 1 1 1 rounded ends surrounded by a marked capsule. It is non-motile, does not form spores, and is readily stained with the ordinary anilin dyes, but not by_ . Gram's method — an important distinction from the Diglococciis pneumonias. In cultivations it forms short rods, long rods, chains, and even filaments, the capsule being absent, but this is regained on passage through a susceptible animal. Cultural characters. — The B. •pneu- monias is aerobic and facultatively anaerobic., and may ..produce indole. It grojws readily on the various culture media from 20° to 37° C, on agar and blood- scrum forming a copious, viscid, greyish growth ; on gelatin, a thick, white, shimmy porcelain -like growth without liquefaction ; and in sub- cultures in gelatin a so-called liaib s4rap_ed^grojy.th is developed (Fig. 46), consisting of a white growth along the needle-track, tapering from above downwards, and at the surface heaped up an cl e x panded/ forminj^tjmiilm ad 3 3 of the nail. On potato a copious culture, seven days whitish growth develops, while mijk_ is curdled and gag-bubbles frequently form in stab-gelatin cultures. It is an active fer- menter of carbohydrates, and the fermentation reactions are given in the table, p. 400. Pathogenic action.— The pneumo-bacillus of Fried- ■ ■ I / Fig. 4.43. — Friedlan- der'spneumo-bacil- lus. Gelatin Btab lander is pathogenic to mice and ^gjrmea-pigs, but rabbits are immune. Post-mortem, the fjflleffi is en- larged, the lungs are congested and consolidated m patches; and the organism is found in large numbers Diagnosis of Pneumonia 437 in the blood. In a small percentage of cases of croupous pneumonia Friedlander's bacillus may be associated with the P. pneumoniae. Friedlander's bacillus may sometimes set up a broncho-pneumonic or bronchitic process, and is occasionally associated with anginal conditions, which are characterised by the formation of a false membrane, with an absence of any general symptoms. A microscopical examination of the membrane will show the orga- nisms surrounded with a capsule and unstaiuable by Gram's method. If a culture be made on serum, the large, round, greyish colonies of the bacillus will b™ recognisable in fifteen to twenty hours, and should be examined microscopically. To obtain a pure culture a white mouse should be inoculated from a "colony j iHnlli die in twenty-eight to sixty hours. Friedlander's pnenmo-bacillus has also been met with in water by Grimbert. Accoixling to him, it is identical with the B. capsulatus of Mori. Clinical Examination (Pneumonia). 1. Make cover-glass specimens from the rusty sputum, and stain some with Loffler's blue, and others by Gram's method with eosin. By a microscopical examination the oval diplo- cocci will l>e readily recognised, the B. pneumonias and B. ppstis being distinguished from the Diplococcus pneumoniee by being decolorised by Gram's method. The latter organism is the only one, moreover, which is likely to be ordinarily met with. 2. If the diplococci are found to be fairly abundant in the sputum, and other organisms nearly absent, an attempt may be made to cultivate by inoculating several glycerin-agar and serum tubes and incubating at 37° C. for forty -eightnours. 3. If the diplococci are scanty, or so mixed with other organisms that it is difficult to identity them, and probably impossible to obtain a pure culture, a drop or two of the 438 Manual of Bacteriology sputum should be injected into the peritomml onvU-^f a mouse or rabbit. The animal will die in fix>m tw^ntp^Ti,, thirty-six hours, and the Diplocoecus pneumonise will be found plentifully in smears prepared from the blood or lung-juice, and pure cultures can be readily obtained by inoculating glycerin-agar tubes with the blood or lung- juice. 4. The culture or inoculation method, preferably both, will probably have to he adopted for the recognition and isolation of the Diplocoecus pneumonias in pus from empyemata, abscesses, etc. 5. Friedh'inder's pneumo-bacillus can be ueadily isolated by making gelatin-plate cultivations, in which its colonies form white, shining, heaped-up points. Influenza. A minute bacillus was first described in this disease by Pfeiffer in 1892, who found it in large numbers in the bronchial secretion. In order to isolate the organism a patient with bronchial expectoration should be chosen ; he rinses his mouth and gargles his throat with hot water several times, and then, after couarhina\ the expectoration is obtained. A little of this expec- toration is washed by shaking in a test-tube with sterile salt solution, thou repenting the washing with sterile salt solution in a second and finally in a third test-tube. By means of a platinum needle a number of glycerin-agar and blood-agar tubes are inoculated with the sputum after the last washing, and incubated at 37° C. Morphology. — The influenza bacillus is one of the smallest bacilli with which we are acquainted. It is a minute rod 0*5— 1"5 /j. in length, and is non-motile and non-sporing. It does not stain by Grain's method, and not very readily with the ordinary dyes, dilute carbol- fnchsin or prolonged staining with Loffler's blue Influenza 439 yielding the best results, the poles tending to stain more deeply than the centre. In the sputum it occurs singly, in short chains, in small groups, or in larger masses, Vicing must numerous early in the acute stage of the disease. Cultural characters— -The W.ilhis is strictly aerobic, and no growth occurs on media at 22 C. On glycerin agar and blood-serum at 37° C. it forms jver^small, tronapareai^drop-like oolonieg_in from twenty-four to forty-eight hours, which, according to Kitasato, never become confluent. There is no growth on potato. The organism g^rows best on inedia containing blood, such as agar smeared with sterile human, rabbit's, or pigeon's blood. In broth it grows at the surface in fine white flakes which subsequently sink. It soon dies out in cultivation, but according to Klein can be kept alive for some weeks in gelatin incubated at 37° C. The liquefied gelatin remains {\jcJC clear, the growth forming a ' delicate flocculent % precipitate at the bottom. Preparations from cultures show long twisted chains and threads of bacilli, aco-reo-ated so as to form dense networks and convolu- tions. These chains or threads are' composed of bacilli placed end to end, and united by a continuation of the cell-membrane. Involution forms occur. It is stated to grow better in association with the M. pyo- genes, var. aureus, than alone. The organism does not seem to be able to live outside the body for any length of time, and is readily destroyed by desiccation, weak antiseptics, and by a temperature of 60° C. acting for five minutes. Pathogenic action. — Canon stated that he obtained this bacillus Prom the blood in a number of cases, but many other investigators have failed to find it. Klein also obtained it in six cases out of forty-three examined. 440 Manual of Bacteriology According to Pfeiffer the bacillus is pathogenic ojdy to monkeys and rabbits. Klein, however, was unable to obtain any definite effects in these animals by the injection either of sputum rich in bacilli or of pure cultures. The influenza bacillus is met with in all uncomplicated cases of influenza in the nasal and bronchial secretions, often almost in pure culture, and in the bronchial tubes and lung in the pneumonic complications accompanying the disease. The organisms disappear with conva- lescence, and are not met with in other diseases. Klein1 appears to consider that the pneumonia often complicating the disease is probably directly due to the bacillus. The typical influenza pneumonia is of the lobular type with a cellular rather than a fibrinous exudate. True lobar pneumonia, due to the Dijilococcus pneumoniae, may, however, often complicate the influenzal attack. The organism also occurs in bronchitis, broncho-pneumonia, and whooping-cough. Although the typical influenza may be due to the B. influenzae, many febrile conditions attended with pulmonary catarrh and frequently termed " influenza " are not due to this organism. In an epidemic simulating influenza occurring in Essex in 1905, the examination was negative as regards streptococci, B. diphtherias, and B. influenzas, but the M. ccttarrhalis was present in numbers in most cases (twenty -two out of twenty-four). This organism was originally isolated by Seifert in a small epidemic of infectious bronchitis, afterwards by Pfeiffer in cases of broncho-pneumonia in young children (see p. 260). Two other Gram-negative cocci were also isolated from three other cases (see table, p. 261). i " Further Report on Epidemic Influenza," 1889-92, hoc. Gov, Board Report, 1893, p. 85. Influenza and Pertussis 441 Clinical Examination. In cases of influenza, accompanied with bronchitis or pneumonia, the influenza bacillus may be met with in large numbers in the sputum, and their presence may aid in confirming the diagnosis. Cover-glass preparations may be stained with carbol-methylene blue. Whooping-cough (Pertussis).1 An influenza-like bacillus has been isolated by Koplik, Czaplewski and Hensel, Davis and others in this disease, but the researches of Borclet and Gengou have shown that it is distinct from the influenza bacillus. . The B. pertussis is a minute bacillus, very like the B. Uj^/l^i^ influpnzie, non-motile, non-sporing, and Gram-negative. It is QJj^^ ^ scanty in the bulk of the expectoration, but is abundant in rv.' the viscid exudate, rich in leucocytes, coming from the depth Y ^*4U- O of the bronchi, and voided at the end of a paroxysm of coughing. It is best isolated on a medium consisting of defibrinated blood (human or rabbit"), thoroughly mixed with an equal volume of 3 per cent, agar containing a little extract of potato made with 4 per cent, acpaeous glycerin. It forms on this a fairly thick whitish streak, the subjacent blood being hsemolysed. It may also be grown in serum or blood broth in shallow layers. After acclimatisation to artificial media it will develop on the ordinary laboratory media. The B. pertussis is agglutinated feebly by the blood of patients, but complement-fixation is marked. Monkeys are stated to develop a typical whooping-cough on iuiicjolaijon^ out tie ordinary laboratory animals are susceptible only to massive intra-peritoneal' or intra- venous inoculation, death ensuing from a septicamiic process. Attempts have been made to treat the disease with a vaccine. 1 See Bordet, Brit. Med. Journ , 1909, vol. ii, p. 1062. 142 Manual of Bacteriology CHA.PTEB XIII. AnAEKOBTC ORGANISMS. TETANUS MALIGNANT CEDE MA B LACK QUARTER — BACILLUS WELCTTII (AEROGKNKS CAPSULATOS, ENTERITID1S BPOKO- G ENEs) BACILLUS CAUAVERIS SPOROOICNES CLOS- TRIDIUM BUTYRICUM. Tetanus. Tke causation of tetanus was for a long time involved in mystery. No obvious or characteristic changes being met with after death, the disease was regarded by many as "functional." Others believed that a primary lesion of the central nervous system might be the cause of the affection, while a few classed it with the specific diseases. It had' long been noticed that wounds soiled with earth were specially prone to be followed by tetanus, and Sternberg in 1880, and Nicolaier in 1884, producedjgtanus in rabbits by introducing a little garden earth beneath the skin. The latter observer found at the seat of inoculation and In his impure cultures — for he was unable to obtain pure ones — a distinctive bacillus, and he was able with these cultures, and with the pus from the seat of inoculation, to induce tetanus in other animals. Carle and Eattone subsequently showed that the bacillus of Nicolaier was present in the tissues of, and secretions from, the wound, in cases of traumatic tetanus in man, and that inoculation with the pus from such a wound produced tetanus in the lower animals— observations which were confirmed by Eosenbach in 188-5. The bacillus was The Tetanus Bacillus 443 isolated in pure culture by Kitasato in 1889 by taking the impure cultures obtained from the wound in a case of trau- matic tetanus, heating to 80° C, and plating the heated cultures, the plates being incubated anacrohicalh^ in hydro- gen. The Bacillus tetani. Morphology. — The Bacillus tetani is a straight,_ slender rod with rounded ends, but under cultivation the rods may grow into longish filaments. It is some- what niotije, an cl possesses a large number of flagella, three or four of which are generally thicker than the rest.1 Spores are freely formed ; they are spherical and develop at one extremity of the rod, and their diameter being much greater than that of the rod, the spore-bearing organism has been likened to a " pin" or " drum-stick " (Plate XVII., a). It stains with the ordinary anilin dyes, and also by Gram's method. " Drum-stick " bacilli are not necessarily tetanus ; other anaerobic bacilli, e. g. B. putrificiis (colt), may also have large terminal spores. Cultural characters. — The B. tetani is a strictly anaerobic orp-anism. and will not grow in the" presence of,_a trace of free oxygen, nor in an atmosphere of carbon dioxide. It can be cultivated in deep stabs in glucose agar and gelatin, or in broth by Buebner's method, or in an atmosphere of hydrogen (p. 73). In a gelatin stab-culture at 22° C. The growth radiates from the central puncture, and the gelatin is slowly lKjuefied. In a glucose agar stab-culture it forms feathery, radiating outgrowths from the central puncture, a small amount of gas being formed (Fig. 47). Broth becomes turbid with the formation of some gas 1 Kanthack and Connell, Journ. Path, and Bad., vo] Lv 1897 p. 452. ' ' 14 f Manual of Bacteriology and the development of a foul odour; there is no film formation. The colonies have a central opaque portion surrounded by diverging rays. It grows on serum without liquefaction and in_ milk without curd- _ Jino\ The tetanus bacillus remains alive for some time, possibly indefinitely, in cultures, and the spores retain their vitality for years in the dried state, withstand a temperature of 80° C. for an hour, but are killed by boiling for live minutes. Carbolic acid (1 : 20) does not destroy the spores under about fifteen hours. Occtirrenoe and watJioaenic action. — Man and the horse are most subject to tetanus ; cattle and sheep are rarely affected, while the fowl, frog, triton, snakes and tortoise are immune. Mice, guinea-pigs and rabbits are all very susceptible. The bacillus is present in the superficial layers of the soils in many localities, but not in all, and this accounts for the fact that tetanus is rare in some places and frequent in others. Curiously enough, some of the savage inhabitants of the Solomon V?.-JL-2$™H»\£& have made nS±^jm^mL tare in glucose arrows, the poisonous nature of which agar, seven days to t^^rbearing earth. The arrows are tipped with a viscid fluid, then rubbed in the soil from a mango swamp, and afterwards dried. Individuals wounded with these arrows often develop tetanus. Tetanus spores are frequently present in the dejecta of cattle, horses, and other animals, and occasionally of man (p. 445). Tetanus 445 The bacillus is confined to the seat of inoculation, or at most is met within the nearest lymphatic glands, so that the p-mim-al symptoms are due to the abs_or.p^ tion of toxin! "The researches of Ransom and "Meyer have shown that the tetanus toxin is mainly absorbed by the nearer trunks (see also p. 166). The organisms associated with the tetanus bacillus in earth are probably of considerable importance in the production of the disease, for it has been shown that if the tetanus bacilli and their spores be carefully i washecLso as to remove all adherent toxins, they fail to set up tetajius^nJ^GirlatiOT, while if the same washed bacilli be injected, together with a little lactic acid, tetanus follows, the explanation being that the bacilli arc unable to multiply unless the surrounding tissues arc damaged 'and phagocytosis is prevented. The associated organisms in the wound probably effect this, and do not act by producing a condition of anaerobiosis as has been suggested. Semple1 has recently found that tetanus spores are occasionally present in the human intestinal tract (Hamilton suggested that teta- noid organisms in the intestinal tract might be the cause of the so-called idiopathic or rheumatic tetanus). He injected guinea-pigs with washed spores, and tetanus did not ensue, but the tissue at the site of inoculation, examined five to seven months later, still contained the living spores. Semple suggests that such latent spores may in some instances be disturbed and become active by the hypodermic or intra-muscular injection of quinine, owing to the tissue necrosis and inhibition of phagocytosis produced by the drug. Toxins. — Cultivated anaerobically in broth, the tetanus bacillus forms a most potent extra-cellular toxin, so that if the culturlPbe filtered throuedi a 1 So. Mum. Gov. of India, Nu. 13, 191] . 446 Manual of Bacteriology porcelain filter, 0-001 c.c, 0-0001 c.c., or even 0-0000 1 o.O. of the filtrate is a fatal dose for a guinea- Tetanus toxin broth contains a tetanising substance, termed tetano-spasmin, and also a hemolysin, tetano- lysin. The toxin lias a, special affinity for nerve-tissue (sec p. 160). Injected into animals such as the mouse, guinea-pig and rabbit, the toxin broth produces tonic, not clonic, spasm, and with small doses the muscles at or near the seat of inoculation tend first to be affected, so that the spine may be curved, the leg paralysed, etc. (Fig. 48). By treatment with carbon disulphide, tetanus toxin broth becomes practically •nmi-tn\iV,J though it still retains its power of i in m un isin g__p n inoculation and of combining with antitoxin — that is to say, bodies are formed analogous to the toxoids of diphtheria toxin. Brieger, from impure cultures of the tetanus bacillus, obtained two basic bodies which he termed "tetanine" and " tetano-toxin," the former producing tetanic symptoms in mice, and the latter tremor, paralysis, and finally convulsions. Brieger also isolated teta- nine from the amputated limb of a tetanic patient. Brieger and Frankel obtained a tox-albumin from bouillon cultures which induced tetanus in guinea- pigs. Brieger and (John subsequently investigated the tetanus poison obtained by precipitating veal-broth cultures with ammonium sulphate added to saturation, and purifying by re-dissolving, precipitating the protein with basic lead acetate, and removing other soluble impurities by dialysis. The purified product forms yellow flakes, soluble in water, but not giving the Millon and xanthoproteic reactions. It is not pre- cipitated by most metallic salts, and is.. not carried clown by Roux and Yersin's method of precipitation Tetanus Antitoxin 1 1 ' with calcium phosphate. It contains no phosphorus and only traces of sulphur. Of the most active preparation O'OOOOOOOS grm. killed a mouse. In a case of tetanus examined by Sidney Martin, an albumose, chiefly deutero-albumose, was extracted from the blood. Injected into an animal, it produced depression of temperature, followed by progressive wasting, but no spasm or paralysis. Antitoxin. — If an animal is cautiously injected with Fig. 48. — Guinea-pig inoculated with a small close of tetanus toxin, showing paralytic condition of right hind leg due to spasm. tptanuF t.o*w. commencing the treatment with a weakened toxin, and increasing the dose very gradually, a high degree of immunity is ultimately obtained, and the blood-serum acquires marked antitoxic properties The toxin is obtained by growing the tetanus bacillus in bouillon in an atmosphere of hydrogen for about three weeks, and filtering through porous porcelain. To obtain an active serum treatment has to be pro- longed, ajiorse immunised by the writer requiring six months. The antitoxic serum so obtained is by far 448 Manual of Bacteriology the most active of any of the sera, and is now reooc- nised as the proper remedy to use in cases of tetanus in man. The antitoxic treatment of tetanus is not nearly so successful as that of diphtheria, and for this reason : in diphtheria,, in a large proportion of the cases, a local manifestation is present to aid diagnosis before any serious absorption of the toxin has taken place, whereas in tetanus the disease is only recognis- able by the symptoms induced by such absorption. Nevertheless, it can hardly be doubted that it is our duty to employ the antitoxin not only in the fully developed disease, but also in certain cases as a prophylactic. As the toxin is at once fixed by the nerve-tissue, thg_antitoxin should be injected into the central nervous system in order to obtain immediate action. The antitoxin may be standardised by the Roux or by the Behring method (see p. 203). Recently a method analogous to that used for standardising diphtheria antitoxin has been introduced.1 Clinical Examination. The symptoms of tetanus are usually so obvious Unit a bacteriological examination is not needed to establish the diagnosis, aud unless there is an evident wound it will be difficult, if not impossible, to detect the tetanus bacillus. (1) Prepare several smears of the pus or discharge, and stain by Grain's method. *~ Examine niicroscopicaTIyTTookiiig for the spore-bearing rods or " drum-sticks." A " drum -stick bacillus is, however, not necessarily the tetanus bacillus (see p. 443). (2) If " drum-sticks " be found, an attempt may be made to isolate the bacillus by making anaerobic plate cultivations i On the standardisation and therapeutic use of tetanus antitoxin, see Hewlett's Serum Therapy, 1910. Malignant (Edema 449 from the discharge, after heating it in capillary pipettes to 80° C. for half an hour. (3) Inoculate mice and guinea-pigs with the heated dis- charge. If they die with tetanic symptoms, treat the pus at the seat of inoculation as in (2). Malignant OEdema. Malignant oedema is met with in man in connection with wounds soiled with septic mattei", compound frac- tures, contused and lacerated wounds, etc. Usually there is a jmtref active and cedematous condition of the tissues with subcutaneous emphysema. Animals also occasionally suffer from the disease, wliich can be pro- duced artificially by inoculation with dust, dust from straw, the upper layers of garden earth, and decompos- ing animal and vegetable matter. If a guinea-pig be inoculated snbcntaneously with a little garden earth, it will very likely die in forty-eight hours. Post mortem, the subcutaneous tissues around the seat of inoculation will be found to be cedematous and blood-stained, with more or less development of gas. The internal organs are only slightly altered, but the spleen may be somewhat enlarged. The juice from the seat of inoculation will be found to contain a mixture of organisms, but in the blood and organs few will be found. Under the capsule of the splpcm, hnw_ ever, long slender rods may be seen ; these are the bacilli of malignant oedema. Morphology. —The bacillus of malignant oedema is a long and slender rod, several of which may be united ti /» into a thread. It is motile, possesses several flagella. t^CtLtsMi and is readily stained by the ordinary anilin dyes, but IT^T^* not byjfrajais method. It spores Wly at tempera- Q&UU mm TuTis above 20° C, the spores being large and central.^ l Cultural characters. —The bacillus of malignant cedema^P^1^ *f* 450 Manual of Bacteriology fyMOJcA*^*' is_strictly anaerobic. In a deep stab in glucose-agar it + forms a thick line of growth in the needle track, with irregular outline and greyish-white in colour. There is profuse development of gas, accompanied by a fonl_ ^dourr, and attended with disruption of the medium into several portions. -* The bacillus of malignant oedema is an organism which has to be distinguished from anthrax, and there should be no difficulty in doing this. Post mortem, the spleen is rarely found much enlarged in malignant oedema, the organism is not very abundant, is almost entirely absent fromlhe blood, "~ and is onlyfound under the capsule of the spleen, not at its centrer it, however, several hours have elapsed since death occurred, the organism may have wandered into the blood and the centre of the spleen. The bacillus of malignant r oedema is motile under anaerobic conditions, the anthrax « ' bacillus non-motile ; the former occurs as a long slender filament, which on staining is seen to consist of two or three long segments ; it does not stain by Gram's method (except by Claudius's modification), and is strictly anaerobic. Bacillus botulinus. In certain forms of meat poisoning (see Chap. XXI) van Ermengem isolated an anaerobic bacillus, the B. botulinus. It is chiefly met with in ham and sausage. and the symptoms are caused by the absorption of toxin, which has a special effect on the nerve centres. The organism is a large G-ram -positive anaerobic bacillus, often occurring in pairs or in short chains. In glucose gelatin it forms a whitish streak in the line of the stab, with lateral out-growths, liquefaction of the medium, and gas- formation. The cultures have a rancid odour, due to butyric, acid production. The colonies in gelatin are semi-transparent spheres. The optimum growth is from 20°-30° C. The B. bohdinus in broth cultures Wins a. potent extra- cellular toxin. The toxin is also produced in the infected ham , sausage, etc. With the toxin an antitoxin can be prepared. Emphysematous Gangrene. Bacillus Welchii.1 Probable synonyms.— B. aiirogenes capsulatus (Welch and Nuttall), Graimlo-bacillus saccharo-butyricus immobilis lique- faciens (Grassberger and Snlmt.teTifrohV B. enteritidis svoro- 7 ijenes (Klein^-B. peifrhujens (Veillon and Zuber), gas- phlegmon bacillus (Friinkel), bacillus of acute rheumatism (Achalme : see " Rheumatism "). This organism was originally described by Welch _and Nuttall under the name B. aiirogenes capsulatus, and r occurs in conditions accompanied by much development Ar**^ ** of gas in the tissues, as in cases which might be described either as phlegmonous erysipelas or as emphysematous gangrene, especially after injuries. ^\j^y(iLl — is also met with occasionally in perforative peritonitis t and in various septicemic and pyemic conditions, in th puerperal state/ complicated stricture, etc. The B. Welchii is widely distributed, and has been cultivated from the soil, dust, and contents of the intestine. It has either been described under a variety of naines, or a group of closely related bacilli may / exist. Gas-bubbles found in the blood and internal fp^^^\) organs (" foamy organs ") at an autopsy seem generally to be due to this organism, but may occasionally* ^tu'ia Med.-Chir. Trans., vol. lxxvii, 1894, p. 263. PLATE XVIII. CZ3 Tofaee page 160. Survival of the Comma Bacillus 4,61 the comma bacillus lived from three to five days in dry soil, but only one day in an artificially dried soil, while in moist soil it lived from twenty-eight to sixty-eight days. In peat, however, it was invariably dead within twenty-four hours. In sterilised salt solution (0'75 per cent.) the comma bacilli were alive on the 159th day, and in fresh urine (sterilised) they lived fourteen days at 37° C. and twenty-nine days at 22° C. In sterilised distilled water the cholera spirillum usually rapidly dies, as a rule within twenty-four hours. The addition of sodium chloride greatly increases the length of time it may remain alive, a survival of five or six weeks having been recorded. In ordinary — st.pn'HsRd potn.blfi wn.ters \t roav survive many months. In unsterilised potable waters its survival is greatly influenced by the presence of salts ; in some cases it dies out rapidly ; in others, especially in those containing a large proportion of salts, it may remain alive for some time. Houston1 found that cholera vibrios die very rapidly in raw Thames, Lee, and New River waters as the result of storage in the laboratory. At least 99'9 per cent, perish within one week, and it was not possible to isolate any, even from 100 c.c. of the water, three weeks after infection. Klein2 found that the cholera vibrio could retain its vitality for at least fourteen days in unsterilised sea-water, while from the interior of oysters, kept in water infected with the vibrios, it was obtained up to nine days after infection. In sterilised sewage the cholera spirillum multiplies and survives for months; in unsterilised sewage it may survive for two to four weeks (Houston). Pathogenicity —The disease is spread mainly by infected water j milk, salads, vegetables and flies are 1 Metropolitan Water Board, Fifth Rep. on Research work, 1910. 3 Rep. Med. Off. hoc. Gov. Board for 1896, p. 135. 462 Manual of Bacteriology also other sources of infection. The organism has been found in the dejecta of contacts not suffering From the disease, and it may sometimes persist for long periods after convalescence. The relation of the cholera spirillum to the disease has been a very vexed question in the past, but the outcome of the voluminous researches which have been made is to confirm Koch's work. The organism is found in all_cases of cholera^ and several instances of laboratory infection from cultures have been recorded. None of the lower animals suffers from or contracts a disease in any way comparable to Asiatic cholera, so that the test of animal experiments cannot be applied except in the_casft of young- suckling rabbits (see below, "Anti^seriun"). By first neutralising the acidity of the gastric juice by an injection of sodium carbonate solution into the stomach, then diminishing peristalsis by an injection of tincture of opium into the peritoneal cavity, and finally injecting a broth culture of the cholera spirillum into the stomach, Koch succeeded m inducing in guinea-pigs a condition somewhat similar to cholera in man— namely, indisposition with falling temperature, weakness of the extremities, and death m forty-eio-ht hours. Post mortem, the small intestine was congested and filled with a watery fluid containing laro-e numbers of the cholera spirillum. Injected into the peritoneal cavity of mice, guinea-pigs and rabbits, it usually produces death from a general septicaemia. Metchnikoff1 ascribes the immunity of animals to intestinal cholera as largely due to the inhibitory action of the other organisms present in the digestive tract In man digestive distarbancesj^^ predisposing _canse olH^lfficT^^ ^Ann. de VlZTltotnr, vii, pp. 403, 562 ; vol. viii, pp. 2o7, 529. Occurrence of Vibrios 463 gastric juice is also probably a means of defence (see "Water"). The blood-serum of an animal immunised by injections of the cholera spirillum gives a typical agglutination reaction with recent cultures of the organism. The reaction can also be obtained with the blood-serum of cholera patients sometimes as early as the first day of the disease, but it is probabl3r of little use for diagnostic purposes, as the course of the disease is generally so rapid. Occurrence of the vibrio. — That the cholera spirillum is associated with the disease seems to be beyond any doubt, and so constant is its presence in true cholera that all investigators, even those who at one time opposed Koch's views, rely on its detection for the bacteriological diagnosis. The matter, however, has become complicated owing to the detection in various natural waters of pathogenic spirilla which, although not identical with the cholera spirillum of Koch, resemble it so closely that it is difficult to classify them as anything but varieties of the cholera spirillum. In certain epidemics in India variations have also been noted in the cholera spirilla that have been isolated. 'Sanarelli1 ^isolated from the Seine and Marne thirty-two spjriUa, of which four were almost indistinguishable from cholera, except that they were only slightly pathogm-.i^ but by passage through a series of animals their patho- genic power was much enhanced. Sanarelli believed that these were the descendants of true cholera spirilla that had gained access to the rivers during some previous epidemic of cholera. At the same time it is to be noted that vibrios may also be present in the normal intestinal tract of man and animals, and may therefore gain access to streams (Sanarelli). Dunbar Ann. de VInst. Pasteur, vii, p. 693, and ix, p. 129. 464 Manual of Bacteriology similarly, from the Elbe, Rhine, and other rivers, isolated a number of spirilla which could not be distinguished from the cholera spirillum (Spirillum Mwers). It was afterwards noticed that some of these under certain conditions of oxidation and temperature became phos- phorescent,1 but Rumpel3 has also found that cultures of the genuine cholera spirillum may exhibit phosphor- escence, so this cannot be used as a differential character for the separation of non-choleraic forms. Neisser isolated a spirillum, which he termed Vibrio Berolinensis, which agreed with the cholera spirillum in every par- ticular except that the colonies in a gelatin plate were invisible to the naked eye in forty-eight hours. Heider found in the Danube a spirillum, named by him the Vibrio Danubicus, which resembled the cholera spirillum closely, but its colonies were somewhat different, and it was more actively pathogenic to mice. Ivanoff similarly obtained a spirillum which could only be distinguished from cholera by the finer granulation of its colonies and more distinct spiral form. Lastly, there is the Spirillum Massowah, isolated from an epidemic of cholera at Massowah, which differs from the Koch spirillum in having two terminal flagella at each end. Cunningham has also described several spirilla differing but slightly from the cholera spirillum. Applying the Pfeiffer and aVfl urination tests, to the spirilla in question, the following results were obtained. In the first place, each of the organisms gives a complete positive reaction to both tests with its own serum ; this, of course, is only to be expected. Pfeiffer found that, using his reaction, the variety Ivanoff gave a positive reaction with cholera serum, and Durham found that Ivanoff and Berolinends reacted completely i Centr. f. Bakk. (lte AM.), xviii, P- 424 (^scher). Munch, med. Wochenschr., 1895, No. 3. El Tor Vibrios 46$ with cholera serum. Conversely, positive reactions with cholera spirilla were obtained with Massowah, Daniibicus, and Elwers sera, while Massowah and Elwers react completely to each other. From these considera- tions it would therefore seem probable that some of these spirilla — Sanarelli, Berolinensis, and Ivanoff — may be varieties of the Koch spirillum. The Massowah spirillum is usually considered not to be a true cholera vibrio. Ruffer1 in 1905 at El Tor isolated vibrios, which may be distinguished as " El Tor vibrios," from the intestine of pilgrims returning from Mecca and suffering from various diseases (dysentery, diarrhoea, pneumonia, rheu- matism), but among whom there had been no cholera, nor had they been in contact with cholera. These vibrios were subjected to detailed examination by the agglutination, saturation and fixation tests, and Pfeiffer's reaction with Berlin cholera-immune serum, and also by the hemolysis test. Vibrios isolated from a previous epidemic of cholera (referred to as Group 1), and other vibr jos isolated from cholera and other stool (Groups 3 and 4), were also compared with the El Tor vibrios. Buffer's results were as follows : Group 1 (undoubted cholera vibrios).— Those which react positively to the, four principal tests with cholera serum— namely, the agglutination, saturation, and Nation tests, and Pfeiffer's reaction. They do not htemolyse, even when remaining in contact with red corpuscles for three days at the temperature of the laboratory. Group 2.— The second group contains the vibru agglutinated by and giving the saturation andPfeiffer 1 Researches on the Bacteriological Diagnosis of Cholera. Sanitary Maritime and Quarantine Council of Egypt, Alexandria, 1907. (Also Ent. Med. Journ., 1907, vol. i, p. 735.) V 30 •10 s 466 Manual of Bacteriology reactions with cholera serum, but not fixing the cholera- immune body. These vibrios are strongly hemolytic. This group consists of the El Tor vibrios only. Group 3.— The third group is formed by vibrios which are not agglutinated by immune serum, do not give the saturation or Pfeiffer's reaction, but fix the cholera-immune body. These vibrios also haemolyse, but feebly and late, often only after thirty-six to forty- eight hours. Group 4.— The last group is formed by strongly hemolytic vibrios not reacting at all to cholera-immune serum. . , Buffer concludes that the El Tor vibrios are not genuine cholera vibrios. He says : " The only possible classification is to group together all the vibrios reacting in the same way to all tests, separating them from those .Inch, under the same conditions behave in a different way. If this method be applied to he vibrios found at El Tor, there is no difficulty m dis- tinguishing them from the true cholera vibrios, m spite of Several of the reactions of both being similar. And it follows also that the agglutination, saturation and peer's tests are not in themselves of absolute diagnostic value for cholera vibrios." leufield and HaendeV however, after a r+ examina- tion of some of these vibrios, consider that they are S Cholera vibrios. The matter therefore remains ^le^ound that the cholera vibrio kept in sea- wafer showed marked ^f^^^^ ^rXtr^ —of not • some monociliate, others multicihate ' ! Arleit. a. d. Kai, G^Keitsa^e, xxvi, 1907, p. o36. Cholera Toxins 467 It may be that, like tho B. dysentaritv, the cholera vibrio is not a single definite organism, butthat cholera may be caused by any one of a group of closely allied" vibrios. Toxins. — Brieger in 1887 obtained cadaverin and putrescin and two other basic bodies from cholera cultures. Brieger and Frankel isolated a tox-albumin, and G-amaleia a ferment-like body. Hueppe believes that the cholera poison is a tox-albumin formed in the culture medium, but that immunising substances are derived from the bacterial cells. Rontaler compared the chemical products of the ordinary cholera and of the Massowah spirilla, and could find little difference between then. Wesbrook1 obtained albumoses and other bodies from alkali-albumin, egg, and Uschinsky medium, cultures. This observer also found aerobic cultm*es of the cholera spirillum to be much more toxic than anaerobic ones. Pfeiffer found that cholera cultures killed with chloroform vapour "contained a toxic substance fataLto~ gninfla-pjgsjn smaJJ__doses, with extreme collapse. He believed the substance to be an integral part of the bacterial cells. Metchnikoff,2 Roux and Salimbeni demonstrated the existence of a soluble cholera-poison in a very ingenious manner. Collodion sacs of 2 c.c. to 3 c.c. capacity wore sterilised, filled with peptone solution, inoculated with the cholera spirillum, and closed. The closed sac was then introduced into the peritoneal cavity of a guinea-pig, which died in three or four days from the effects of the soluble toxins dialysing through the walls of the sac (see also next page). 1 Journ. of Path, and Bad., vol. iv, 1896, p. 1. 2 Ann. de I'Inst. Pasteur, x, 1896, p. 257. 468 Manual of Bacteriology Bran and Dernier1 obtained a toxic filtrate l>y culti- vating the cholera vibrio in a medium consisting- of horse-serum with an addition of 10 per cent, of defibrinated horse-blood. Macfadyen obtained a highly toxic endotoxin by triturating cholera cultures with liquid air. Emmerich"* strongly supports the view that the cholera intoxication is not a toxin intoxication, but is due to nitrite poisoning, the nitrites being produced by the reducing action of the vibrios on nitrates present. Anti-serum. — By growing the cholera spirillum in a shallow layer with Jree access of oxygen in a peptone o-platin-saft medium, Metchnikoff and his co-workers obtained a toxic fluid after three or four days' growth. During incubation the fluid becomes concentrated to about one eighth by evaporation. After filtration, _ 0"25 c.c. killed a 300-grm. guinea-pig in eighteen hours. Goats having been inoculated with increasing- doses of this toxin, commencing with 10 c.c. and reaching 200 c.c. in six months, become immunised and yield an antitoxic serum, 1 c.c. of which will neutralise four times the lethal dose of toxin. Metch- nikoff had previously found that young suckling- rabbits suffer from an intestinal cholera when fed with cultures, so that the effect of the cholera antitoxin in preventing intestinal cholera could be tested on these animals. Experiment showed that of the treated rabbits, 51 per cent, survived, of the untreated only 19 per cent. Salimbeni employed a serum prepared in this manner in the treatment of cases of cholera m the Russian epidemic, 1910. Animals may be inoculated with dead and living i Ann. de I'lnst. Pasteur, xx, 1906. ' Lancet, 1906, vol. ii, p. 494. 3 Munch, med. Wochenschr., 1911, No. 18, p. 942. Cholera Vaccine 469 cultures and an immune serum so prepared, but no practical value has yet attended the use of anti-sera in the treatment of cholera. Macfadyen immunised a goat with cholera-celT~juTce, and obtained a serum of which -tttj c.c. protected a guinea-pig- against thi-ee lethal doses of cholera culture. The writer prepared an anti-en dotoxic serum in this manner, with which a few cases of cholera were treated in Russia.1 Vaccine. — Ferran in 1885 first prepared a vaccine by making cultures (mixed) in broth from cholera stools and injecting 0"3— 05 c.c. subcntaneously, but the reports of the commissions sent to investigate the method were unfavourable. Halftone subsequently pi-epared a vaccine against cholera from cultures of the Koch spirillum, which seems to be efficacious in preventing the disease. For example, a number of labourers were inoculated during an epidemic, and among the inoculated the mortality was only 2-25, whereas among the uninoculated it was nearly 19 per cent. In another instance amongst 654 uninoculated there were 71 deaths, a mortality of 10-86 per cent., while among 402 inoculated there were only 12 deaths, a mortality of 2"99 per cent., and a reduction in mortality of 72-47 per cent. I" the ldaffkine method two vaccines are made use .of. The first or weak vaccina is prepared from cultures of the cholera vibrio attenuated by growing on tlie surface of agar, with free aeration, for several generations. The second or strong vaccine is prepared by enhancing the virulence of a cholera culture by a succession of passages through the peritoneal cavity of guinea-pigs. The virulence of this culture must be maintained in the same manner. 1 Lancet, 1.910, vol. ii, October 22nd. 470 Manual of Bacteriology For making both vaccines, " standard " agar cultures are employed. These are tubes in wliich the sloping surface of agar measures 15 cm. in length, and the cultures are incubated for twenty-four hours. The whole growth on such a tube is emulsified in 8 c.c. of broth or salt solution ; the dose of this is 1 c.c, and the vaccines (living, or killed by heat, or carbolised) are injected into the flank, the second or strong being given seven to ten days after the first or weak. Besredka,1 by making a mixture of cholera culture and cholera-immune serum, allowing this to stand for twelve hours, heating to 56° C. for one hour and then injecting subcutaneously, claims that an immediate and lasting (six months) immunity may be produced. Strong2 prepares a vaccine from autolysed cultures. The cholera vibrio is grown on surface agar for twenty- four hours at 37° C. ; the growth is then washed off with sterile water, the suspension is kept at 60° C. for twenty-lour hours, and then at 37° 0. for two to five davs, and is finally filtered through a porcelain filter. . Clinical Diagnosis. Some of the rice-like flakes should be picked out of the stool and well rinsed in sterile salt solution. 1. From one of the whitish, slimy, rice-like flakes in the evacuations or the intestine films are prepared, stained in Loffler's blue, washed, dried, and mounted. If on examina- tion large numbers of curved rods lying in groups parallel to one another are observed, the diagnosis of Asiatic cholera may be made with some degree of certainty. Koch states that this is so in quite half the cases, especially the acute ones. (Single, or a few, vibrios are of no diagnostic signifi- cance ; they may occur in normal and diarrhoea stools. The i Ann. de VInsi. Pasteur, 1902, p. 918. » Bureau of Gov. laboratories, Manila, Hull. No. 16, 1904. ( Bibhog.) Diagnosis of Cholera 471 presence of numbers of vibrios having the " fish-in-stream " arrangement is alsojioLabsolutely characteristic .J~ ~ 2 Gelatin and agarplates should be prepared from an emul- sion of rice-like flakes. Agar plates are best prepared by smearing the flake over the surface of the solidified agar. The plates are incubated at 22° C and 37° C. respectively. In the gelatin plates the characteristic colonies of the cholera vibrios should be recognisable in about twenty-four hours, in the agar plates in from twelve to sixteen hours. The likely colonies should be examined microscopically and peptone- water and other cultures prepared from them. A better medium to employ is Dieudonnc's blood alkali agar. Equal parts of defibrinated ox-blood and normal caustic soda solution are mixed and sterilised in the steamer. Of this 30 c.c. are mixed with 70 c.c. of ordinary peptone- agar (neutral to litmus), previously melted. Plates are poured and the plates are kept at 60° C. for half an hour, and are then allowed to stand for twenty-four hours for ammonia to vaporise. On this little else than the cholera vibrio develops (except cholera-like vibrios, which develop equally well). 3. With other rice-like flakes several peptone- water cultures should be prepared and incubated at 37° C. This is best done in the small Erlenmeyer flasks containing a shallow layer (1-2 cm. deep) of Dunham's peptone-water, without wool plugs, but capped with a piece of sterile filter-paper. In eight to ten hours the upper layers of the fluid should be examined microscopically for the presence of commas, and gelatin, agar and Dieudonne agar plates and subcultures in peptone water are also made by inoculating from the surface layer of fluid. The peptone-water culture may then be tested for the presence of indolq by carefully adding a few drops of pure concentrated sulphuric acid. In cases of Asiatic cholera the indole reaction can be obtained as early as eight hours after inoculation. 4. To vibrios that have been isolated, the agglutination, saturation, and fixation tests and Ffeiffer's reaction should be applied, a high-grade authentic cholera-immune serum 472 Manual of Bacteriology being- used. The haemolysis test should also be applied (p. 189). Agglutination tests are, however, somewhat variable with different strains. 5. If the case has lasted any time the agglutination reaction may be applied, testing the patient's serum on a known strain of cholera vibrio, but this is of doubtful value. Spirillum Metchnikovi. Isolated by Gamaleia from the intestinal contents of chickens dead of an infectious gastro-enteritis which occurred in certain parts of Russia. The disease, although resembling chicken cholera in some respects, is quite distinct from the latter. This spirillum forms curved rods and spiral filaments, generally slightly shorter, thicker, and more curved than the Koch spirillum. It is decolorised by Gram's method, and is best stained with weak carbol-fuchsin. It is readily cultivated and is aerobic and facultatively anaerobic. In gelatin plates it forms small whitish colonies, visible within twenty hours, which grow more rapidly than the cholera vibrio, and in two or three days produce marked areas of liquefaction. In a stab-culture in gelatin a whitish granular growth occurs along the line of puncture with liquefaction, much like that of the Koch spirillum, but the rate of growth and the liquefaction are more rapid (Plate XVIII., c). Grown in eggs by Hueppe's method typical appearances are produced." After ten days the white becomes trans- formed into a yellowish limpid liquid, while the yolk, though retaining its form and consistence, is quite black. On surface agar a thick cream-coloured layer develops; on potato the growth is brownish, and milk is coagulated. It grows freely in broth and peptone water, the fluid becoming uniformly turbid, and a slight Finkler-Prior Spirillum 473 film forms on the surface, and these cultures give a marked indole reaction on the addition of sulphuric acid alone, in this respect resembling the Koch spirillum. The S. MetchniJeovi is pathogenic to chickens, pigeons and guinea-pigs, but not to rabbits or mice except in large doses. It is, however, more pathogenic to guinea-pigs than the cholera vibrio. Pigeons are killed by intra-muscular inoculation, and fowls are susceptible to feeding, whereas the cholera vibrio is not fatal to pigeons and fowls under these conditions. It is not agglutinated with cholera- immune serum. Abbott1 isolated a pathogenic spirillum from the Schuylkill River, Philadelphia, which resembles the 8. Metchnikovi closely, and is probably identical with it. Spirillum Finkleri (of Finkler and Prior). Isolated from the stools in certain cases of cholera nostras, but its aDtiological significance is doubtful. It occurs as short, thickish, curved or straight rods, and sometimes as spiral filaments. It is aerobic and facultatively anaerobic, does not form spores, and does not stain by Gram's method. In a gelatin stab-culture a yellowish growth forms with rapid liquefaction (Plate XVIII., d). On agar a thick, slightly brownish, moist layer develops. Serum is rapidly liquefied. On potato a slimy brownish growth occurs even at room temperature. It grows in broth and peptone water, producing a general turbidity. It does not as a rule give the indole reaction with sulphuric acid alone, but the ordinary laboratory cultures after three to four days' growth occasionally give a slight reaction. Jt is stated to be pathogenic to guinea-pigs by intra- peritoneal inoculation. 1 Journ. of Exper. Med., vol. i, 1896, p. 119. 474 Manual of Bacteriology Spirillum tyrogenum. Obtained by Deneke from old cheese, and frequently spoken of as Deneke' s spirillum. It forms curved rods and spiral filaments somewhat closely resembling the Koch spirillum. It grows well on the ordinary culture media at room temperature, bvit development is usually slight or absent at 37° C. In a gelatin stab-culture a yellowish growth occurs with liquefaction, which is much more rapid than that of the Koch spirillum, but less so than than of the Pinkler- Prior spirillum. On agar a thinnish, brownish, somewhat membranous and coherent layer slowly develops at room temperature. On potato a yellowish growth occurs. It is stated to be slightly pathogenic to guinea-pigs by intra- peritoneal inoculation. Spirillum rubrum. A chromogenic spirillum obtained by Koch from the putrefying tissues of a mouse. In a gelatin stab-culture a dark red growth slowly develops along the line of puncture without liquefaction ; at the surface, however, the growth is colourless. In broth at 37° C. it grows freely, producing a general turbidity with a red deposit at the bottom of the tube ; there is no film formation. In such a broth culture large numbers of typical spirillar filaments can be seen, which are thin ami delicate, of varying length, and actively motile. It is non-pathogenic. Vibrios are common in the mouth, and may be met with in the discharge of septic ulcers. Streptothrix Infections 475 CHAPTER XV. STREPTOTHRIX INFECTIONS ACTINOMYCOSIS MYCETOMA LEPTOTITRIX BUCCALIS CLADOTHRIX DICHOTOMA MYCOSIS TONSILLARIS. Streptothrix Infections (Streptothricosis).1 The Streptotrichese ave a group of thread-forming organ- isms showing true, but not dichotomous, branching. Their exact position in the botanical scale is uncertain ; by some they are considered to belong to the higher Schizomycetes, forming a connecting link between these and the Hypho- mycetes ; others place them among the latter, and others mate them a separate and distinct group. The Streptotrichea^ form a filamentous network, or mycelium, the individual threads of Avhich show branching, while their terminal portions undergo segmentation, with the formation of rounded bodies regarded as spores. The mycelial network, unless old, stains by Gram's method, and occasionally pos- sesses " acid-fast " properties.2 The leprosy bacillus apparently sometimes grows as a streptothrix, and the tubercle, glanders, and perhaps diphtheria, bacilli may belong to this group. Pathogenic streptothrix forms are not uncommon, the best known being those causing actinomycosis of the ox and other animals and of man, the white variety of mycetoma, the S. Eppingeri, more or less acid-fast, originally isolated from a cerebral abscess, and also causing a variety of madura foot, S. Nncardii of the ox, and 8. canis of the dog. Doubtless 1 Sco Musgrave, Clegg and Polk, Philippine Journ. of Science, vol. hi, 1908, p. 147 ; Fonlerton, Lancet, 1910, vol. i, p. 551, el seg. - Sec Birt and Lcislunan. Journ. of Hyg., vol. ii. Pt. ii, 1902. 476 Manual of Bacteriology cases of streptothrix infection in man may occasionally be missed, as the clinical chai-acters are those of tuberculosis. Actinomycosis. In man, actinomycosis in its clinical history and pathological lesions closely resembles tuberculosis, with which in the past it was frequently confounded. In cattle, actinomycosis has long been known, but its exact pathology was involved in considerable doubt until the researches of Bollinger in 1876. It forms tumours chiefly affecting the tongue, jaw, face, and throat, and was described under such varied names as wen, scrofula, scirrhus, osteo-sarcoma, cancel-, wooden tongue, etc. The tumours after a time break down and discharge, the tongue often protrudes from the mouth, the saliva drips, and the animal becomes much emaciated. On cutting info a " wooden tongue," or wen, a grating sensation is felt, such as that experienced in cutting a turnip or unripe pear; on examining the section little rounded, yellowish, frequently almost caseating areas will be noticed, resembling old tubercles. On making sections and examining with a low power it is found that these rounded areas are composed of masses of small round-cells, with occa- sionally giant-cells, surrounded by a capsule of fibrous tissue." The growth may be so soft as to be practically purulent, and abscesses varying in size from a pin's head to that of an orange may be present in the affected areas. Like tubercles, the growths may become caseous, calcified, or fibrous. In the growth or in the pus from abscesses, when examined fresh with a low power, yellowish or yellowish-white granules will be found here and there, which may be very minute, or as large as a small pin's head, and are somewhat soft PLATE XIX. a. Actinomycosis boms. Section of tongue. Gram, x 350. b. Mycetoma.. Section of tissue, white variety. Gram. x 350. Toface page 176. Actinomycosis 477 in consistence and on slight pressure flatten out. Examined with a high power, these granules are found to contain round; ovoid, or reniform bodies which have a rosette-like appearance, 'a more or less structureless centre with club-shaped bodies radially arranged around the periphery (Plate XIX., a). These peculiar structures are the cause of the disease, and are the form assumed in the animal body by an organism belonging to the streptothrix group termed the Actinomyces, or Streptothrix hovis, or, from its appearance, the ray fungus. Sections of the diseased tissues show the structure of the organism still better. Gram's method usually C\£XA*C gives good results, and it will generally be Tound that « the following appearances can be observed: Surrounded by the round-cells are the reniform or ovoid bodies, situated at the periphery of which are radially arranged, club-shaped structures deeply stained with the gentian violet, while the central portion is unstained and struc- tureless, or contains granular matter or calcareous particles. Various appearances may be met with in different parts of the section, according as the actino- mycotic nodules are cut through their centre or peri- phery ; when the latter is the case, the clubs are shown in transverse section and appear as closely packed, deeply stained dots. Sometimes, however, in addition to the clubs, the centre of the rosette is occupied by numerous interlacing filaments, also stained by the gentian violet. In man, actinomycosis is often associated with suppuration. If a little of the pus be examined it will probably contain tiny yellowish or sulphur-yellow granules, which, microscopically, are found to consist of tufts of fine tangled filaments, the ends of which may be continued into little swellings or clubs. In teased-up ■478 Manual of Bacteriology specimens, or in sections stained by Gram's method, an appearance is observed very different from that of the bovine variety, viz. tufts of interlacing filaments stained by the gentian violet, but a complete absence of purple clubs (Plate XX., a). Clubs, however, are frequently pi"esent around the periphery of the filamentous tufts in a stunted condition, and they do not usually stain by Gram's method. These clubs are often seen better in fresh specimens of the pus or in unstained sections, or by staining with orange-rubin, or the Ehrlich-Biondi reagent (Plate XX., b). The conditions in cattle and man, at first sight so very different, are thus seen to be similar, a similarity which is further established by the occasional occurrence in cattle of filamentous tufts, staining by Gram's method, within the rosettes, and by the clubs in man now and then taking on the gentian- violet stain. Cultural characters. — The cultivation of the Actino- myces can be perfoi'ined by collecting the pus from a case of the disease in sterilised tubes, and subsequently turning it out into a sterilised capsule and picking out the actinomycotic granules with sterilised needles, planting these on the surface of glycerin agar, and incubating at 37° C. A certain number of the tubes will probably be uncontaminated, but in others a growth of the Micrococcus pyogenes var. aureus or other pyogenic organism, which is not unfrequently asso- ciated with the Actinomyces, may occur. In the uncontaminated tubes a growth begins to appear in a few days in the form of little colonies of a tough membranous consistence, somewhat wrinkled, greyish, and shining, while the agar beneath them becomes stained brownish. The growth increases and the colonies coalesce, forming a brownish, wrinkled, mem- branous expansion, sticking firmly to the agar and PLATE XX. a. Actinomycosis hominis. Section of liver showing- a mycelial mass. Gram, x 500. b. Actinomycosis hominis. Section showing- a ring- of stunted clubs. Gram, x 350. Same material as fig-, a above. To face jxii/e 178. The Actinomyces 479 difficult to remove or break up, while the agar becomes stained brown throughout; later on the membranous growth may become dappled with yellow as though powdered with flowers of sulphur, but occasionally remains whitish. In gelatin little spherical feathery tufts develop, and sink to the bottom as liquefaction progresses. On potato a remarkable growth develops; at first blemish, it afterwards becomes almost black, and is very thick or heaped up with a much wrinkled surface, while later on it has the appearance of being sprinkled with flowers of sulphur (Fig. 49). In broth delicate woolly flocculi form. Films from young agar cultures show masses of tangled fila- ments, which appear to be more or less branched, and stain well with the ordinary anilin dyes and by Gram's method; with the latter the filaments often appear somewhat beaded, but no trace of rosette formation or even of clubs is ever found in cultures (Fig. 50). In pus, especially human, the filaments can sometimes be seen if stained by Gram's method with orange-rubin. Inoculated into the peritoneal cavity of rabbits and guinea- pigs the cultivated organism reproduces the disease, numerous antinomycotic nodules forming in the perito- neum and elsewhere. There is much doubt as to the mode of spread of, and the infection of man with, the disease. It does not seem to be particularly contagious, and diseased and healthy animals are often placed together without bad result; it can, however, be conveyed by '0. Fig. 49. — Actino- myces. Potato cul- ture, three months old. 480 Manual of Bacteriology direct inoculation; for calves inoculated intra-peritoneally with portions of diseased tissues die after some weeks or months, with an abundant development of actino- mycotic nodules, as shown by the experiments of Jone and Ponfick. Crookshank also infected a calf with the material from a human case. Feeding experiments give negative results. The view generally held is that the organism occurs on cereals, straw, or roots, and jrIG so.— Actinomyces. Covor-^lass preparation. Gram, x 750. gains access to the system through slight scratches or wounds in the mucous membrane of the mouth, pharynx, or larynx. In man no source of infection has been traced, though cases have been reported where the disease has occurred after eating grains of barley, etc. The disease is met with not only in cattle, but also m horses and swine. In the last-named animals con- siderable calcification may be present in the nodules, and it may be necessary to decalcify with dilute nitric or hydrochloric acid before the rosettes can be stained. Actinomycosis 481 It is inmortaitt to note that tuberculin may cause a reaction in actinomycosis, similar to that which occurs in tuberculosis, and as Fhe actinomycotic lesions are very like those which are found in the latter disease, mistakes may easily be made, and can only be avoided by a microscopical examination. It is of con- siderable practical importance to distinguish actino- mycosis from tuberculosis, for in many cases of the former, both in man and in animals, iodide of potassium exerts a specific curative action. By some several species of Actinomyces are believed to exist, but Homer Wright* considers that but oue species of micro-organism is the setiological agent, both in man and animals, the A. bovis. " Farcin des bceufs," a disease of cattle occurring in Guadeloupe, and characterised by infection first of the skin and afterwards of the lymphatic glands and viscera, is due to the 8. Nocardii. Clinical Examination. 1. Pour out the pus or discharge into a large capsule or Petri dish so that it forms a thin layer, look for any yellowish or other granules, pick them out with a needle, and place on a clean slide in a drop of 50 per cent, glycerin. If no granules can be found a little of the discharge may be spread on a slide with a drop of 50 per cent, glycerin. Cover with a cover-glass, and apply a little pressure. Examine with a -f-in. objective. If any actinomycotic tufts are present they will be seen as yellowish or pale brownish, spheroidal, ovoid, or remform masses, and with a -J -in. objective will be found to have a radiating structure from the presence of the clubs. 2. Stain cover-glass specimens of the discharge, by Gram's method, with eosin. The actinomycotic tufts will generally be found to consist of little masses of tangled filaments stained 1 Jcjurn. Med. Research, 1905. 31 482 Manual of Bacteriology violet and surrounded by a pink zone which has an indistinct radial Lug structure. N.B. — In most instances the. clubs in Actinomycosis horuinis do not stain by Grain's method. The reverse is the case in Actinomycosis bovis. 3. Sections of actinomycotic tissue are best prepared by the paraffin method. If frozen, the actinomycotic nodules arc very apt to fall out, Sections may be stained by any of the following' ways : O) By Grain's method, with eosin or orange- rubin. (&) With the Ehrlich-Biondi triple stain. Stain for from half an hour to two hours. Place in methylated spirit until the sections appear greenish, theii pass through absolute alcohol and xylol. The clubs are stained yellowish-brown, and are sometimes shown in human cases when unstained by Gram's method. (c) By Plaut's method. Stain in warm carbol-fuchsin for ten minutes, rinse well in water, stain in a, saturated solution of picric acid in methylated spirit for five to ten minutes, rinse well in water, place in 50 per cent, alcohol for ten minutes, pass through absolute alcohol and xylol. (J,) Good preparations may be obtained by staining in Ehrlich's hematoxylin and counter- staining with orange rubin. This may also show the clul is when they are unstained by Gram's method. Madura Disease or Mycetoma. Madura disease, otherwise known as madura toot, mycetoma, or the " fungus disease of India," is a chronic local affection generally attacking the foot, occasionally tin. hand, some- times extending up the leg, but rarely to the trunk, the disease occurs in certain districts in India, and full descrip- tions of it have been given by Vandyke Carter and by Lewis and Cunningham. A " madura " foot appears enlarged, and numerous sinuses with raised mammilated apertures open on the surface (Fig. 51). On making a section into the diseased tissues the bones are found to be more or less carious, while Madura Foot 488 the soft structures are tough and hypertrophied from the occurrence of chronic inflammatory changes. Numerous small cavities are present, sometimes filled by yellowish granules resembling fish-roe, and hence termed " roe-like particles," at others containing black particles of irregular shape, coal-like consistence, and variable size, exceptionally as large as a marble or walnut. The presence of the white or black granules, which may be discharged from the sinuses before mentioned, divides the disease into two classes — the so-called white and black varieties. Lewis and Cunningham - Fig. 51.— A foot affected with madura disease. (White variety.) have also described a third variety, in which the granules are red like cayenne pepper. Vandyke Carter1 first called attention to the similarity between the white variety and actinomycosis in their micro- scopical characters. In sections stained by Cram's method more or less crescentic or reniform bodies are noticeable, divided into wedge-shaped areas, which contain masses of fine filaments stained purple. Surrounding the crescentic bodies is a zone of radially arranged elements, many of which are fan-shaped owing to branching j they are indistinct, as they do not stain with the gentian violet, but they are very sug- > Boniba/y Med. and Phys. Boo., vol. ix, 1886 (new series), p. 80. Also Hewlett, Trans. Path. Soc. Lond., vol. xlii, 1893. 48-t Manual of Bacteriology gestive of the club-shaped structures present in actinomy- cosis, and they resemble the Actinomycosis hominis inasmuch as they do not stain by Gram's method (Plate XVIII., b). By staining with hematoxylin and orange rubin, or with the Ehrlich-Biondi triple stain, here and there in the radial zone well-defined clubs ran be demonstrated. It seems, therefore, that the radial zone is composed of degenerate club-shaped structures, and the disease evidently closely resembles actinomycosis, but seems to be due to a different species of streptothrix. Prom a case of the white variety Boyce1 cultivated a strepto- thrix which differed somewhat from the Actinomyces, as it -row slower, produced no pigment, and on agar formed white raised colonies with radial grooves, not unlike the tiny barnacles found on wooden piles in the sea. Vincent2 also isolated a streptothrix, perhaps identical with that of Boyce, which differed from the Actinomyces in growing feebly in broth, in not liquefying gelatin, and in not being inoculable in the rabbit. He describes it- as forming on glycerin agar umbilicated colonies, first white and afterwards red. Shattock3 suggests that the red, cayenne-pepper-like grains occasionally met with in mycetoma may be due to colonies of the strepto- thrix which have produced their pigment, Microscopically, this organism (Streptothrix madurx) is identical with the Actinomyces. Musgrave and Clegg in a case of the white variety isolated a streptothrix (S.frecri) differing from the 8. madurse, but identical with the 8. Eppingeri. The relation of the black to the white variety of madura disease has been somewhat debated. Kanthack* described the black variety as being probably a late stage of the white. It seems however, that the co-existence of the two conditions in the same specimen is very rare, and Boyce and Surveyor, after a critical examination of a large number of specimens, 1 Hygienische Rundschau, 1894, No. 12. 2 Ann. cle I'Inst. Pasteur, 1893. :< Trans. Path. Sot: Lond., vol. xlix, 1898, p. 294. 4 Journ. Path, and Bact., 1892. 5 Proc. Boy. Soc. Lond., 1893, and Phil. Trans. Roy. Soc. Lend. Mycetoma and Mycosis 485 came to the conclusion that the black variety is a distinct disease, and clue to an organism belonging to the group of the higher fungi, the black particles or masses being the lignified mycelium or " sclerotium " such as is met with in ergot. By planting out the granules from an early case of the black variety Wright succeeded in cultivating a hypho- mycete.1 It formed long branching hyphte, but no spore- bearing organs were produced, and inoculation experiments on animals were negative. It grew on potato as a dense, widely spreading, coherent, velvety membrane, in colour pale brown with white periphery. Small drops of brown, coffee- coloured fluid appeared on the surface, and the potato became brown throughout. On agar the growth formed a meshwork of widely spreading greyish filaments ; in old cultures (also in potato infusion) black hard granules, or " sclerotia," were observed. In broth little balls of radiating filaments developed. Brumpt has distinguished no less than eight varieties of mycetoma. It would seem that there are probably several conditions, both in actinomycosis and in mycetoma, having a general resemblance but differing slightly, and dependent upon different species of streptothrix. Mycosis tonsillaris (Mycosis pharyngis leptothricia) . A chronic disease attacking young adults, resistant to treat- ment, and characterised by the presence of small, white, tough, adherent excrescences on the mucous membrane of the pharynx. Microscopically, the patches consist of collections of epithelial cells and debris, infiltrated with leptothrix filaments and bacteria. The disease, however, seems to be a keratosis, in- fection with the organisms being secondary. But occasionally a true " mycosis " apparently occurs, readily amenable to treatment, and due to a leptothrix.- 1 Joum. Exp. Med., vol. iii, 1898, p. 421. 2 See Gla scfow Mp(fi<. 1 (Tiibliog.). Thrush 491 The botanical position of the organism is uncertain ; by some it is regarded as a true fungus. It is stated to occur on decaying vegetable matter and to be the cause of epizootic lymphangitis in the horse— a disease having a superficial resemblance to farcy — in the pus of which oat-shaped bodies are found, the " crypto- coccus " of Rivolta. Clinical Examination (Pathogenic Yeasts, etc.). The cells cau be well seen in the fresh state in the teased-up tissues mounted in water or glycerin. Curtis recommends staining in carbol-thionine blue, and for sections, picro-carmine. Busse's method for sections is as follows : 1. Hematoxylin solution for fifteen minutes. 2. Wash in distilled water. 3. Counter-stain in weak carbol-fuchsin (1 : 20) for thirty minutes to twenty-four hours. 4. Decolorise in 95 per cent, alcohol for fifteen seconds to one minute. 5. Absolute alcohol, xylol, mount in Canada balsam. Gilchrist recommends treating the sections with 10 per cent, caustic potash solution and examining in 50 per cent, glycerin without staining. Bray ton recommends that small pieces of the tissues should be excised from the growing margin, treated with ether for two to five minutes, macerated in 20 to 30 per cent, caustic potash solution for five to ten minutes, and then examined without staining. Cultures may be rea-dilv obtained, with a little care, preferably on beer-wort gelatin or maltose agar. Thrush. Thrush is due to an organism [Oidium or Monilia albicans) which is usually classed among the Iivpho- mycetes. It forms the whitish patches so frequently 492 Manual of Bacteriology seen on the mucous membrane of the mouth and pharynx in children and in those suffering- from wasting- diseases but a general infection has occasion- ally been produced by it. If one of these patches is removed and teased up, it will be found to consist of masses of tangled mycelial threads with yeast-like budding. The organism can be readily cultivated on all the ordinary laboratory media, and will also grow on slightly acid media such as wort gelatin. It pro- duces whitish, membranous, adherent growths, in which it appears morphologically under two forms — as masses of tangled filaments or hyphae and as yeast-like cells. On acid media the latter exclusively occur, on alkaline the former predominate. It liquefies gelatin, stains by Gram's method, produces an alkaline reaction by the formation of ammonium carbonate, and does not ferment lactose. Inoculated on to a damaged mucous membrane the " thrush" patches appeal', subcutaneously it pro- duces an abscess, and injected into the peritoneum a general infection, followed by death and accompanied by a sero-purulent peritonitis. Clinical examination. — The patches may be teased up and examined in the manner described for the Hyphomycetes (p. 502). Cover-glass preparations may lie stained with carbol-fuchsin or by Gram's method. Fermentation. The yeasts are of great importance in inducing many chemical changes, especially alcoholic fermentation, beer and wine being almost exclusively due to their activity. Taking brewer's yeast, Saccharomyces cerevisite, as a type, the yeast cell is observed to be slightly ovoid in shape, measuring 8 to 9 /z. in diameter. The protoplasm is granular, contains one or more clear spaces or vacuoles, frequently Fermentation VX'> bright, retractile globules of fatty matter, and is surrounded by a cell wall of cellulose. It has been repeatedly stated that a su lcus is present, but this is doubtful. When the yeast-cell is freely supplied with nutriment, reproduction by gemmation proceeds rapidly, and a whole string of cells may form owing to the daughter-cells budding again before they have separated from the parent. When the cell is starved, gemmation ceases, fat-globules and vacuoles increase in number, and the cell may finally become little more than a large vacuole, the protoplasm forming a thin coating over the inside of the cell wall. Within the vacuoles are often seen minute spherical bodies of a doubtful nature in rapid move- ment. In ordinary circumstances endospore formation does not occur, but by deprivation of nutriment, as by growing on a block of plaster-of-Paris, the cells develop spores. First the cell becomes divided by the development of membranes, the so-called "partition- wall formation," into several chambers in which the spores form. In the different yeasts the number and arrangement of the spores vary ; in the 8. cere- visise the typical number is four, arranged close together, three on one plane and one resting on these, like a pyramid of billiard balls. The spores are of considerable importance in the identifica- tion of species of Blastomycetes, as the form of the cells alone and the growths on culture media are not sufficiently dis- tinctive. In fact so little can these two characters be relied upon that in order to isolate in pure cultivation it is necessaiy to grow from a single cell. This can be done by making a miniature plate cultivation with wort- gelatin on a large sterilised cover glass, and, after the layer of gelatin has set, mounting, gelatin downwards, on a large cell on a glass slide. The cover-glass should be divided into small squares by cross- lines etched on the glass and numbered. The preparation is carefully examined with a } or } inch objective, and the positions of single isolated cells are noted. This is not a difficult matter on account of the comparatively large size of the yeast-cells, and their position is determined by the numbered squares on the cover-glass. The preparations are 494 Manual of Bacteriology kepi in a nioisl chamber in a warm place, and when visible colonies have developed, those which are derived from a single cell can be inoculated into tubes or flasks of a suitable culture medium. It is found that the various yeasts form spores in different periods of time when grown under similar conditions, and on this fact is based what is known as the analysis of yeast — a most valuable method, which we owe to Hansen. The chief " diseases " of beers and yeast— i.e. abnormal fermentations giving rise to inferior products — are due to admixture of certain " wild yeasts," as they are termed, with the brewer's yeast, chiefly the 8. ellipsoid* us and 8. pastorianus ; and, in order to detect these " disease " species, the analysis consists in determining at what time ascospores appear. The mode of procedure is as follows : The yeast is sown in a flask of sterile wort, and incubated at 25° C. for twenty-four hours. The yeast revives, and from the deposit of young cells two cultures are made on plaster- of-Paris blocks. These cultures are kept, one at 25° C, the other a1 15° C, and are. examined twice daily. In an un- contanii iiated brewing yeast ascpspores should not lie detected in less than thirty hours iu the culture kept at 25° C, and seventy-two hours in that kept at 15° C. The plaster-of- Paris blocks are sterilised by careful flaming in the Bunsen, and are then placed in sterile glass capsules with lids, con. taining sufficient sterilised water thoroughly to moisten the whole of the blocks; unless this is done no growth occurs. By this method of analysis as little "wild yeast" as one two-hundredth of the whole can be detected. Besides the distinct species of yeasts, there are also a number of varieties employed in brewing, etc., differing but slightly in morphological and cultural characters, yet giving rise to varied products. These varieties may be divided into two groups — the surface, high or top, and the sedimentary, low or bottom, fermentation forms. In this country beer is brewed by fermenting an infusion of malt (" wort ") with yeast, which, during fermentation, rises to the surface, and belongs to the first group ; while the German beers are Yeasts of Fermentation 495 obtained by yeast, whirl' sinks to the bottom, and belongs to the second group. The floating of the yeast, in the high fermentation process seems to be due to the attachment of minute bubbles of carbonic acid gas to the cells, and it has not yet been possible to convert the one form into the other. Characters of some of the more important yeasts.— Hansen divided the important yeasts into groups having the same general characters, and distinguishes the varieties in each by Roman numerals (I., II., etc.) CEKBVisiiE Group.— These are the yeasts producing the normal fermentations resulting in beer, etc. They are round or slightly ovoid cells, and four ascospores are produced. In old cultures long sausage-shaped or even filamentous cells may be met with. 8. cerevism I. and II. — These are bottom fermentation forms in use at the Old Carlsberg Brewery ; the cells of No. II. are rounder and slightly larger than those of No. L, and ascospore formation is more abundant. There is also a top fermentation form described by Hansen (8. cerevisim I. top), which is the yeast employed in the breweries of London and Edinburgh. The yeasts of the cerevisiie group can invert, cane sugar, select dextrose from lsevulose, and ferment maltose, but they cannot ferment lactose, nor decompose malto-dextrin. Pastoeianus Group. — These are wild yeasts. The cells are elongated or sausage- shaped, and six or eight ascospores are produced in a cell. 8. pastorianus I. — A bottom fermentation yeast producing a 1 litter taste in beer. 8. pastoriamis II. — A feeble top fermentation form. Surface cultures on yeast-water gelatin have smooth edges, which clis- guishes it from the next species : ;S. pastorianus III. — A top fermentation form producing turbidity in beer. Surface cultures on yeast-water gelatin have woolly margins. Ellipsoideus Group.— These are wild yeasts. The cells are usually ovoid or pear-shaped, sometimes round, rarely elongated. Manual of Bacteriology Five or six ascosporea are produced in a cell. 8. ellipsoideus I. — A bottom fermentation yeast, occurring on ripe grapes. 8. ellipsoideus II. — A bottom fermentation yeast causing turbidity in beer. Both the -pastor lu ii us and ellipsoideus groups resemble the cerevisise group in their chemical actions, but they are able in addition to decompose malto-dextrin. 8. anomalus is a yeast forming small ovoid cells. It is curious in that the spores are hemispheres with a projecting rim at the base like a bowler hat. Another point in the identification of species of yeasts is the period of formation of films. If the yeast is grown in wort with free access of air and is undisturbed, e. cj. in a beaker capped with filter-paper, after a varying period a film composed of a zoogloea mass of cells appears on the surface. If yeast, or disintegrated yeast-cells, be injected into animals, the blood acquires specific agglutinative properties, agglutinatiug the yeast-cells of the species with which the inoculation has been carried out,1 On the yeasts of fermentation, see Jorgensen, Micro- organisms unit Ferme ntation, 4th ed„ 1911 (C. Griffin & Co.), (full bibliog.) ; Klocker, Fermentation Organisms. Examination of Yeasts. The yeasts can be readily examined in the fresh state in hanging-drop preparations. The cells should be young or they will not be of the typical form ; a two or three days old culture in wort or grape-sugar solution may be used. The yeasts grow well at 20°-30° C. on the ordinary gelatin, agar, and potato, but wort gelatin or agar is to be preferred. The elongated cells, common to all old cultures of yeasts, may be obtained from the films which form on wort cvdtures in wide flasks or beakers after two or three weeks. i See Macfadyen, Oentr.f. Bakt. {V- Abt), xxx, 1901, p. 3(58. Examination of Yeasts 497 In order to stain yeasts, a dilution of the culture should be made in a watch-glass of water, so that the cells may he isolated, as they become distorted if groups form in the preparations. If the yeast has been grown in wort, it is best, before staining, to pour off the fluid from the deposit of cells at the bottom of the flask or test-tube, add some physiological salt solution and shake, then allow the vessel to stand for an hour for the cells to sediment, aud the process of washing may be repeated once. Films may be prepared in the ordinary way and stained for five minutes in Loffler's methylene blue, washed in water, dried, and mounted. Or the films, after air-drying, may be fixed by immersion in equal parts of alcohol and ether for ten minutes, dried in the air, and stained as before. The preparations can also be stained in gentian violet or fuchsin, or by Gram's method. Ascospores may be double stained by preparing films of a sporing culture in the ordinary way, staining with carbol- fuchsin for two minutes, rinsing in water, decolorising in alcohol for half to one minute or longer if necessary, rinsing in water, counter-staining with Loffler's blue for five minutes, washing, drying, and mounting. The spores are red, the remainder of the cells blue. 32 Manual of Bacteriology CHAPTER XVII. 1 THE HYPHOMYCETES — ASPERGILLOSIS — RINGWORM. I.. The Hyphomycetes. The Hyphomycetes are an important group of the true fungi, and include those forms which are commonly known as moulds. They are multicellular individuals, composed of filaments which may be simple or branched, jointed or unjointed. These filaments are termed hyphre, and are formed by the end-to-end union of elongated cells. When the hyphee project upwards into the air they are known as aerial hyphre, and when downwards into the fluid or medium on which the organism is growing as submerged hyphre, and the compact tufts or masses resulting from interlacing hyphre are termed mycelia. A mycelium may form a hard hgnified mass or pseudo-parenchyma, which is known as a sclerotium such as is met with in ergot and in the black variety ot mycetoma. In addition to being multicellular, the higher development of the Hyphomycetes is seen in the specialisation of certain parts for the function of reproduction. Although all the species multiply asexually, in most, if not m all, a sexual method occurs also. Mucor mucedo, Penicilhvv, glaucum, and Aspergillus niger may be taken as types and more fully described. Mucor mucedo. Mucor mucedo, the common white mould which appears like tufts of cotton-wool on various substances, may be obtained bv exposing some moistened bread or horse-dung to the an Mucor Mucedo 499 for a short time, and then keeping it moist under a bell -jar. It consists of a mycelium composed of hyphse, and its fluffy appearance is caused by aerial hyphse. The aerial hyphse are at first of even diameter throughout, but later on their free ends become swollen and ultimately form spherical bodies, which become filled with spores and are known as sporangia. In the early stage the whole organism forms but a single cell, the protoplasm of which is granular and contains vacuoles and numerous small nuclei. As it grows, and the sporangia form, these become separated by a septum from the hyphse, and when it becomes older still the mycelial hyphse may be divided into elongated cells. The development of a sporangium takes place as follows: The distal end of an aerial hypha swells, and immediately below the swollen part a division occurs in the protoplasm aud a cellulose septum is formed, so that the swollen part is separated off from the rest of the hypha, forming the rudimentary sporangium . The sporangium continues to grow, and its protoplasm undergoes multiple fission into numerous ovoid masses, the spores, each of which becomes surrounded with a cellulose capsule. The septum separating the sporangium from the hypha projects upwards into the interior of the sporangium as a club-shaped knob known as the columella. When the sporangium is ripe the slightest touch causes its wall to rupture, so liberating the spores. When placed under favourable conditions the spore germinates, and the buds increase in length and ulti- mately form hyphse. Occasionally a process of conjugation occurs. Two adjacent hyphse send out lateral branches which come in contact with one another, and a septum forms in each, separating a small portion of protoplasm from the rest of the hypha. The apposed walls of the two cells become absorbed and the contents mingle. The mass of protoplasm so formed becomes surrounded with a thick cell-wall, giving rise to an inactive spore-like body known as a zygospore, which under favourable conditions develops like an ordinary spore. Certain Mueors form appreciable amounts of alcohol. 500 Manual of Bacteriology Penicillium glaucum. This forms the bluish-green mouldy patches familiar to everyone. It is by far the commonest of all species, and may be obtained from moist bread or jam or by exposing a gelatin plate to the air for a short time. If the mouldy patch be rubbed a fine greenish dust comes away. This dust consists of myriads of spores ; if a little of it be transferred with a moistened needle to a gelatin plate, or, better still, to a hanging-drop preparation, the growth of the organism can be studied. After two or three days little white specks will be observed, which microscopically are found to consist of tufts of delicate interlacing hyphae ; these, becoming interwoven, ultimately form a tough mycelium. The patches of growth are circular, and the hyphae will be found to radiate from the centre. As the patch increases in size it changes in colour, becoming bluish- green, though the margin for some time still remains white. Prom the upper surface of the mycelium delicate aerial hyphae grow upwards, and from the under surface short submerged ones project downwards. The hyphae are composed of elongated cells arranged end to end, the cell- walls of which consist of cellulose enclosing a more or less vacuolated protoplasm containing several nuclei. The aerial hyphae are unbranched filaments, but as develop- ment proceeds the distal ends branch dichotomously, the branches remaining short and nearly parallel to one another, so that a kind of brush is produced. The ultimate branches are known as sterigmata. The ends of the sterigmata become constricted so that little globular masses, the spores, are formed; this process is repeated until a chain of spores results, 'the proximal one being the youngest. A spore when placed under favourable conditions germinates, a little bud appearing, elongating, and forming a hypha, just as m Mnrur. Brefeld by sowing spores on moist bread, inverting the bread, and examining at intervals, observed a sexual method of reproduction in Penicillium. Two sets of spiral cells develop on a thick hypha, they intertwine, then' contents Aspergillosis probably mingle, and from the union or carpogonium a tube- like hypha develops, which becomes surrounded and enclosed bv branching- hyphse from the mother cell. By further development and thickening- of the cell- walls a sclerotmm forms; it is a hard solid body, yellowish in colour, and resembles a grain of sand, the carpogonium being at the centre. If place! in favourable conditions the sclerotia germinate after some time. Two forms of hyphae are pro- duced, one thick, the other thin ; the latter become much twisted. The thick hyphse become branched, and ultimately a number of pear-shaped bodies are produced. The contents of these bodies then become broken up and form spores ; the bodies are known as asci and the spores as ascospores. From the ascospores the ordinary mycelial form again develops.1 Aspergillus niger. Aspergillus (several varieties) is occasionally met with; it can be recognised by the rounded sporangia with radial markings which are supported on aerial hyphae given off from the mycelium. A process of sexual reproduction occurs very like the one observed in Peiiicilliiim. Aspergillus niger grows well on the ordinary laboratory media, producing on potato a powdery, sooty growth after a time. With the exception of the ringworm and allied, fungi, which produce parasitic skin affections, the Hyphomycetes are not of very great pathological importance. In the ear and nose mucors and aspergilli may be met Avith, but in these situations they are epiphytes rather than parasites, and the same species occur in bronchiectases and pulmonary vomica?. Occa- sionally, however, a pneumono-myeosis has been met with, the mycelium of the fungus ramifying in the lung tissue and setting up irritative and other changes. Pneumono-myeosis " or "pulmonary aspergillosis" is 1 See Brefeld, Quart. Juum. Microscop. Soc, vol. xv, p. 342. 502 Manual of Bacteriology especially a trade disease among- bird-reavers. Grain is taken into the mouth and the bird is fed with it, and in the course of this operation the mould spoi'es are inhaled. The course of the disease is much like chronic bronchitis or pulmonary tuberculosis. The species met with in this condition seems generally to have been the Aspergillus fumigutus. The black variety of madura disease, as already stated (p. 484), is due to a fungus form. Cultivation and Examination. The Hyphomycetcs can be cultivated on the ordinary labora tory media, but wort-agar, or wort-gelatin, potato, bread, or maltose agar is to be preferred. They can be examined by removing a portion of the growth, teasing up gently with needles in a little 50 per cent, alcohol containing a trace of ammonia, removing the surplus fluid with blotting-paper, and mounting in Karrant's solution or in glycerine jelly. If desired, they may be stained by the irrigation method with fuel) sin. In the tissues they may be stained with hematoxylin or methylene blue, or by Gram's or by Weigert's method. Ringworm. The ringworm fungi must probably be included in the group of the Hyphomycetes. Human ringworm, formerly regarded as a single disease, has been proved to comprise at least two affections through the researches of Sabouraud. These two forms are dis- tin For current literature on Trypanosomes and trypanosome diseases see Sleeping Sickness Bureau Bulletin (Royal Society). The Trypanosomata 517 brates, and a blood-sucking invertebrate is ahnosl invariably concerned in their transmission. In the case of each patho- genic trypanosoine, some indigenous wild animal, tolerant to that form, serves as a reservoir from which infection is derived. A trypanosoine has a slender, flexible, flattened body, one extremity of which is pointed, the other passes into a single flagellum. A delicate undulating membrane passes along one edge of the body. The organism lives in the plasma, in which it is actively motile, the flagellated end being usually anterior, and measures 15-30 ^ or even 40-50 fi, in length. The proto- plasm of the organism is .finely granular, and near the centre of the body is a large macronucleus, and generally between it and the non-flagellated end is a smaller micronucleus or blepharoplast. From the latter 'a chromatin filament starts, runs along the free edge of the undulating membrane and passes into the flagellum. Eeproduction takes place by longi- tudinal division, occasionally probably by transverse division, and amoeboid and plasmodial masses may be found in the internal organs and bone-marrow. The trypanosomes have great morphological similarity, which renders them practically indistinguishable by structural characters. They can usually be differentiated into three forms — indifferent, male, and female — which in some cases may all occur together, but only become fully differentiated in an invertebrate host. The males are slender, active, only slightly granular, and with an elongated nucleus ; the females are bulky, sluggish, granular, and have a rounded nucleus ; the indiffereut forms are intermediate. The males usually soon die off unless they conjugate; the indifferents are more hardy, the females most so. The sexual forms conjugate in an invertebrate host, but if the males have died off, both male and female forms may be reproduced from the females by a process of parthenogenesis. Trypanosoma gambiense. In human trypanosomiasis and sleeping-sickness of West and Central Africa, a trypanosome, Tr. Gambienae is the causative agent (Plate XXI. , a). It is usually pre- 518 Manual of Bacteriology sent, though scanty, in the blood, hut can often he found in numbers in the fluid aspirated from the en- larged cervical glands. In the later stages, when cerebral symptoms ensue, it is found in the cerebro- spinal fluid, but scantily, centrif legalisation being neces- sary in order to demonstrate the parasites. The Tr. Gamhiense is pathogenic to monkeys, and to a less extent to white rats and guinea-pigs. Cattle and cer- tain antelopes may act as reservoirs for the parasite. It is conveyed by a tsetse-fly (G. palpalis), possibly by other tsetses. The tsetse (and possibly other biting flies) may rarely convey the disease by direct inoculation. Generally a cycle of development is passed in the tsetse. The stages of this are not known with certainty, but Roubaud has observed multiplication of the parasites in the fly and the development of Herpetomonas forms. According to the observations of Kleiue and Bruce, the flies become infective about thirty-four days after feeding and remain infective for 70-80 days, anl probably for the rest of their lives. In Rhodesia, a human trypanosome has been found which is probably distinct from Tr. gamhiense. The G. palpalis does not occur in the district, and the macronucleus of the parasite is situated between the blepharoplast and the posterior end. In Brazil another human trypanosome-like parasite has been discovered by Chagas (Tr. or ticliizotrypaimm cruzi), and is conveyed by a bug (Conorhimis megistus). Tr. Brucei is the causative parasite of nagana or tsetse-fly disease of horses in Africa. Nagana is met with in large tracts of country in Zululand and West Africa. It especially attacks the ecpiines — horse, mule, and ass — in which it is very fatal. The annuals become anaemic and emaciated, there is a discharge from the eyes and nose, staring coat, swelling of the legs and neck, and fever. The animal dies two Nagana and Surra 519 to six weeks after infection. Oxen are also attacked, but a small proportion recover. The dog, cat, rabbit, guinea-pig, mouse, and rat may be infected by inocula- tion with the fresh blood of a diseased animal. In infected animals the trypanosome is generally abundant in the blood and spleen. The Tr. Brucei can be cul- tivate,!, though with difficulty, on rabbit-blood agar— melted sterile agar cooled to 45° C. + sterile defibrmated rabbit's blood warmed to 45° C, mixed and allowed to solidify in the sloping position {Novy and McNeal). The disease is conveyed through the bites of a tsetse-fly (Glossina morsitans). The trypanosome is believed to live in the big game, from whence it is transmitted to horses entering the infected localities. The blood loses its infective properties usually within twenty-four hours of being withdrawn. Surra attacks horses in Burma, Mauritius, and the Philippines, and is pathogenic to the same animals as nagana, and in the blood a parasite {Tr. Evansi) similar to that in nagana, but more active, was observed by Evans. Surra is probably spread by certain biting- flies belonging to the Tabanidx. The tsetse flies (Glossina) belong to the house-fly order (MusciclBe), and have a general resemblance to a house-fly, but when at rest the wings fold completely over each other. The proboscis is long and straight and the wing venation is characteristic, especially the fourth longitudinal vein, which makes two bends. Instead of laying eggs, the female extrudes a single full-grown larva. They are confined to Africa and Arabia; some sixteen species have been differentiated, and they occur in the vicinity of water on the edge of forest land ("fly-belts"). Tr. equinum attacks horses in South America, causing weakness and paresis of the hindquarters (" mal de caderas"). Cattle are immune, most other animals susceptible. 520 Manual of Bacteriology Tr. Theileri, the largest trypanosome known (50-60 p in length), is found in cattle in South Africa, and is not patho- genic to any other animal. Tr. dimorphum occurs in two forms, large and small, in horses in Africa. Is pathogenic to most animals. Dourine, a venereal disease of the horse met with in North Africa, Spain, and Hungary, is due to the Tr. equijperdum, which is conveyed by direct contact, and is mainly confined to the lesions, being scanty in the blood. It is pathogenic to the ordinary laboratory animals. In rats a non-pathogenic trypanosome was found by Lewis (TV. Lewisi). It is especially met with in sewer-rats, but also occurs in field-rats (Crookshank). It is somewhat shorter and thinner than the Tr. Brucei, and there are other small differences between the two forms. With the exception of rats and mice, and to a less extent guinea-pigs, other animals cannot be infected with the Tr. Lewisi. It may be kept alive for long periods in the blood placed in a refrigerator, whereas the Tr. Brucei soon dies under the same conditions. The two forms do not protect against each other. The Tr. Lewisi is readily cultivated on rabbit-blood agar and is probably transmitted by the rat-flea, in which it seems to penetrate into the epithelial cells of the gut and undergoes a process of multiplication.1 A number of other trypanosomes have been found in the lower animals, birds, fish, reptiles, and amphibians. A large and characteristic one is generally present in the blood of the eel. The trypanosomes are usually agglutinated when mixed with the serum from an infected animal. Hewlett was unable to obtain any toxic or immunising sxibstance from ground-up trypanosomes (Tr. Brucei) 2 Levaditi and Twort3 have found that the filtrate of broth cultures of B. suhtilis is markedly trypanocidal in vitro but not in vivo. i Minchin and Thompson, Brit. Med. Jown., 1911, vol. ii, p. 361. ^ Proc. Boy. Soc. Lond., b. vol. lxxxiv, 1911, p. 56. '•' Comp. Bend. Soc. Biol., vols, lxx and lxxi, 1911. Leishmaniosis 521 Examination of Trypanosomes, etc. The trypanosomes, if numerous, are readily observed in the fresh blood. A very shallow cell may be formed on a slide by ringing with melted paraffin. For stained preparations the Leishman stain (see " Malaria ") or the Heidenhain method (p. 514) may be employed.1 Leishmaniosis. This term is applied to a group of diseases, caused a b C Fig. 56.— a. The Leishman-Donovan body. b. The flagellated form developing- in citrated blood, c. Seven parasites in a large mononuclear leucocyte. (After James, Patton, and Eogers.) by a similar parasite, and widely distributed in tropical and sub-tropical countries of the old and new world.2 In kala-azar or tropical splenomegaly, a disease met with in India, Assam and the East, a small parasite, the Leishman-Donovan body, occurs in large numbers in the spleen and liver, also in the lymphatic glands, lungs, and intestinal submucosa, and in large mono- nuclear leucocytes and endothelial cells. The bodies are small (2-3 /t), round, ovoid, or oat-shaped masses of 1 For a special method of staining, see Plimmer, I'roc. Roy. Soc. Lond , B. vol. lxxix, 1907, p. 102. 5 See Hewlett, Practitioner, 1911, July, p. 109. 522 Manual of Bacteriology protoplasm, apparently encapsuled, and contain two chromatin masses — one large and oval, staining pale red with Leishman's stain, the other small and rod-sliaped, and staining deep red with Leishman (Fig. 56, «). They sometimes occur in masses (Fig. 56, c). Leishman considered the bodies to be degenerate trypanosomes, but the organism is now considered to belong to a distinct genus, and is termed Leiahmania Donovani. Kogers succeeded in cultivating it in citrated blood at 20°-25° C, in which it develops into a flagellated form like Herpetomonas (Fig. 56 , b).1 The parasite is not inoculable into animals, and it is probably transmitted to man by a bug (? a Gonorhinus). The bodies are well shown in smears stained with the Leishman stain. In Oriental sore, or Delhi boil, a parasite practically identical with the Leishman-Donovan body is present, but as the two diseases run a totally different course, it is probably a distinct species {L. tropica). On cultiva- tion it develops a flagellated form. The disease has a seasonal prevalence, and Wenyon suggests that it is conveyed by a mosquito, Stegomyia, sp. In N. Africa Nicolle has observed a Leishmaniosis of children due to another species (L. infantum). It is transmissible to the dog and monkey, and can be cultivated. The disease has recently been found all along the Mediterranean littoral. Spirochaetosis. Diseases caused by infection with spirochaetes. -The spiro- chaetfe are delicate, undulating, or somewhat spirillar, filiform parasites, occurring in the blood of man, mammals, birds, shell-fish, etc. The filaments taper to a point at the ends, are flexible and motile, coiling and uncoiling, are described as 1 Brit. Med. Joum., 1907, vol. i, p. 427 et seq. Spirochaetosis 523 having two nuclear masses, and some possess an undulating membrane, like trypanosomes, but in the smaller forms no definite structure can be made out. They are now generally regarded as protozoa, but some still consider them to be bacteria. Bacterial cells are never pointed, nor do they show the coiling movements of spirochaetes ; motility is produced bvrlagella, which are absent from most spirochaetes (statements to the contrary are due to errors of observation and technique), and periodicity is not exhibited by bacteria. Spirochaetes multiply by longitudinal fission, while fission in bacteria is transverse ; they react in some cases to drugs (e. g. " 606 ") like trypanosomes, are much more sensitive to the action of immune sera than bacteria are, and are transmitted by insects. No spirochaete has yet been cultivated. Schaudinn believed that many so-called spirochaetes may be connected with the trypanosomes. In S. plicatilis he described the presence of a thread-like nucleus and of chromidia, and of an undulating membrane, but flagella are absent. In the little owl minute slender trypanosomes occur ; these later penetrate leucocytes, and develop into relatively very large trypanosome forms (which have been termed Leucocytozoa) . These intra- corpuscular forms are male and female gameto- cytes, the male being smaller and more slender than the female. If taken into the gnat's stomach, the male gameto- cytes give rise to eight microgametes by a process of sporula- tion, which fertilise the macrogamete, and the resulting zygote ultimately forms by sporulation an immense number of spirochaetes. In the case of a Halteridium parasite of the little owl (Athene noctua) , Schaudinn claimed to have shown that it is a stage of a trypanosome (T. noctuse) which is disseminated by the common gnat. His observations have not been con- firmed, and Novy and McNeal believe that Schaudinn was dealing with a double infection of both a trypanosome and a Halteridium, not that one was transformed into the other. S-pirochaeta recurrentis (Ohermeieri) . — Pound in the blood-plasma, not in the corpuscles, in relapsing fever 524 Manual of Bacteriology during the Eebrile paroxysms. It is very slender and delicate, measuring 12 to 16 fi in length, and actively inutile. It is said to be conveyed by the bed-bug or by pediculi — but this is uncertain — and is inoculable into monkeys, and, less readily, into rats. It has not been cultivated (Plate XXI., b). It is probable that the spirochaetes of relapsing fever in different countries are distinct species. Spiruchaeta Duttoni. — Found in the blood-plasma in African relapsing, or tick, fever. It closely resembles the recurrent/is, but is more readily inoculable into rats, mice, and guinea-pigs, and the one does not protect against the other. It is conveyed by a tick, Ornithodoros moubata. Blood spirochaetes have been found in many animals, e. g. cattle (8. Theilcri), mice {8. maris), fowls (8. galli- narum), and geese (S. anserina). Spirochaeta pertenuis. — Castellani1 has found in the yaws (frambcesia) granubmata a delicate spirochaete resembling the 8. pallida of syphilis closely, but even more delicate and difficult to stain than the latter organism, and named the 8. pertenuis. It is present also in the spleen and lymphatic glands in the disease and in inoculated monkeys. Rabbits can be inoculated in the testicle. Some observers have supposed yaws to be a mani- festation of syphilis, but (1) syphilitic patients can be inoculated with yaws ; (2) syphilis may supervene on yaws; (3) Neisser and Castellani have shown that monkeys inoculated with syphilis are not immune to yaws, and vice versa; and (4) Castellani- has shown that the yaws antigen and anti-bodies are distinct from the syphilis antigen and anti-bodies. i Brit. Med. Journ., 1907, vol. ii, p. loll. 3 Journ of Hygiene, vol. vii, 1907, p. 558. PLATE XXI. b. Spirochaeta recurrentis (Obermeieri). Smear of blood. x 1500. To face page 52-1. Syphilis 525 Spirochaetes are also present in the ulcerating granuloma of the pudenda of Guiana (Wise) and Australia, in malignant growths, in ulcers, in the mouth (p. 474), and in Vincent's angina (p. 311). Blood-smears may he stained with Irishman's stain. (On Spirochaetosis, see Nuttall, Journ. Roy. Inst. Public Health, vol. xvi, 1908, p. 449.) Duval aud Todd1 state that multiplication of S. Dnttoni takes place in vitro in a culture medium made with hens' eo-o-s and mouse hlood. Leishmau believes that certain chromatin bodies present in the eggs and nymphs of the ticlcs are the developmental forms of the spirochaetes. Trichomonas vaginalis. — This parasite is found in the acid vaginal mucus in 50 per cent, of those examined. It must not be mistaken for a spermatozoon. It is a pear-shaped body, measuring 12 to 30 fx in length, and from the blunt end three flagella are given off. A much smaller species, T. intestinalis, measuring 4 to 15 fx, has been met with in the intestinal canal of man in conditions associated with diarrhoea. Syphilis. Various bacterial organisms have been described in this disease, e. g. by Lustgarten, Eve and Lingard, Van Niessen, de Lisle and Jullien, etc., and bodies regarded as protozoa by Siegel, de Korte, and others. In March, 1905, Schaudinn2 noted the constant presence of a spiriform organism or spirochaeta (S. pallida, or Tre- ponema or Spironema pallidum) in various lesions in acquired and congenital syphilis. The T. pallidum varies from 6 to 15 u in length, averaging 8-9 /< (Plate 1 Lancet, 1909, vol. i, p. 834. 2 Arbeit, a. d. Kaiser. Gesundheitsamte, xx, 1905. 526 Manual of Bacteriology XXII. , a and h). It is much more attenuated than the majority of spiroehaetes, having a maximum thickness of 0'3 n, has from three to twelve, usually from six to eight, twists, forming a close, regular, and narrow spiral, is actively motile, possessing a single delicate flagellum at either end, and it may have an undulating membrane. It stains feebly and with difficulty. Another spirochaete, the S. refringens, frequently accompanies, and must not be mistaken for, the T. pallidum in ulcerating lesions ; the former is more retractile and coarser, has fewer twists and forms a wider spiral, and stains deeper and more readily than the latter. The T. pallidum is found generally in all primary and secondary lesions of syphilis, e. g. the primary sore and adjacent lymphatic glands, in the papular and roseolar eruptions, in condylomata and mucous patches. It has also occasionally been found in the spleen and blood. In congenital syphilis the T. pallidum is met with in the bullous eruptions, blood, and organs, and is particularly abundant in the spleen and liver (Plate XXIII., a). Tertiary lesions are generally considered to be non- infective, and the T. pallidum is usually difficult to find in them. It has, however, been detected in the peri- pheral portions of gummata and in syphilitic aortitis, and may persist in the body for years after the primary lesion. The T. pallidum is now universally regarded as the specific organism of syphilis, being present not only in the human lesions but in experimental lesions of inocu- lated apes (see below). It must be recognised that spirochaetes are of frequent occurrence in various non- syphilitic ulcerating and other lesions, e. g. m the mouth and in pyorrhoea, in yaws and ulcerating granu- loma (in these two they may be specific forms), m ordinary ulcers and in carcinomatous tumours. Generally PLATE XXII. face pctffe 526. Treponema Pallidum 527 the T. pallidum can be distinguished microscopically from the other species, but care is necessary. The T. -pallidum has not been cultivated in vitro in spite of numerous attempts. By placing material from a rhesus monkey inoculated with syphilis into collodion sacs aridintroducing^them into the peritoneal cavity of another monkey, and examining the contents ol the sacs a month after the operation, a great multi- plication of the organism was found to have taken place.1 Metchnikoff and Roux (also Grunbaum) found that thVchimpanzgeJsjgry susceptible to syphilis, and can realuylbe inoculated from man, the T. pallidum being found in the lesions. Macacus rhesus is also somewhat susceptible, like- wise the M. cynomolgus and the Chinese bonnet monkey, but not the mandril. By several passages through a rhesus monkey the syphilitic virus becomes attenuated, so that in man it produces merely a local lesion.2 Bertarelli3 states that syphilis may also be inoculated on the eye of the rabbit. Attempts by Metchnikoff and Roux to prepare an anti-syphilitic serum by inoculating apes and goats with syphilitic virus jDroved unsuccessful (as did earlier experiments with other animals by Hericourt and Richet). The syphilitic virus as ordinarily introduced into man by sexual intercourse probably takes some hours to become generalised, for Metchnikoff found expeinmentally in apes that if the seat of inoculation were treated with" a calomel ointment up to eighteen hours after inoculation infection was prevented. The syphilitic virus does not pass through a Berke- 1 Levaditi and Mcintosh, Ann. de I'lnst. Pasteur, xxi, 1907. 3 Metchnikoff, Journ. of Prev. Med., 1906, August. 3 Centr.f. Bald. (Orig.), xliii, 1907. Manual of Bacteriology I'cld filtei', and hence is not ultra-microscopic. It is readily destroyed by heat (52" C.) and antiseptics. Treatment with mercury and with " 606" (salvai*san) cause diminution or disappearance of the spirochaetes. Examination for the T. pallidum. 1. 'Examination in fresh preparations. — Scrapings' from the deeper layers of the chancre, etc., may be emulsified in physiological salt solution and examined microscopically, particularly with dark-ground illumination (p. 146) with special condenser. Another useful method is the Indian-ink method. A scraping is obtained from the lesion as above, and the fluid thus obtained is placed on a slide and an equal quantity of ink added. The ordinary commercial Indian inks may be nsed, Griinther Wagner's being particularly good (p. 82). The serum and the ink are then rapidly and thoroughly mixed and smeared over the slide so that a pale bvown colour results. The material dries in a minute or slightly less, and may be examined directly with the oil-immersion lens, or the wet preparation may be covered with a cover-glass and examined. The preparations keep for a considerable time. The pre- paration shows the red blood-cells as large clear circular areas in a brownish-black field, the bacteria and debris present appearing as white rods, dots, etc., and spirochaetes, if present, as clear white spirals (Plate XXII., a). It is particularly important in using this method that in so far as possible serum alone be used, and that a minimal amount of mucous material or fibrin be mixed with the ink. The presence of mucus results in the taking up of a large amount of the colouring matter of the ink, with the result that a smear of the requisite colour and thickness cannot be made. If too much serum is used the albuminous material appears to precipitate the colour from the fluid and a finely granular appearance is seen microscopically, which is practically worth- less for diagnostic purposes. Again, if too much ink is used, PLATE XXI II a. Trepon&ma pallidum. Section of liver of fetus (congenital syphilis) Levaditi's method, x 1500. ''• Coccidium oviforme. Section of rabbit's liver, x 350. To face pctf/e 528. Treponema Pallidum 529 the surface of the smear is increased iu size to such an extent that the task of examining it thoroughly is greatly lengthened. Coles1 notes a useful point in the recognition of the trepo- nema, namely, that if the number of turns of the spiral of the syphilitic spirochaete in the length of the diameter of a red blood-cell be counted, these will be found to be from six to seven. The distance from the top of one spiral io the next is from 1 to T2 /x. The red blood-cells measure about 7'5 /x in diameter, so that on the average six or seven turns will be equal to that of a red blood-cell. The treponema varies in length from 6 to 15 or even more, and consequently contains from six to fourteen and sometimes twenty or more turns of the spiral. This measurement of the length of the spiral is usually possible, and is of the greatest value in identifying the treponema. 2 Stained preparations.— Smears from chancres, etc., may be stained by the Giemsa method. The Griemsa stain is a solution of an azur-blue eosm. compound in equal parts of glycerin (Merck, puriss.) and methyl alcohol (Merck or Kahlbaum) . The smears are fixed for ten minutes in absolute alcohol. The preparations are then stained in a dilute solution of the Griemsa solution for two to twenty-four hours, Avashed in distilled water, dried, and mounted. (The dilute Griemsa is prepared by adding one drop of the Griemsa stain to a cubic centimetre of distilled water, and rendering alkaline with one drop of 001 per cent, potassium carbonate solution.) The pi-eparations may also be stained in the undiluted Griemsa stain for half to six hours. Leishman's solution may also be used. Sections may be stained by Levaditi's method : (1) Fix pieces of tissue about 1 mm. thick in 10 per cent, formalin for twenty-four hours. (2) Wash in water, and harden in 96 per cent, alcohol for twenty-four hours. 1 Brit. Med. Journ., May 8th, 1909. 34 530 Manual of Bacteriology (3) Wasli in distilled water for some minutes (until pieces sink). (4) Place in 3 per cent, silver nitrate solution at 37° C. for three to five days in the dark. (5) Wash in distilled water for some minutes, and then place in the following solution at room temperature for twenty-four to forty-eight hours . Pyrogallic acid 2-4 grm. Formalin 5 c.c. Distilled water 100 c.c (6) Wash in distilled water, dehydrate in absolute alcohol, clear in xylol, embed in paraffin, cut, and mount. The spirochaetes are stained black or brown (Plate XX1IL, a), the tissues yellow. Some have asserted that the spirochaetes seen in the tissues after staining by this method are artifacts or are composed of filaments of elastic tissue.1 3. The Wassermmn reaction or wtig.en test? — This has been largely applied in the diagnosis of syphilitic conditions, and as a, confirmatory test of the presumably syphilitic nature of such conditions as tabes dorsalis and general paralysis of the insane. The test is based on complement-fixation (p. 190). In this method an organism (the " antigen ") fixes its homo- logous immune body, and the complex then tabes up com- plement; this is demonstrated by the use of aJuemoMic system (p. 191). ' As a matter of fact, however, the Wassermann reaction, as it is preferably termed, is apparently not a true antigen reaction,3 for the substance used as antigen is soluble in alcohol, and various non-specific bodies may be employed as antio-en. Moreover, the substances which act as amboceptor and fix the complement are probably lipoid in nature, and are derived by a peculiar degeneration or breaking clown of the i See Saling and Miihlens, Gentr.f. Bakt. (Orig.), xlii and xliii. * One of the best resume's on this subject is by Gomes, Archwos do Institute Bacteriologico Camara Pestana, vol. Hi, Faso. II, p. 143. (In French. Lisbon , 191 1 . Full bibliography ) . * See Emery, Lancet, 1911, vol. i, p. 564. The Wassermann Reaction 531 tissues in syphilis. Again, the reaction is not confined to syphilis : it may also be obtained with the syphilitic " antigen " in trypanosomiasis, yaws, Jeprosy, and the early stage of scarlatina. This does not, however, militate against its value in the diagnosis of syphilitic conditions. In the original method a fresh salt-solution extract of the liver of a syphilitic fetus was used as the " antigen." Levaditi employed a similar extract of the dried and powdered liver. Now an alcoholic extract of the liver is made use of. The test-substance was the blood-serum or cerebrospinal fluid of "tEe" patient, inactivated by heating to 56° C for half an hour. The complement" was guinea-pig serum, and the hemolytic system sheep's corpuscles, and a serum hsemolytic for these corpuscles. This is probably the best method, but is now not generally^ employed on account of the difficulty of obtaining syphilitic/ qj^ fetal liver, and other substances act nearly as well as the) \\ (JL/K antigen, e. g. alcoholic extract of heart-muscle, sodium tauro- 1 CAA^4 ^ cholate, glycocholate, or oleate, lecithin, cholesterin, etc. J At present alcoholic extract of heart-muscle1 (human or guinea-pig) is commonly employed. The complement may be_ that contained in the serum to be tested, and the hsemolytic system may be (1) normal human serum and sheep's cor- puscles,- (2) the serum of a rabbit injected with human corpuscles, and human corpuscles, or (3) serum hsemolytic for sheep's corpuscles, and sheep's corpuscles. The following is the method devised by Emery,3 and has the advantage of comparative simplicity. (a) The hemolytic serum and corpuscles. — A rabbit is in- jected intra-peritoneally two or three times at intervals of a week with 10 c.c. of a 50 per cent, suspension in salt-solution of well-washed human corpuscles. The serum is prepared for use by bleeding the animal to death, and collecting the 1 Heart-muscle is peculiar in that it contains a large amount of lipoid substances. - Fleming's method (Lancet, 1909, vol. i, p. 1512). Normal human serum is generally (not always) hsemolytic for sheep's corpuscles. ;! Lancet, 1910, vol. ii, September 3rd. 532 Manual of Bacteriology blood aseptically. The serum is then standardised with human corpuscles and fresh human serum, so as to determine I he minimal hsemolytic dose under the conditions of the experi- ment. It is then diluted with sterile physiological salt solution in the proportion indicated in the standardisation experiments, pipetted off into sterile vaccine bulbs (|-1 c.c. in each) sealed and heated to 60° C. for half an hour. If there is any doubt as to its sterility the heating should be repeated on two other successive days. The method of standardisation will be seen from an example. The requisites are: (1) A 20 per cent, suspension of human red corpuscles in salt solution ; it should have been re- washed at least three times. (2) Fresh normal human serum. (3) Physiological salt solution. (4) The serum to be tested (" immune serum ") and previously heated to 60° C. for half an hour. The following mixtures were prepared: (a) Suspension 1 vol. + serum 5 vols. + immune serum 1:0 1 vol. (6) „ » » 1:3 » (c) „ » » 1:5 » (d) „ „ „ 1 = 10 » (e) „ » 1 = 20 » (/) „ , » 1 = 50 » Of) » » » 1:100 » The mixtures of the ingredients are made by means of an ordinary Wright's opsonic pipette with a unit mark about 1 inch from the end, and the serum dilutions prepared in a similar way. The mixtures are placed in small test-tubes about | in. internal diameter, well stirred with the pipette, and incubated at 37° C. for one hour. (They are also thoroughly stirred after half an hour.) The following was the result: With a, b, c and d there was complete ha^molvsis ; with e there whs a trace of haemolysis and much agglutination ; with /there was agglutination, but no haemo- lysis • and with g there was partial agglutination. This serum was only diluted 1 in 4 for use, so as to make sure of there being an excess of immune body in the conditions of the experiments. The five volumes of normal serum contain a large excess of complement. The Wassermann Reaction 533 (6) Patient's serum.— The blood for the test is collected in bhe ordinary way from a skin puncture in a Wright's capsule (Fig. 35, d, p. 225) ; about j cubic centimetre of blood is ample, » 'veil if the test has to be repeated. To obtain plent y o£ serum it is advantageous (a) that the blood shall not be allowed to cool after it is collected, and (6) that the clot shall be separated from the sides of the vessel in which it is con- tained, so as to allow of free retraction. To meet the former indication it is a good plan to put the pipette in the incubator as soon as possible after it has been filled ; to meet the latter shake the clot as soon as it has formed towards the curved end of the pipette and back again. (c) The "antigen" used is an alcoholic extract of normal human heart, prepared by grinding up a weighed amount of heart muscle with four times the number of cubic centimetres of absolute alcohol that there are grammes of muscle, allowing the mixture to stand twenty-four hours, and repeating the grinding and maceration for another twenty-four hours, after which it is heated to 60° C. for one hour. It must be quite clear when used, a little being withdrawn from the top layer by means of a pipette. It is diluted with nine times its volume or more, as determined by experiments, of salt solution for use. Before use and occasionally afterwards it is im- portant to test this extract. To be of value it should — (a) give complete absorption of the complement in a known case of late secondary or early tertiary syphilis, even when one volume of fresh normal serum from a healthy person is added ; and (b) used in the conditions of the test about to be described should cause very little absorption (or destruction?) of com- plement in a normal blood. This is tested simply as follows : Two tubes are prepared, of which the first contains one volume of normal serum, four volumes of salt solution, and the second one volume of the same serum and four volumes of the diluted extract. These are incubated together for half an hour, and to each is then added one volume of the immune serum pre- pared as above, and an excess (five volumes is usually enough) of 20 per cent, emulsion of washed human corpuscles. The incubation is continued for one hour, the tubes being stirred 534 Manual of Bacteriology once or twice. At the end of that time they are centri- fugalised, and a definite quantity of the clear blood-stained fluid is pipetted off and examined in a hsemoglobinometer. In a good extract there should be no difference between the two, and this is sometimes the case. Usually there is a slighi difference, which naturally tends to interfere with the accuracy of the react ion. If there be a marked difference between the amounts of haemoglobin liberated in the two tubes, the extract should be discarded or re-tested at a higher dilution. (d) Salt solution. —A 0'85 per cent, solution of sodium chloride in distilled water. (e) The corpuscles. — A 20 per cent, suspension of human corpuscles, washed three times in the salt solution. The apparatus required are (1) a Wright's pipette, some- what wide, i. e. 1 mm. internal diameter, with a 1-unit mark about one inch from the end, and a 4-unit mark ; (2) a series of small test-tubes like Durham's tubes, i. e. about | inch diameter and H to 2 inches long ; (3) an incubator. The ordinary blood-heat incubat >r may be used, but a water-bath is preferable. Emery has modified Hearson's opsonic incu- bator for the purpose, which is very convenient, but a tin filled with water, and having on top of it a piece of paraffined cardboard pierced with holes of appropriate size to contain the small test-tubes will, with a little care in regulating the temperature to 37°-38° C, serve every purpose. All the constituents, antigen solution, sera, salt solution, etc., are contained in larger tubes, and are kept warm in the bath, as well a,s the small tubes for the tests : for each test two small tubes are required. The process is carried out as follows : Prepare a Wright's pipette with a 1-unit and a 4-unit marks. Place in one tube 4 units of salt solution ; this is to serve as a control in order to make sure that the serum to be tested contains sufficient free complement and that the hsemolytic system is in working order. In the other tube 4 units of the diluted extract are placed and the pipette is carefully washed out. One unit of the serum to be tested is now added to each of the two tubes, that containing the salt solution receiving The Wassermann Reaction 535 its addition first, then that containing the extract; this is to a void carrying over a little extract into the control. In each case the fluids are thoroughly mixed by repeatedly sucking them into the pipette and expelling them, and in each case it is advisable to see that the mixture forms a continuous column. The pipette is then rinsed out with salt solution, and the process is repeated with as many sera as are to be tested. The hemolytic system is then prepared. Take one unit of suspension of red corpuscles which have been washed three or four times, and mix them with four times their volume of the prepared rabbit's serum. This will be more than enough to saturate them with amboceptor and also with agglutinin. Place them in the incubator or bath so as to hasten the com- bination of the corpuscles and the antibodies. The final stage of the test consists in the addition of these sensitised and agglutinated corpuscles to the tubes containing the diluted serum. When a bath is used the combination in the extract tube will be complete in five minutes after it has reached 38° C, and almost complete in two and a half minutes Of course if the tubes are incubated in air these times are greater, since the tubes and their contents take an appreciable length of time to become heated to this temperature; but when the two substances are placed in a narrow tube sur- rounded with warm water the combination is very rapid, and if several sera are being tested, the reactions will be complete in the first tubes by the time the mixtures have been made in the last tubes. If haemolysis occurs the reaction is negative, i. e. the serum is not syphilitic; if no haemolysis occurs the reaction is positive, i. e. the serum is syphilitic. The examination of a very large number of cases of syphilis by different observers indicates that the test is of very con- siderable value and diagnostic significance. In conditions such as tabes dorsalis and general paralysis of the insane which on other grounds are generally regarded as due to syphilis, 52 per cent, give the reaction. A positive reaction may be said to show a positive, and probably active, syphilitic 536 Manual of Bacteriology infection, but a negative reaction does not necessarily exclude syphilis. A course of mercurial treatment may render the reaction negative. (4) Porges reaction,.— It syphilitic serum be added to a solution of lecithin or other lipoid substances, in many cases it gives a white precipitate. Normal or non-syphilitic serum gives no precipitate. This has been tried extensively as a substitute for the Wassermann reaction, but it is not so deli- cate. Class IN. — Infusoria (Ciliata). The Infusoria are protozoa the locomotive organs of which consist of cilia, and in which the nuclear apparatus is differen- tiated into a vegetative macronucleus and a generative micro- nucleus. The cytoplasm is enclosed within a cuticle, an oral aperture is present in the form of a slit or pore, and waste matter is extruded by a pore, constant in position, but, as a rule, visible only when in use. A contractile vacuole is generally present. Eeproduction usually takes place by fission, which is preceded by division of the two nuclei, the micronucleus by mitosis, the macronucleus by direct division. The Infusoria are not of much pathological importance, but are common in ponds and ditches, e. g. Paramecium and Vorticella. Balantidium (Paramecium) coli. This is an intestinal parasite of swine, occasionally met with in man in conditions associated with chronic diarrhoea and dysentery. It is somewhat ovoid in shape, the ends being bluntly pointed, is covered witli cilia, measures 65 to 85 n in length, and has a superficial resemblance to the ordinary Paramecium. According to Saville Kent, the Balantidium coli is to be distinguished from the ordinary forms of water paramecia by the following characters : The Bal. coli is somewhat spindle- Balantidium Coli 537 shaped or ovoid, and bluntly pointed at each end, one and a half to twice as long as broad, measuring fa in. to fa in. in length; the paramecium is more cylindrical, four times as loug as broad, measuring fa in. to ¥V in. in length. The oral aperture in Bal, coli is near one extremity (Fig. 57) ; iu Paramecium it is situated at about the middle of the ventral surface. In Bal. coli the cilia round the oral aper- ture are as long again as those over the body generally ; in paramecium the whole of the cilia are of the same length The Bal. coli seems undoubtedly soiriefiines tp Jje_n, cause of dvsen- ^ tery.1 Fig. 57.- -Balantidium coli. Examination of Flagellated and Ciliated Forms. (1) These may be examined fresh in the fluid in which they are present, by mounting on a slide, and covering with a coverglass, one edge of which rests on a bristle to avoid pressure. (2) Permanent mounts may be made by the Heiclenhain method (p. 514). (3) Films may be made in the ordinary way, and stained with weak carbol-f uchsin or Leishman's stain. (The organisms are apt to be distorted.) (4) The following method, devised by Eousselet (Joum. Quelcett Microscop. Clul, 2nd series, vol. vi, no. 36, p. 5, March, 1895) for preserving rotatoria, may be tried. In those forms which are non-contractile, kill by adding a drop of i per cent, osmic acid, wash immediately in water, and preserve in 2| per cent, formalin. Contractile forms maybe first narcotised 1 Strong and Musgrave, Jolms Hopkins Soup. Bull., vol. xii, 1901, p. 31 ; Bureau of Gov. Laboratories, Manila, Bull. 26, 1904. 538 Manual of Bacteriology by adding a drop or two of 2 per rent, cocaine solution, then killed with the osmic and preserved as before. Class IV. — Sporozoa. The sporozoa are exclusively endoparasitic protozoa, the adult lacking organs for locomotion or the capture of food, and multiply by some method of sporulation, often very com- plex. Binary fission is almost unknown in this group. A parasite during the. nutritive or " trophic " phase, when it is absorbing nutriment and growing at the expense of its host, is termed a" trophozoite; when it is mature and ready for sporulation it is termed a sporozoite or schizont. The spores are of various kinds, and may develop outside the body or in a second host. Order. — Coccidiidea. The Coccidiidea, with a single exception, are intra-cellular during the trophic stage, and present a dimorphism or alterna- tion of generations; the one is endogenous and asporular, determining the reproduction of the parasite within the host, the other exogenous and sporular and permitting of infection. GoccuVial Disease of Babbits. This is a disease caused by a sporozoon, the Coccidium oviforme or cuniculi, and often met with in warrens and hutches ; in some of the former as many as 90 per cent, of the animals may be affected. The young animals suffer most, and become infected when they cease to suckle and commence to eat green food, the adult animal as a rule resisting the disease. The affected animals waste, suffer from enteritis, and a large proportion die in from one to three weeks, the condition being known as « wet-snout" among the keepers. The parasites occur in the intestine, bile-ducts, and liver in We numbers. Each parasite is ovoid in shape, measuring Coccidiosis of Rabbits 539 36 in length and 22 /x in breadth, is enclosed in a firm translucent cyst, which encircles a very granular protoplasm. Sometimes this protoplasm becomes condensed so as to form a spherical muss lying l ive within the cyst (Fig. 58, c). In the intestine and bile-ducts the parasites are attached to the epithelial cells, and in the liver, if the animal lives beyond the acute stage, set up some remarkable changes. The affected liver is studded with greyish-white nodules varying in size from a pin's head to a pea. On making sections and examin- Fm. 58. — Coccidium Oviforme of rabbit: a, Coccidium attached to an epithelial cell, h-g, Stages in the life-cycle, h, Free spores. ing them microscopically, it is found that these nodules consist of dilated bile-ducts filled with a much hypertrophied and convoluted mucous membrane, which forms branched projections covered with cubical epithelium, among which the parasites occur in great numbers (Plate XXIII., b). A curious fact is that subcutaneous or intra- venous inoculation, or inocu- lation int.. the liver of a healthy rabbit with the coccidiafrom another rabbit, falls to induce the disease. The coccidium has a complicated life-history, and infection only seems possible in one of the stages. In order to study the life-cycle the parasite must be placed under suitable con- 540 Manual of Bacteriology ditions, and an infusion of rabbits' freces, kept at the ordinary temperature, is perhaps as good a cultivating medium as any, the changes being watched by means of interlamellar films. When the coccidia are observed under these conditions, the first change is apparently the formation of micro- and macro- gametes, fusion of these, and the formation of a zygote or oocyst (Fig. 58, b). The protoplasm of this then condenses so as to form a sphere lying free within the cyst (c), a stage sometimes observed in the animal. The sphere then divides into four smaller spherules (cZ). Each spherule becomes elongated, and again divides into two somewhat crescent- shaped bodies, around each pair of which a new, somewhat spindle-shaped capsule forms (e and /). In this condition the parasite is very resistant, and may remain alive for six months, undergoing no further change unless introduced into another animal. If a young rabbit swallows with its food these crescentic spores, the enclosing capsule is dissolved, and each crescent becomes a rounded amoeboid mass, and this again divides up into many crescentic spores (g and h). These spores are apparently motile, and enter the epithelial cells of the intestine, gall-bladder, and bile-ducts, where a process of growth and differentiation occurs, and the fully developed parasite is ultimately reproduced. Coccidial disease, or, as it is sometimes termed, psoro- spermosis, is occasionally met with in animals, as the sheep, and a wasting disease of young pheasants due to coccidia has been described by McFadyean.1 In man, coccidial disease has been described (but rarely) in the liver, gall-bladder, ureter, etc.2 Eixford and Gilchrist3 described two cases of protozoan infection of the skin and organs, accompanied by great destruction of tissue and ending in death. The organisms were spherical, 7 to 27 in diameter, surrounded by a thick capsule, enclosing granular bioplasm (0. ! mm it is). 1 Journ. Comp. Path, and Therapeut., 1895. 2 Journ. Comp. Path, and Bad., 1898, June, p. 111. 3 Johns Hopkins Hosp. Reps., vol. i, 1896, p. 209. Malaria 541 The Euffer-Plimmer bodies of cancer were at one time believed to be coccidia (p. 561). The term "psorospermosis" has been applied to human infection with coccidium, Sarcosporidia (p. 561), etc. Examination. (1) The coccidial forms are readily examined in the fresh state. The only bodies they are likely to be mistaken for are certain ova. (2) Paraffin sections of rabbit's liver containing coccidia may be stained much in the same way as tuberculous tissues —viz. warm carbol-fuchsin ten minutes, decolorise cautiously in 5 per cent, acid, arid counter-stain in methylene-blue. Sections may also be stained in the Ehrlich-Biondi stain for one to two hours. Order. — Haemosporidia. The general characters of this group are : (1) Life at the expense of the red blood-corpuscles, at least during a portion of the life-cycle. (2) Endogenous multiplication by spores, by which the life-cycle is repeated within the host. (3) Development of a form which becomes free in the plasma, and which is the commencement of a sexual cycle to be completed in a second host. (4) Inoculability, but only from one animal to another of the same species. The group includes the malaria parasite and similar para- sites in mammals and birds, the hsemogregarines, Drejpanidium of the frog, and perhaps the Piroplasmata. Malaria. Malaria is caused by parasitic protozoa, placed in the genus Plasmodium {Hxmamceba), the credit of the discovery of which must be given to Laveran, who Manual of Bacteriology described the parasite as occurring in four phases, viz. (1) spherical bodies, (2) flagellated bodies, (3) crescentic bodies, and (4) segmented or rosette bodies. The parasites cannot be cultivated, but inoculation of healthy individuals with the blood of malarial patients reproduces the disease, and the same structures or parasites are found in the blood of these infected pei'sons. Inoculation experiments on all animals except man have proved negative, and in the latter the inoculation must be intra-venous. In the various forms of malarial fever the parasites have the same general characters, though there are distinct differences between them, by which they can be recognised and the type of fever differentiated. In each there is an endo-corporeal cycle within the host, through which the recurrent attacks are developed; there is also an extra-corporeal cycle of development outside the body of the host, whereby the infection of fresh individuals becomes possible. Each of these cycles needs separate description. If the blood of a malarial patient is examined an hour or two before, or at the very commencement of, the febrile paroxysm, the parasite will be recognised as a pale, ill-defined mass of protoplasm within the red corpuscles, of which a variable proportion are infected, the size of the parasite varying in the different types of fever. When some hours old a variable number of blackish pigment-granules of melanin make their appearance. These subsequently coalesce into smaller groups, and the latter again into one or two larger, more or less centrally disposed, masses. The parasites exhibit more or less amceboid movement, and the melanin granules are frequently in a state of tremor. Later on most of the parasites (now schizonts) become divided into a variable number of segments, The Malaria Parasite 543 which separate and become spherical, the blood-cor- puscle breaks down, the spherical bodies or spores are set free, and a certain number of them, again becoming- attached to red corpuscles, develop into the first stage of the parasite. The melanin grannies and some of the spores are ingested by phagocytes, and the melanin is deposited in the spleen and liver for a time. The parasite, termed a phtsmuducm, or better, an amcebula, contains a vesicular nucleus and a nucleolus, and the melanin granules are present in the surrounding protoplasm. When segmentation occurs, each segment contains a portion of both the nucleolus and the proto- plasm. The maturation of each " brood " of parasites is coincident with a fresh febrile paroxysm. In the sub- tertian (pernicious) forms of malarial fever there exist in the blood for some time after the subsidence of the acute paroxysms well-marked non-motile, crescentic or sausage-shaped bodies, with rounded ends, the so-called " crescentic bodies" or "crescents"; their longer diameter is greater (-f) than that of a red corpuscle, their protoplasm is finely granular, and contains at about the centre several well-marked pigment-granules. In the crescentic forms the extremities of the crescent often appear to be joined by a delicate membrane (Fig. 64, / and j) ; this is the remains of the blood- corpuscle in which the parasite has developed. When a "wet" specimen of malarial blood from a case of pernicious or sub-lertian malaria is kept under observation (p. 555), it not infrequently happens that after a time the so-called flagellated "bodies" make their appearance. These consist of a central proto- plasmic mass attached to which are from one to six delicate flagella measuring 20-30^ in length (Fig. 59, c). The flagella are actively motile and disturb the cor- puscles, but the body itself does not move much. 544 Manual of Bacteriology Frequently one or more of the flagella break away and swim free, remaining active for several hours. The flagellated bodies are never seen in the freshly drawn blood, and Ross has found that flagellation does not occur if the finger be pricked through a spot of vaseline, the blood remaining covered with the film of grease. Careful observation has shown that the flagellated bodies develop from "crescents" in sub- tertian malaria, and from special rounded parasites, Fig. 59.— Development of the malaria parasite in the mosquito. a /,, and c, the male gametocyte; d, e, and/, the female gametocyte j /, fertilisation of the female gametocyte by a miorogamete. (After Ross and Fielding-Ould.) difficult to distinguish from the schizonts, in the benign tertian and quartan fevers. Various theories were held in the past as to the nature of these flagellated bodies. Through the brilliant researches of Ross, which have been confirmed and extended by observers in all parts of the world, it is now known that these cells are sexual elements. The flagellated body represents the male cell or "male gametocyte," the flagella (» gametes ") being analogous to the spermatozoa of higher animals. The female The Malaria Parasite 545 cells or female gametocytes or gametes are non- flagellated, and are fertilised by the entrance of one of the flagella of a male gametocyte. This fertilisation tahesplace in the stomach (middle intestine) of certain species of mosquito, and after fertilisation a series of changes ensues resulting in the forma- tion of spore-like bodies, which are injected when the insect bites its victim, and thus the infection of fresh individuals with the malaria parasite takes place. The first demonstration of the nature of " flagellated bodies" was given by Opie and MacCallum on the Halteridium, a parasite of pigeons (p. 556), and this forms a good example of the value of abstract research to practical medicine (see p. 556). Ross also followed the development of the malaria-like Proteosoma of sparrows, etc., in the mosquito, Culex fatigans. The development of the malaria parasite of man in the mosquito is as follows, according to Ross and Fielding- Ould.1 It is not known what determines whether an amcebula will become a sporocyte or a gametocj^te. When the sexual cells or " gametocytes " are ingested with the blood by the mosquito, they pass into the middle intestine. Within a few minutes the corpuscles enclosing them break down, the parasites are set free, and quickly become spherical or ovoid (Fig. 59, c, e, and /). One or two spherical granules are often attached to the naked parasites, and may represent polar bodies (Fig. 59, c and /). Very soon the male cells become flagellated (Fig. 59, c), and before long the flagella or " microgametes " break away from the parent cell, and by their own motility make their way through the liquor sanguinis. Should one come in contact with a female cell or " macrogamete," it fuses with the latter, uniting with the nucleus (Fig. 59,/), 1 Thompson Yates Laboratories Report, vol. iii, pt. vol. ii, 1901, p. 183. 35 r, m; Manual of Bacteriology fertilisation is completed, and a " travelling vermicule " or " ookinet " results ; this passes into the outer wall of the mosquito's stomach, where it becomes encysted and forms a "zygote" (Fig. 60, a, b). At this period the zygote is about 7-8 /jl in diameter. It' development proceeds, it acquires a distinct capsule and begins to Fig. 60. — Development of the malaria parasite in the mosquito. (After Eoss and Fiekliny-Ould.) grow rapidly, and when mature at the end of a week or more, according to the temperature, is 60 fx in diameter, and projects into the body-cavity of the. insect (Fig. 60, b). Its substance next divides into eight to twelve portions, or " zygotomeres," then each zygotomere becomes a spherical body, or "blastophore (Fig. 60, c), and each blastophore develops upon its The Malaria Parasite 547 surface a number of spindle-shaped, radially disposed bodies, or "zygotoblasts" (Fig. 60, d). When the zygote reaches maturity the blastophores disappear, leaving its capsule packed with large numbers {" thousands ") of free zygotoblasts. The capsule then ruptures, and the zygotoblasts are poured into the Fig. 61.— Diagram of the asexual and sexual cycles of the malaria parasite. body-cavity of the mosquito. The " blasts " measure 12-16 /x in length, taper at each extremity, and possess a central nucleus (Fig. 60, e), and they make their way to all parts of the body of the host, and accumulate in the salivary or poison glands, whence they are discharged by the middle stylet (hypopharynx) of the proboscis, when the insect " bites," into the circulation 548 Manual of Bacteriology of a fresh vertebrate host. Here, presumably, (he blasts become attached to erythrocytes and develop into amcebulae. The diagram1 (Fig. 61) represents in graphic Eorm the asexual and sexual cycles of repro- duction of the malaria parasite. So far as is known, malarial infection is conveyed only through the bite of infected mosquitoes of the sub-family Anophelinae. It has been repeatedly proved that infected mosquitoes convey infection, and thai IF mosquitoes be excluded human beings may live in the most malarious districts without contracting the disease. Mosquitoes (Culicidte) are distinguished from other mos- quito-like insects by the fringe of scales on the wings. The common mosquitoes belong to the sub-family Culiciiue. The Anophelime are usually less abundant (but there is great variation in different districts), and bite mainly at night; the females alone are blood-suckers. Some species breed in natural collections of stagnant, others in slowly running fresh, water well supplied with lowly forms of vegetable life. If the head of a mosquito be examined with a hand-lens, three sets of appendages will be noticed. In the middle is the stout pro- boscis containing the stinging and suctorial apparatus ; situated at the base of this are two palpi, one on either side, and outside these again are two antennae, which are more or less hairy. In Anophelinae, both male and female, the palpi are as long as the proboscis ; in the female Gulex (also in Stegomyia and many other genera ) they are short and stumpy. In Anophelinae the scales on the veins of the wings are usually arranged in alternating light and dark patches, giving a speckled or dappled appearance, different as a rule from anything seen in Gulex. (Some Culices have a similar arrangement, and it is wanting in A. maculipennis and bifur- catusJ) The front or costal margin of the wing in Anophelime is almost always marked with dark blotches. Anopheles, as a 1 This figure is reproduced by permission from Daniels' Laboratory Studies in Tropical Medicine (Bale, Sons &. Danielsson, 1908). Benign Quartan Parasite 549 whole, is a more slender insect than Gulex, and when at rest its body is all in one line, whereas Culex is angular or hump- backed. The important species known to carry malai ia are Anopheles mactilipennis in Europe, 1ST. Africa, and 1ST. America, A. bifurcatus in Europe, Myzomyia funesta and Pyretophorus costalis in Central and W. Africa, and GelUa argyrotarsis in tropical America. Other species, e. g. Myzorhynchus sinensis, Gellia Kochii, and others, are less important carriers. (On Mosquitoes, see Theobald, Brit. Museum Monograph, and Allbutt's System of Med., ed. 2, vol. ii, pt. 2; Giles, Handbook of the Gnats and Mosquitoes ; Daniels, Laboratory Studies in Tropical Medicine, ed. 3, 1908.) There are probably at least three species of malaria parasite 1 occurring in the various types of malarial fever in man, though some authorities {e.g. Laveran) regard the forms as varieties of a single species, and the following are the differential characters between them : (1) Benign quartan fever (Fig. 62). — The quartan parasite [Plasmodium malarias) completes its asexual life-cycle in seventy-two hours ; there are two complete days without an attack, and reckoning the day of the previous attack, an attack occurs every fourth &&y, hence the name " quartan." It commences as a small amoebula, which is feebly motile. It enlarges, becomes pigmented, and motility ceases, the pigment-granules being numerous and coarse. The parasite finally occupies nearly the whole of the corpuscle, which, however, is but little altered (a-d). Towards the end of the apyrexial period the pig- ment collects in the centre, and segmentation takes place with the formation of a symmetrical rosette (e), and afterwards of six to twelve spores (/). The 1 Hewlett, Trans. Fourteenth Internal. Congress of Hygiene, vol ii 1908, p. 141. 550 Manual of Bacteriology quartan parasite does not form crescents, and the flagel- lated bodies (fc), which are rarely seen, are developed from large pigmented parasites. (2) Benign, or spring, tertian fever (Fig. 63- Plate XXIV., a).— The benign tertian parasite {Plasmo- dium vivax) completes its asexual life-cycle in forty- eight hours, an attack occurring every other day or reckoning the day of the previous attack, every "third clay. In the early stage it resembles the quartan, but shows much more active amoeboid movement. The pigment-granules are also finer than in the quartan, Fig. b2. — The quartan parasite: a, b, c, d, amcebnlce; e, sporocyte ; /, free spores ; g, female gametocyte with so- called polar body; h, male gametocyte. (After Eees.) and incessantly change their position. The parasite finally invades (lie whole corpuscle, which becomes enlarged and pale. Enlargement of the corpuscles is a marked feature in the benign tertian infection (a7). Segmentation takes place, but is nn symmetrical {<•), resulting in the formation of a grape-like cluster of twelve to twenty spores (/). As in the quartan, no crescentic bodies are developed, and the gametocytes (g, h) are similar to, but larger than, the quartan (Plate XXIV., 6). (3) The xstivo-antnmnal, malignant, pernicious, or sub-tertian, fevers (Fig. 64). — This parasite (Laver- PLATE XXIV. b, Malaria. Gametocyte of benign tertian parasite. Smear of blood. x 15U0. To face page 560. The Sub-Tertian Parasite 551 aula mcdnruv) (or parasites, for it lias been divided into three species by the Italian observers, viz. the pigmented and the Depigmented quotidian and the Fig. 63.— The benign tertian parasite: a, b, c, J, amosbulse; e, sporocyte ; ,/', free spores ; g, female gametocyte with so- called polar bodies ; h, male gametocyte. (After Rees.) malignant tertian, but this is not generally accepted) is much smaller than the quartan or benign tertian, and when it reaches the stage of multiplication it dis- Fig. 64. — The siib-tertian parasite : a, b, c, amoebnlae ; d, sporo- cyte ; e, free spores; f, g, h, female gametocyte; j, k, I, male gametocyte. (After Rees.) appears from the peripheral blood and collects in the internal organs, spleen, liver, cerebral capillaries, and bone-marrow. It is actively amoeboid, seems to change its position within the corpuscle, and the pigment- 552 Manual of Bacteriology granules are very fine in the young parasites, but early aggregate into large clumps. The fission forms [d, e) are only met with in the internal organs. Multiple infection of the corpuscles may also occur. 'The cor- puscles often suffer severely from t he infect ion, some being shrivelled and spinous, others dark in colour, ""brassy"; they may also he altered or destroyed without being actually invaded by the parasite. It is in this form that the crescentic bodies appear {f, j). These, however, are not met with at the very commence- ment of the attach, but appear in a week or so, and may not disappear until some weeks after the termination of the attack. This parasite is met with in the sub- tertian, or so-called malignant, types of fever, which are characterised by irregularity of the fever, considerable blood destruction, often accompanied by luemoglobin- uria, and cachexia ; coma is another complication in certain instances, probably caused by massing of the parasites in the cerebral capillaries. The cure of malaria by quinine is regarded as being due to a poisonons action on the parasites analogous to that exerted on numerous protozoa, amoebae, for example, being injuriously affected by so little as a 1-50,000 solution of quinine hydrochlorate. No toxin can usually he demonstrated in the blood of those suffering from a malarial attack, but Eosenau and his co-workers have found that the filtered blood, taken when the temperature is rising, produces a malaria- like paroxysm.1 A malaria-like parasite (Plus. KocUi) occurs in apes, in which it produces fever. The nature of Blackwater fever, so called from the presence of hematuria and hemoglobinuria, has given nse to much discussion. By some it is considered to be a dxsease i See Hewlett, loc. cit., p. 144. Diagnosis of Malaria 553 mi generis, of unknown aetiology. By others it is regarded as a form of malaria, cither of an intense type, or in which the kidneys are especially involved, or as due to malarial infection pVus quinine. It may be that under particular conditions, of the nature of which we are at present ignorant, luemoh xins may be set free and cause haemolysis, t he blood- pigment being eliminated by the kidneys.1 Clinical Examination. The blood of malarial patients may be examined either in the unstained or stained condition. Examination in the unstained condition. — The finger or lobe of the ear is pricked, and a droplet of blood taken up on a clean cover-glass, which is then placed upon a slide, so that the droplet of blood spreads out into a thin layer between the two glasses. The cover-glass may then be ringed with oil or vaseline to prevent evaporation. A little practice is required to judge the right quantity of blood. The preparation should be examined with a TVinch oil-immersion lens. Examination in the stained condition. — To prepare stained specimens the finger or ear is pricked as before, and a droplet of blood taken up on a cover-glass ; another cover-glass is applied, and the two are. separated so that each is smeared with a thin film of blood ; several are prepared in this manner. Manson recommends picking up a droplet of blood on an oblong slip of fine clean tissue or cigarette paper. The charged surface of the paper is then applied to a clean glass slide ; in a second or so the blood will have formed a thin film 1 iet ween the slide and the tissue paper. The latter is then withdrawn, leaving a very thin film on the glass, and may be applied to a second slide, and, m like manner, to three or four in succession. A piece of gutta-percha tissue may be similarly used. Or a droplet of blood may be picked up on a slide near one end, and the edge of a. second slide held at an anode of 45° being applied to it, the blood is spread by pushing the second slide over the first one, or the droplet of blood on a 1 See Hewlett, loc. cit., p. 145. 554 Manual of Bacteriology slide may be spread by touching it. with a needle held Hal on the slide and drawn evenly along the surface of the slide. Whatever method is adopted, the film is allowed to dry in the air, arid may then be fixed (not if Irishman's stain is used) by heat, preferably at 110° C. for one hour, as over- heating ruins the preparations. It is much simpler and better to fix in a mixture of equal parts of absolute alcohol and ether for not less than ten minutes, preferably for half an hour; this gives excellent results. In hot countries a saturated solution of corrosive sublimate may be used. The methods detailed at p. 99 may also be employed. As regards staining, this is usually carried out with Irish- man's stain (No. 13, p. 104). The blood films, unfixed, are flooded with a few drops (5-10) of the stain, which is spread by tilting, no attempt being made to check evaporal ion. After half a minute about double the quantity of distilled water is added, allowed to mix with the stain on the film, a nd staining is continued for live, or in some cases for ten, minutes. The film is then washed in distilled water, some of the water is allowed to remain on the 61m for one minute, and it is then dried ami mounted. .Tenner's or ( viemsa's blood-slain may he similarly used. Staining may also be done in a half- saturated aqueous solu- tion of methylene-blue or in Loffler's blue for half an hour, washing in water, and counter- staining with a very weak eosin solution for a few seconds, washing, drying, and mounting. Manson recommends treating the Elms with a very weak acetic acid-two or three drops to the ounce of water to wash out the haemoglobin, and, after washing, staining in the following solution for half a minute : Borax Mparts Methylene-blue . . . .0-5 part Water . ' . • ■ ■ ■ 100 Parts washing, drying, and mounting in xylol balsam. Hematoxylin (Ehrlich's, or Mayer's hamialum ) is preferable for permanent preparations, and in hot countries, where methylene-blue rapidly fades. The preparations may be counter-stained with a weak solution of eosm. Diagnosis of Malaria 555 Eoss recommends for rapid diagnosis the use of thick blood films, from which the haemoglobin is first removed with very dilute acetic acid ; the films are then stained with Irishman's stain, and examined with a |-inch objective. Practice is required for this method. In order to demonstrate the flagellated organisms Manson recommends the following procedure : Thirty or forty strips of thick 1 .lot ting-paper (3 inches by H inch), each having an oblong hole (| inch by f inch) cut lengthways in the centre, are prepared, moistened with water, and laid on a sheet of window glass. A patient is selected in whose blood the crescentic form is plentiful, and a minute droplet of the blood, about the size of a pin's head, is expressed from a prick. A clean slide is then breathed on, and the droplet of blood picked up on it and spread out with a needle so as to cover an area a inch by \ inch. The slide is immediately inverted over a blotting-paper cell and pressed down sufficiently to secure perfect apposition. The rest of the paper cells are similarly covered with blood-charged slides. In from half to three quarters of an hour the slides are removed and dried by gentle warming, and then fixed with absolute alcohol for five minutes. The alcohol is allowed to evaporate, and the films are treated with a few drops of 15 per cent, acetic acid to dissolve out the haemoglobin. The slides are then washed in water and stained with weak carbol fuchsin (20 per cent.) for six to eight hours, washed in water, dried, and mounted. N.B. — Negative results in the examination for the malaria parasite must be accepted with caution unless repeated. A single uudonbted parasite is sufficient to establish the dia- gnosis. Quinine causes the disappearance of the parasite. The parasites in the sub-tertian fever disappear during the apyrexial intervals (except the crescents), and are most likely to be found at the commencement of the attack — i. e. when the temperature is rising. The parasites of the other forms are larger and more obvious during the apyrexial intervals. [For further particulars on Malaria, and on the demonstra- tion of the malaria parasite, see Daniels' Laboratory Studies in Tropical Medicine, 1908.] 556 Manual of Bacteriology Plasmodium prascox. Syn. Proteosma Grassii, Hsemamceba relicla. This parasite (commonly called " proteosoma ") is met with in sparrows and other birds, in which it invades the red I>1 1- eorpuscles, and its structure ami development are practically identical with those of the benign malarial parasites of man. It grows from a minute granule into an a.iiKchoid [ilasmodium, which ultimately segments and forms a rosette. In some specimens of blood flagellated male gametocytes make their appearance, similar to those of malaria, the flagella break away from the main mass, fertilise other non-flagellated or female cells, and a series of changes ensues analogous to those occurring in the malaria parasite (p. 543). The fertilisation and development of the fertilised cell take place in the stomach of a mosquito (Cwlex fatigans), by which the infect ion is transmitted to other birds. Halteridium Danilevvskyi. This is an elongated, curved parasite (also known as Hssmo- proteus or Msemamceba Dunilewslcyi), found in the red corpuscles of certain birds (pigeon, crow, etc.), and embracing the nucleus (Plate XXV., 6). By some it is included among the malaria-like parasites (Plasmodium). At an early Stage it much resembles the proteosoma,, but as it grows it becomes elongated. pigment*granules appear, and are either scattered throughout the protoplasm or collect in two groups, one at each extremity. Finally, the parasite occupies nearly the whole of the corpuscle, dislocating its nucleus. The fully grown parasites may he differentiated into two forms, one of which remains almost completely unstained when treated with methylene-blue, the other staining deeply with this dye (Opie) . When the blood is withd rawn, the corpuscles disintegrate and liberate the contained parasites, winch assume a circular outline, and a certain number become, flagellated. It is only the not, -staining form which becomes flagellated. These two varieties of the parasite are the male and female PLATE XXV. a. Malaria. A tertian " rosette." Smear of blood. x 1500. b. Halteridium Danilewshji. Smear of pigeon's blood, x 1500. To face page 556. The Piroplasmata 557 cells respectively, and the fertilisation of the female cell by a free flagellum has been actually observed by MacCallum.1 It can hardly be doubted that the development of the fertilised cells lakes place in some insect, but the definitive host has not yet been discovered with certainty. The presence of these parasites induces rise of temperature, deposition of melanin, and changes in, and enlargement of, 1 he spleen and liver, analogous to those occurring in malaria in man. The Halterldium parasite, according to Schaudinn, is a stage in the life-cycle of a trypanosome (see p. 523). The Piroplasmata." Syn. Pyrosoma, Babesia. The Piroplasmata form a somewhat anomalous group, but are usually included in the Hsemosporiclia of the Sporozoa. They differ from the Plasmodia in the following respects : absence of pigment, non-fragmenting of the nucleolus, division into two or four only, and frequency of extra-corpus- cular forms. They cause many diseases in animals, are con- veyed by ticks, but are unknown in man. (A piroplasma was described as the causative organism of Bocky Mountain spotted fever by Wilson and Chowning, but the observations appear to be erroneous, see p. 575). The body of a piroplasma is typically pear-shaped (Plate XXVI., a), but rounded and rod forms occur. Two nuclear masses are present, one larger than the other. The developmental cycle in the ticks has not been worked out, but Koch has observed peculiar rayed forms with P. bicje- minum, and Christopher3 various developmental forms with P. canis. Miyajima states that a piroplasma of Japanese cattle (apparently P.parvum) in blood broth develops into typical trypanosome forms.1 1 Journ. Exper. Med., vol. iii, ]898, pp. 79, 103, 117. 2 See Hewlett. Trans. Fourteenth Internal Cong, of Hygiene, Berlin t2 ' ^T' P' '' MinChin in Allbutt's System of Med., ed. 2, vol. ii, 3 Brit. Med. Journ., 1907, vol. i, p. 7G. ' Philippine Journ . of Science, vol. ii, 1908, p. 37. r,r,s Manual of Bacteriology Piroplasma bigeminum. — This is the parasite of the well- known Texas fever of cattle, a disease which is characterised bv fever emaciation, anaemia, hemoglobinuria, and enlarge- ment of the liver and spleen. The disease causes considerable loss among caltle, and is met with in various parts of the world, America, Australia, South Africa, Malaya, the Philippines, the Roman Campagna, Greece, Eoumania, and North Ireland. In the acute type of the disease a small proportion (1-5 per cent.) of the red corpuscles in the peripheral circulation contain pairs of pyriform bodies 2-4 in length and 1-5-2 /x in largest diameter. One end of each body is rounded, and the body gradually tapers to a point at the other end, and the pair lie "close together, their tapering ends directed towards each other. A dark spherical body is present at the rounded end of the parasite. Some of the young parasites exhibit amoeboid movements when the blood is examined on a warm stage. In the internal organs the parasites are more numerous ; in the kidney and liver 10-25 per cent, of the corpuscles contain them, in the heart-muscle 50 per cent. In the mild type 5-50 per cent, of the corpuscles in the circulating blood may be infected at one time or another, and the parasite appears in some cases as a coccus-like body at the periphery of the corpuscle. This appears to become enlarged and spindle-shaped, then to taper in the middle, divide, and so give rise to the pyriform bodies. Occasionally minute free coccoid bodies are seen in the plasma, and at times two to live minute (0'5 ,x) coccoid ceils are present in the red cells. After death the pyriform bodies seem to become spherical or angular. Sexually differentiated gametes are not known with certainty, but flagellated forms have been described. The disease is transmitted through the bites of ticks (Bhipi- cephalus annulate, B. australis). The female tick, after biting an infected ox and sucking its blood, falls off and lays its e^s ; the eggs hatch in two to six weeks' time, and the daughter ticks transmit the disease to other animals through The Haemogregarines 559 their bites.1 The disease may be to some extent controlled by prophylactic measures designed to destroy the ticks, and to prevent infection thereby. A partial immunity is enjoyed after an attack of the disease, but by repeated attacks the immunity may be rendered absolute. By inoculation with the blood of an affected animal in which the fever has subsided, a transient illness in the inoculated animal is produced together with partial immunity, and by a second or third inoculation the immunity may be much increased. The mortality from such a procedure amounts to 3-5 percent.2 P. parvum causes Ehodesian red-water of cattle. It is not directly inoculable, and is conveyed by the tick R. appendi- culaius. P. equi causes biliary fever in horses. P. canis causes epidemic jaundice in dogs (Plate XXVI., a). It is conveyed by the ticks Hsemaphysalis leach i in South Africa, B. sanguineus in India, and Dermaceutor reticulatus in Europe.3 (On Ticks, see Nuttall, Journ. Roy. Inst, of Public Health, vol. xvi, 1908, p. 385.) Hasmogreg'arina. The Htemugregarines (which must be distinguished from the G-regarines) are unpigmented parasites, not amoeboid, typically having an elongated body or vermicide, occurring in the blood, mostly in cold-blooded vertebrates (Plate XXVI., b), but several species have of late been found in mammals (clog, jerboa, palm squirrel), though not in man. In the dog, the parasite (Leucocytozoon canis) occurs as an elongated, curved "i- doubled-up body in the polymorphonuclear leucocytes. It is encapsuled and contains a single granular nucleus. 1 See Smith and Kilborne, Texas or Southern Cattle Fever, United States Dep. Agricult. Bull. No. 1, 1893. 8 See TidsweU, EepoH on Protective Inoculation against Tick Fever, New South Wales, Dep. Pub. Health, vol. i, 1898; vol. ii, 1900. See Nuttall and Graham- Smith, Journ. of Hygiene vol iv to viii 1904-8. 560 Manual of Bacteriology Encystment with sporulation occurs in the bone-marrow, and a sexual development is stated to occur in a tick. Hcemogregarina (Drepanidium, Lanhesterella) rami rum inhabits frogs (liana esculenta), and possesses both an intra- and an extra-corpuscular phase. In the former the parasite occurs as an elongated gregarine-like body within the red corpuscles, which increases in size until its length is 10- 15 fji. ; it then divides into numerous small or a few large gymnospores. In the first case the spores may number fifty, are 8-5 //, in length, occur in May or June, and are exclusively within the erythrocytes ; in the latter case the spores measure 5-8 \i in length, are five to fifteen in number, and develop within cells in the blood-forming organs. The extra-corpuscular phase, commencing within the corpuscles, ends in an elongated orga nism possessing a vermicular move- ment, and free in the plasma. Similar parasites are frecpient in the lower vertebrates, e.g. snakes (Plate XXVI., I). Order. — Myxosporidia. In this group the trophozoite is amoeboid, and the species are almost exclusively parasites of fish, in the young stage being intra-cellular (" fish psorosperms "). Order. — Microsporidia. The Microsporidia are cell parasites of invertebrates, especially anthropods, and the trophozoite is more or less amoeboid. Nosema bombycis causes pebrine, a disease of silkworms, which is of considerable importance commercially, for the silk industry in France was once threatened with extinction owing to its ravages. The worms do not grow normally, cease to eat, and die, or form abnormal pupse. Within the body of the affected worms a large number of roundish, highly refractile corpuscles are found. Pasteur ascertained that the disease was propagated by healthy worms eating with their food the excreta of infected ones. The moths were thus PLATE XXVI. b. Hseniogregarine of cobra. Smear of blood, x 1000. Tofaae paije 560. Pebrine infected, and laid infected eggs. Bj allowing each moth to lay its eggs separately, and subsequently examining the body of the moth microscopically, he was able to separate the healthy from the diseased, and the eggs of the former were kept, while those of the latter were destroyed. According to Pfeiffer,1 when the worms eat the excreta containing the corpuscles mentioned above, these lose their capsule and form large amoeboid masses which penetrate the muscles and blood- corpuscles. The amoeboid masses then become encapsuled and are yellow and granular. Later on the bright roundish corpuscles form within them. Another disease of silkworms is known as flacherie, but is due to a bacterium, Micrococcus bombycis. It is contagious, and can be transmitted by inoculation. Order.— Sarcosporidia. The parasites belonging to this order are not thoroughly worked out. They complete their life-history in the substance of striated muscular fibres : such are the well-known Miescher's corpuscles. Few instances of this class of parasite are recorded in man, but it occurs in the monkey2 and also in the ox. T. Smith 3 describes the characters and development of a species found in mice. 1 Zeitschr.f. Hyg., vol. iii, 1888, p. 3. 2 De Korte, Journ. of Hygiene, vol. v, 1905, p. 451. :i Journ. Exper. Med., vol. vi, No. 1, 1901, p. 1. 30 562 Manual of Bacteriology CHAPTER XIX. Scarlet Fever— Hydrophobia— Infantile Paralysis— Typhus Fever- Yellow Fever— Dengue — Phlebotomus Fever— Vaccinia and Variola— Malignant Disease. Scarlet Fever. Vaeious organisms have been described in scarlet fever— a bacillus by Eddington, a streptococcus by Frankel and Freudenberg, protozoa by Mallory and others. The disease may be milk-borne, and in the historic Hendon outbreak a streptococcus was claimed by Klein to be the specific infective agent, but the researches of Crookshank and others seem to disprove this. . In 1885 an epidemic of scarlet fever occurred m Marylebone, and was traced to infection conveyed by milk supplied from a farm at Hendon. The infection could not be traced to any human source, and it was therefore concluded that the cows themselves were affected with scarlet fever, and infected the milk. A vesicular eruption was found on the udders and teats of the cows, and this was regarded as the local mani- festation of bovine scarlatina. From the vesicles and crusts Klein isolated a streptococcus which, although closely resembling the Streptococcus pyogenes (as then known), differed slightly from it; on inoculation into calves it produced death, with lesions of the kidney resembling those of the human disease. Klein also Scarlet Fever :,(;:; isolated the same streptococcus in five out of eleven cases of tlie disease in man. The conclusions which Klein and Power came to were, therefore, that scarlet fever is communicable to, and may exist in cows, the milk thereby becoming infected and conveying the disease to man, and that a sti-eptococcus is the specific infective agent. The Hendon outbreak was reinvestigated by Axe and Crookshank.1 Axe found that, so far from there being no source of human infection, case^_ of scarlet TJeyer had occurred near the dairy within a short time of the outbreak, and the eruptive disease of the cow was shown by Crookshank to be cowpox, while the so- called streptococcus of scarlet fever he regarded as a variety of the 8. pyogenes. The existence of bovine ^scarlet fever is entirely discredited by the veterinary profession, both here and on the Continent. In 1901) a milk-borne epidemic occurred in certain districts in London and Surrey, and was traced to milk derived from one farm. The outbreak was investigated and reported on by Hamer and Jones, who again traced it to infection of the cons. Hunting2 reviews the evidence and shows how little there is to support this conclusion, as there is no doubt that the family of one of the employees on the farm were suffering from scarlatina. Scarlatina seems to be inoculable on the chimpanzee and some of the lower apes. Gordon' reinvestigated the bacteriology of scarlatina with special reference to the Streptococcus scarlatina or con- glomeratus of Klein. He found that this organism differs 1 On the Hendon outbreak, see Trans. Path. Sac. Land., 1888 (Refs ) " Journ. Roy. Sanitary Inst., vol. xxxii, 1911, p. 62. a (a) Rep. Med. Off. Loc. Gov. Board for 1898-99, p. 480 • (b) ibid for 1899-1900, p. 385. «>Wtottt. 504 Manual of Bacteriology distinctly in its cultural characters from other varieties of streptococci, and that it occurs constantly in the mucous secretion on the surface of the tonsils and fauces and in the nasal, but not in the aural, discharge in scarlatina. It is also present in a somewhat modified form in the blood and tissues post mortem. It was not found in four non- scarlatinal throats examined. Gordon concluded, therefore, that the h scarlatina or conglomerate is the " specialised and essential ao-ent" of scarlatina. It is pathogenic to mice. °Cumpstoni investigated the biological characters of 101 streptococci isolated from scarlet fever, applying Gordon s tests (p. 248). The majority corresponded with the 8. longus t7Baginsky and Sommerfeld, Class and Jaques also isolated streptococcal organisms in scarlatina, but they possessed no very distinctive cultural characters. _ _ It seems very doubtful if streptococci are the biological agents in scarlet fever ; they are probably secondary mfechve aLts. It is remarkable how frequently diphtheria com- rjlieates scarlatina. . . Mallory detected small bodies, 2-7 ^ in diameter, staining delTca elv but sharply with methylene-blue, and occurring n and between the epiLlial cells of the epidermis and m the ^ph-vessels and spaces of the cerium. He regards hese as protozoa, but others" consider them to be degenerated leuco- CyThe blood in the early stages of scarlatina gives the Wassermann reaction (p. 531). Hydrophobia.2 Hydrophobia attaching man is invariably contracted through the bite of an animal affected with the disease. most frequent in the dog, but the cat, wolf, and i Joum. ofHyg.,™l. 1907'P15f9No 2 1901 p. 260; Marie, La * See Stanley, Joum of Hyg., vol. i.No 2, 1901, P Sage, 1901; Scientific Memoir, Gov. ,/ In^a, Nos. 30 and . Hydrophobia 5(v> are also subject to it, and other animals can be infected by inoculation. The disease may assume two forms — the raging or the paralytic. The latter is not met with in man, unless certain rare forms of acute ascending paralysis [e. g. Landry's) be manifestations of it. In the dog either may occur, but in rodents the paralytic form is almost always the one assumed. In man the incubation period is very variable ; it is never less than about twenty days, and possibly may be as long as two years, or even more ; the average seems to be about ten weeks. In the rabbit, after inoculation from the dog, the incubation period is about two to three weeks. The virus resides in the central nervous system, as was shown by Pasteur. Inoculation with emulsions prepared from the medulla and with the saliva conveys the disease, but the filtered emulsions are usually inactive, and the other tissues and fluids of the body, excepting the lacrimals and suprarenals, are non- infective. Remlinger 1 has found that after very complete trituration the virus may pass through a porcelain No micro-organism has been demonstrated with certainty in rabies. Negri has described the constant presence of structures — the Negri bodies — particularly in the grey matter of the hippocampus major, which he regards as protozoa. They are of varying size, apparently encapsnled, taking a homogeneous purplish colour in smears stained with eosin and methylene-blue, the smallest spherical and structureless, larger ones with a central granule or nucleus, the largest, round, ovoid or elongated, containing several (as many as eight) granules (Fig. 05). They occur abundantly in 1 Ball, de Vlnst. Pasteur, iv, 1904, p. 342. 566 Manual of Bacteriology animals suffering from chronic rabies, but in the acute typo are scanty, though still to be found; in ''fixed virus " (p. 567) they are very small. So constantly are the Negri bodies present in rabies, and absent in non- rabic conditions, that their presence or absence forms a rapid and simple means of diagnosis.1 Babes states that the virus is destroyed at a tempera- ture of 60° C, but the medulla and other infective i Fig. (15. —Smear from hippocampus major of rabid dog: n, nucleus of nerve-cell; b, b, the Negri bodies (eosin and methylene-blue). (After Williams and Lowden.) material retain their virulence for months in glycerin. He has described certain lesions present in the medulla in eases of rabies, the so-called rabic tubercles. These consist of an invasion of the peri- ganglionic spaces by an accumulation of round-cells, with degeneration of the cells of the bulbar nuclei. Van Gehuchten has described as pathognomonic of rabies certain lesions in the sympathetic and cerebro- spinal ganglia, especially those of the pneumo-gastrie. 1 See Williams and Lowden, Journ. Infect. Diseases, vol. iii, 1906, p. 452. Hydrophobia 567 These ganglia consist normally of a supporting tissue holding in its meshes large ganglionic cells with distinct well-staining nuclei, each being enclosed in a capsule lined with endothelium. The changes in rabies consist in atrophy of the ganglionic cells, which become shrunken and no longer fill the enclosing capsule, and their nuclei at the same time become ill-defined and stain badly. A number of new-formed cells also appear within the ganglionic capsules. Ravenel and McCarthy studied twenty-eight cases of rabies in various animals, and consider that these capsular and cellular changes in the ganglia, taken in conjunction with the clinical manifestations, afford a rapid and trustworthy means of diagnosis of rabies, but that the absence of these changes does not necessarily imply that rabies is not present. They also consider that the rabic tubercle of Babes is present sufficiently often to furnish valuable assistance in cases where the central nervous system only is obtainable.1 Pasteur showed that the virus can be attenuated by desiccating the infective nerve matter, and in this way was able to prepare a vaccine which protects animals from otherwise fatal doses of the virus. Advancing a step further, he used his vaccines to treat individuals who had been bitten by rabid animals, but in whom the symptoms had not yet developed, and so inaugurated the present system of anti-rabic inoculation as carried out at the Pasteur and other institutes. To prepare the anti-rabic vaccines, a rabbit is inoculated subdurally with an emulsion made from the medulla of a rabid dog. When the animal dies, a second rabbit is similarly inoculated from the first, and the passage through rabbits is continued until a "fixed" 1 See Journ. Compar. Pathol, and Therapeut., vol. xiv, pt. i, 1901 p. 37. :,t;s Manual of Bacteriology virus is obtained, with which the first symptoms appear on the seventh or eighth clay, and which kills with certainty in about ten days. This having been attained, two or three rabbits are inoculated subdurally every day, so that there is a daily supply of animals dead of the disease. The spinal cord is removed with aseptic precautions, cut into convenient segments, and suspended in bell jars containing a layer of caustic potash at the bottom, which serves to desiccate them. The jars arc dated, and preserved in glass cases in a dark room, kept at a constant temperature of about 23 C. In Paris the vaccine fluids are prepared by triturating portions of the dried cords in sterile broth, so as to form an emulsion — 1 cm. of cord in 5 c.c. of sterile broth, of which 1 c.c. (i. e. 2 mm. of cord) forms a single dose. At the commencement of treatment the cords which have been dried for fourteen clays are used, at the end of treatment those Avhich have been dried for only three days ; the latter are much more virulent, and would communicate the disease but for the previous treatment. The rabbits employed should all be of the same weight (2i kilogrammes in Paris) ; if the rabbits are small, a slightly shorter period of desiccation of the cords would be necessary. The treatment varies in duration according to the severity of the case, which is gauged by the number and situation of the bites and by the species of animal. Bites on exposed parts are regarded as much more serious than those through clothing, and on the face, where efficient treatment is difficult, than on the hands, and wolf-bites than dog-bites. The doses are injected snbcutaneously in the flank, and do not produce much constitutional disturbance. At first there is a feeling of lassitude, and considerable muscular tenderness at the seat of inoculation, which Anti-Rabic Inoculation :,r>: i later on passes oft*. At Lille, where there are only a few eases under treatment at a time, the cords, after drying- for the requisite period, are placed in pure sterile glycerin. In this they retain their virulence unimpaired for about a month. This method docs away with the necessity for the daily inoculation of rabbits, a rabbit being inoculated occasionally as required. The system of dosage employed at the various anti-rabic stations differs somewhat ; the following is that employed at Lille, 2 mm. of cord being emulsified in 5 c.c. of sterile broth, or physiological salt solution : OllDINAltY TREATMENT. Day of Dnys of desicca- treatinent. tion of cord. 1 (two injections) . 14 and 13 2 Si 3 ■i ■ „ 5 6 . 7 8 9 (two injections) 10 n 12 . 12 and II 10 and 9 8 and 7 H 5 4 3 9 and 8 7 and o' 5 4 OlIDINAET TlfE ATMENT. Day of Days of desicca- treatment. tion of cord, 13 . . .3 14 (two injections) . 9 and 8 15 „ .7 and b' 16 . . . .5 17 . . . .4 18 . . , .3 For Seveue Bites, in Addition. 19 (two injections) . 7 and 6 20 21 5 and 4 3 At Buda-Pestli a dilution method has been employed ; instead of drying the cords, an emulsion is made with the fresh cord, and this emulsion is considerably diluted for the earlier doses, dilutions of i in 10,000 to 1 in 6000, corresponding to cords dried for from fourteen to eight days. Other systems of inoculation have also been proposed. Undoubtedly the Pasteur inoculations will protect animals from rabies, the duration of immunity after vaccination in the dog being at least three years. In man the efficacy of the treatment can only be judged by statistics. The mortality after bites by supposed 570 Manual of Bacteriology rabid animals is variously stated, the most favourable being about 16 per cent. (Leblanc). At the Pasteur Institute, Paris, among 2730 cases treated in which the animal which inflicted the bites was proved to be rabid by inoculation experiments, nineteen deaths occurred — a mortality of 0'7 per cent. In 1905, 727 cases were treated, with three deaths; in 1906, 772 cases, with one death ; in 1907, 786 cases, with three deaths, being mortalities of 0"41, 0T3, and 0'38 per cent, respectively. The failure of the treatment may be due to two causes: (1) delay in its commencement, and (2) a short incubation period. The principle of the treatment probably depends upon the long incubation period of the disease, owing to which it is possible to forestall the disease, and to immunise the body by the inocula- tions before *iEs~ onset. If, unfortunately, the infective material should be very virulent, and the incubation period thereby reduced to the lower limit, it may be impossible to do this before the onset of the disease, and the same is the case if the commencement of the treatment be delayed. Pasteur's system of inoculation is useless when the disease has declared itself. By vaccinating animals by the Pasteur method by a long series of injections, and with the most virulent material, the blood-serum acquires <£ anti-" properties, and this " anti-rabic" serum is said to be of service ill the treatment olThe declared disease. Variations from typical rabies have been described both in animals and in man under such names as " chronic rabies," « abortive rabies," etc. Harvey, Carter, and Acton^ describe a spontaneous disease in dogs due to a general infection with B pyocyaneus, which closely simulates rabies. By subdural inoculation the disease is reproduced in rabbits, with paresis i Veterinary Record, July 22nd, 1911, p. 57. Diagnosis of Rabies 571 of the hind legs and death in from sixteen to twenty-one days. The Negri bodies are absent, the course of the disease differs somewhat from rabies, and the B. pyocyaneus can be isolated from the brain and blood. Diagnosis of Rabies. In a case of suspected rabies in a dog the animal should not be killed immediately, but should be kept under observa- tion until it dies, or for three or four weeks, and then killed. 1. Moderately thin smears on slides are made from (a) the cortex in the region of the fissure of Rolando (the crucial sulcus in the dog), (fe) the hippocampus major, (c) the cerebellum. They are dried in the air, fixed for five minutes in methyl alcohol, and then stained in weak Giemsa (1 drop stain, 1 c.c. distilled water ; with 1 drop of 1 per cent, potassium carbonate solution to every 10 c.c. of the dilute stain) for three hours. The stained films are then washed in running tap-water for one to three minutes, dried with filter- paper, and examined for the Negri bodies. Or the moist films may be fixed in methyl alcohol, and without drying stained for one minute in a mixture of 10 c.c. distilled water, 3 drops of a saturated alcoholic solution of basic fuchsin, and 2 c.c. of Loffler's methylene blue. Eosin- methylene-blue mixtures may also be used. The cytoplasm of the bodies stains orange, pink, red, or magenta, the central nuclei are granular, and appear bluish or purplish. 2- If_..t,ie Negri bodies cannot be detected inoculation should be performed. The brain should be removed as soon as possible, and if it cannot be manipulated immediately, should be placed in sterile glycerin. From the middle of the floor of the fourth ventricle a small piece about the size of a pea is removed ; this is triturated and thoroughly emulsified in a sterile watch-glass by means of a sterile glass rod with a bulbous end, a little sterile broth being used to make the emulsion, and sufficient being added to measure about 10 c.c. The hair on the head of a good-sized rabbit is cut close, the t 572 Manual of Bacteriology animal is anaesthetised with ether, the skin on the scalp reflected and a trephine hole made through the skull. The centre of the trephine hole should be in the middle line, and on the line drawn between the posterior corners of the eyes, the diameter of the trephine being about T\ inch. A little of the emulsion is drawn up in a small syringe, having a fine needle, and two or three drops are injected beneath the dura mater. The operation is carried out with antiseptic precautions, the wound closed, and a little wool and collodion dressing applied. If the material injected be from a rabid animal, the hist symptoms will be noticed in fr.un ten to fourteen days. The inoculated animal loses control over its hind legs and throws them about peculiarly when running. This increases, and in another day or so the animal is apt to fall when running, and in another day or two the hinder extremities become paralytic, and the animal is unable to move, and dies shortly. The onset of symptoms is hardly ever delayed beyond twenty-one davs. Van Gehuchten's method— The ganglion is placed m abso- lute alcohol for twelve hours, the alcohol being changed once ; it is then embedded, and sections are cut. These are stained for five minutes in Nissl's methylene-blue and mounted. Or the material may be fixed in 10 per cent, formalin before staining. The capsular changes are best shown by staining with hsematoxylin and eosm. Babes' method.— A piece of the medulla or cord is hardened in alcohol and stained with anilin red, and sections are prepared. Infantile Paralysis.1 Infantile paralysis or acute anterior poliomyelitis occurs sporadically and also in epidemics. Various organisms have been described in this disease, but recent researches, particularly by Levaditi, Land- . See Levaditi, Journ. Boy. Inst, of PulUc Health, vol. ^ 1911, . PP- * and 65 (Bibliog.); Flexner and others, Journ. Amer. Med. Assoc., 1910-1911. Infantile Paralysis 573 steiner, and Flexner, have proved that the virus is a filter- passer. Injection of emulsions of the affected cord into the brain, spinal cord, peritoneal cavity, and blood-stream of monkeys reproduces the disease with the same clinical and pathological features as in man. The disease can be carried on from monkey to monkey by inoculation, but does not seem to be transmissible to other animals. The salivary and some of the lymphatic glands contain the virus. Flexner has observed a case of spontaneous infection in the monkey, and found that the naso-pharyngeal mucosa is infective, so that this is probably the channel of infection in man. Human cerebro-spinal fluid was not found infective in some instances, but monkey cerebro- spinal'fluid is infective (infectivity in this case may depend on the stage of the disease). Human ascitic fluid inoculated with the filtered fluid from emulsions of cord became turbid, but no organism could be detected microscopically. Monkeys which have recovered from an attack are refractory to inocula- tion. A certain degree of active immunity may be established by subcutaneous injection of the virus. The serum of immunised and recovered animals possesses considerable neutralising power for the virus. Attempts are now being made to prepare a curative serum. Some cases of the acute ascending pai'alysis of Landry may be forms of this disease (see also p. 565). Buzzard, from a case of the latter disease, isolated a coccus which induced a rapidly spreading palsy on subdural inoculation into rabbits. 574 Manual of Bacteriology Typhus Fever.1 Many organisms have been described in tin's disease. Nicolle, in Tunis, lias found that typhus fever of man is communicable to the chimpanzee by inoculation and from the anthropoid to the Chinese bonnet monkey. Nicolle and Conseil have found it possible directly to infect the Macacus sinicus and rhesus monkeys from human cases. Nicolle ascertained that the blood is virulent from the commencement of infection and continues so until the day after the temperature becomes normal. The dog aud rat are quite refractory. The disease appears to be transmitted by the body-louse (P. vestimenti) , not by the flea, as suggested by Matthew Hay. The blood from a mild case does not produce immunity on injection, nor does a mild attack itself induce any appreciable immunity. On the other hand a severe infection induces considerable immunity. Nicolle and Jasggy have not detected any microbe in affected persons or animals. As the polymorphonuclear leucocytes suffer considerably during the attack, under- going fragmentation of the nucleus and necrosis, it is suggested that the micro-organism may be intra- leucocytic. Other researches have been carried out in America on the typhus of Mexico, known locally as "Tabardillo." Anderson and Goldberger first showed that the Macacus rhesus monkey could be directly infected with Mexican typhus. Ricketts and Wilder have confirmed this, and find that typhus blood is not infective if passed through a Berkefeld filter, indicating that the micro-organism is of appreciable size. They also find that the disease is conveyed by the body-louse, and, moreover, that the 1 See Hewlett, Practitioner, July, 1911, p. 112 (Kefs.). Typhus Fever 575 infection is hereditary in the louse, the second genera- tion of lice derived from infected lice apparently being- still infective. Neither bugs nor fleas conveyed the disease. In the blood of typhus patients Ricketts and Wilder detected a small bacillus, measuring 2/u in length by 0'6/bi in breadth, tending to stain at the poles and belonging to the group of the hemorrhagic septicemic bacteria. It is not numerous, and is found from the seventh to the twelfth day of the disease. It is also found in infected lice, but could not be cultivated. A similar micro-organism was also observed in Mexican typhus blood by Gavino and Girard, and by Campbell, and the latter also finds that the blood is not infective if passed through a Chamberland F filter. Ricketts and Wilder also discuss the relationship between typhus fever and Rocky Mountain spotted fever.1 Some years ago Wilson and Chowning made observations on a typhus-like fever occurring in limited tracts of country near the Rocky Mountains and ascribed it to a Piroplasma. Subsequent research, however, failed to confirm this, though the disease appears to be conveyed by a tick, and not by fleas, lice, etc. There are clinical differences between typhus and Rocky Mountain spotted fever; moreover, the guinea-pig is susceptible to the spotted fever but not to typhus, and a monkey immunised to typhus is susceptible to spotted fever. Ricketts believes that the spotted fever is due to a bacillus which can be found in the ovary of the tick and is agglutinated by the serum in dilutions of 1-500. Cathoire has made observations /on.' complement fixation in typhus. Using as an antigen' an alcoholic 1 The name is an unfortunate one, for this disease is quite distinct from spotted fever "-epidemic cerebrospinal meningitis. 576 Manual of Bacteriology extract of typhus spleen, marked complement fixation was obtained with the serum of typhus cases. Yellow Fever. As far back as 1889 Sternberg described a bacillus — " Bacillus X" — in yellow fever, a facultative anaerobic organism, very pathogenic to rabbits. In 1897 Sanarelli described his Bacillus ictero'ides, which later investi- gation has proved to be an organism belonging to the Gartner group (see p. 394). Reed and Carroll3 critically examined the B. ictero'ides and its relation to yellow fever. Their conclusions were that the Bacillus X belongs to the colon group, the B. icteroides to the Gartner group, that the B. ictero'ides and hog-cholera bacillus produce the same lesions in animals and mutually protect against each other, that the B. ictero'ides causes in swine all the symptoms and lesions of hog cholera, and that the blood of hog cholera agglutinates the 35. ictero'ides in a much more marked degree than does the blood of yellow fever. Reed, Carroll, and Agramonte,3 having thus shown the serological position of the B. icteroides to be untenable, directed their attention to the transference of yellow fever through the agency of mosquitoes. Finlay, of Havanah, suggested many years ago that yellow fever might be propagated through the inter- mediary of a mosqmto-Stegomyia calopus (fasciata)- and with this species these investigators worked. They allowed mosquitoes to bite yellow-fever patients at Various stages of the disease, and the infected mosquitoes were subsequently allowed to bite eleven » Ann. cle I'Inst. Pasteur, xi, 1897, pp. 443, 673, and 753. ■ Journ. Ex-per. Med., vol. v. pt. iii, p. 215. a Philad. Med. Journ., October 27, 1900, p. 790. Yellow Fever 577 individuals, two of whom contracted yellow fever. \ It is true this is not a very convincing experiment, but it is to be noted that during the period of fifty-seven days among a population of 1400 non-immune Americans there were only three cases of yellow fever, and that two of these had been bitten by contaminated mosquitoes within five days of the commencement of their attacks. The matter was put to the further test of exj^erimeut in the following- manner.1 Under the same observers a camp was established with several tents each occupied by one to three non-immune individuals, and precautions were taken to prevent the introduction of yellow fever from outside. Five individuals were bitten by infected mosquitoes, and four out of the five contracted yellow fever, no other occupants of the camp being attacked by the disease. Subsequently several non-immume individuals were exposed to yellow fever infection from soiled linen, yellow-fever discharges, etc., in a mosquito- proof hut from which mosquitoes were excluded, with entirely negative results. These experiments prove, therefore, that yellow fever is conveyed by mosquitoes only, and further work by Americans and Cubans, and by French and Brazilian Commissions, have entirely confirmed these researches and conclusions. It has been found that to convey infection, it is necessary for the mosquitoes to bite the patient during the first three or four days of the illness, but they do not become infective until about the twelfth day after feeding, and then retain their infectivity indefinitely. .All these facts point to a protozoon as being the causative organism, but none has been found with certainty. The Americans have shown that the blood-serum after filtration through a porcelain filter is still infective ; the organism, therefore, is probably ultra-microscopic,' 1 Jouru. Amur. Med. Assoc., February 16th, 1901, p. 431 37 578 Manual of Bacteriology at least at one stage. Seidelin1 describes extremely small rounded bodies with a minute chromatin point and feebly staining- protoplasm, without pigment, in the blood corpuscles. Somewhat similar, but larger, bodies may also be present in the organs and free in the plasma. Dengue. No organism, bacterium or protozoou, has been demonstrated in this disease. The intra-venous inocu- lation of filtered dengue blood into healthy individuals is followed by an attack; the organism is therefore probably ultra-microscopic. The disease can be trans- mitted by a mosquito, Gulex fatigms, and this is probably the common mode of infection.2 Phlebotomus Fever. A fever of short duration (three days) occurs in South Austria, the malady being somewhat like dengue. It is known locally as " pappataci," and an apparently identical disease has been described by Birt3 in Malta under the name of " phlebotomus fever." Investiga- tion has shown that this disease is conveyed by the bite of a dipterus fly, the sand-fly (Phlebotomus vappatasii.) « Canary fever" "Shanghai fever "Chitral fever," and the seven days continued and " sand-fly " fevers of India are probably of the same nature. The virus in phlebotomus fever passes through a Berkefeld filter. , , , Further research must decide whether these and dengue are distinct diseases or whether they are all manifestations of dengue. i Journ. Pathol, and Bacterial., vol. xv, 1911, p. 282. = Ashbum and Craig, Philippine Journ. of Seience, vol. u, 1907, p. W. » Journ. Boy. Army Med. Corps, 1910, August. Variola and Vaccinia 579 Variola and Vaccinia. The specific contagia of these two diseases have not ye1 been discovered with certainty. Variola is inooulable on man, the calf and the monkey, vaccinia on the rabbit in addition. A lara'e number of observations have been made with vaccine lymph, bat no distinctive bacterium lias been obtained except by Klein and Copeman. Usually the ordinary pyogenic organisms and many saprophytic forms can alone be isolated. Klein observed the presence of a bacillus in vaccinia, which was subse- quently more fully studied by Copeman.1 It was found in vaccine vesicles at an early stage, but at maturation could no longer be detected. It is a very fine bacillus, and these observers were unable to cultivate it. Subsequently Copeman found a similar organism in variola, and succeeding in cultivating the bacillus from both sources in eggs, and from such egg- cultures was able to inoculate calves. Klein," by storing variola crusts in 50 per cent, glycerin and so getting rid of the saprophytic forms, has cultivated an organism which he terms the Bacillus alius variolse. Morphologically it closely resembles the bacillus observed in vaccine lymph; it forms small white, opaque, coherent colonies on agar, but grows very feebly on gelatin. Involution forms occur, and it seems to belong to the group of diphtheria and xerosis bacilli. On inoculation into calves some approach to, but not typical, vaccinia was produced. Moreover, the inoculated calves were not immune to subsequent vaccination. Copeman,3 by inoculating collodion cap- 1 Milroy Lectures on Vaccination, 1898. 2 Rep. Mad. Off. hoc. Gov. Board for 1896-97, p. 267. :' Brit. Med. Journ., 1901, vol. i, p. 450. 580 Manual of Bacteriology sules filled with beef broth with glycerinated vaccine lymph, in which the extraneous organisms had died out, and inserting in the peritoneal cavity of rabbits, observed zoogloea masses made up of bodies resembling spores which he regards as the resting stage of the specific microbe. De Korte finds that the vesicles, both in variola and in vaccinia, are sterile before maturation, and regards the bacterial forms that have been isolated as secondary infections. The failure to isolate a bacterial form has induced many observers to seek for a parasitic protozoon in variola and vaccinia. L. Pfeiffer in 1887 observed roundish or ovoid bodies in the lymph in both diseases, which he regarded as sporozoa. Guarnieri found small bodies, about half the size of the nucleus, in the epithelial cells of the skin in the prepustular stage of variola {Cytori/ctes variolse). Small shining amoeboid bodies were also noticed in the epithelial cells of the corneaa of guinea-pigs inoculated with vaccine lymph. L. Pfeiffer confirmed (luavnieri's work, and also described these amoebiform parasites in the blood in variola and vaccinia, and of vaccinated calves. J. Clarke, and Ruffer and Plimmer in this country described somewhat similar appearances. Ruffer and Plimmer describe the supposed protozoon as a small round body, about 3 in diameter, lying within a clear vacuole in the protoplasm of the epithelial cell. Councilman, Magarth, Brinkerkoff, Tyzzer, and Calkins1 in America have found the Guarnieri body in variola and vaccinia in man and animals, and regard it as a protozoon and the causal agent of these diseases. , t, t, ,r,.i vi 1Q04 v> 173; Philippine Joum. of i Joum. Med. Research, vol. xi, iWi, p. ±fii; Manual of Bacteriology a micro-organism in this disease. Pekelliaring and Winkler isolated a coccus producing a while growth and resembling the M. pyogenes, var. albus, very closely. Hunter obtained a similar coccus from two cases. A G-rarn-positive coccus was also isolated by Okata aud Kokubo Erom the blood and urine. Eost described a small motile sporing bacillus which he isolated from the blood and cerebro- spinal fluid in cases of beri-beri. The org.misin is also present in rice, and can be cultivated in rice-water, ascitic fluid, or on blood-serum. Inoculated into fowls it produced paresis and death. Hamilton Wright suggests that the disease is due to an intoxication, the result oE a gastro-duodenal infection with a large Gram-positive bacillus (unisolated). Daniels believes that the epidemiology of the disease is best explained on the hypothesis of a protozoan infection conveyed by lice. The writer and De Korte1 also suggest a protozoan infection, the organism perhaps being eliminated in the urine. Other views are that beri-beri may be a peripheral neuritis due to arsenical poisoning, or that it is caused by the absence of certain nutritive elements from polished rice, and the evidence in favour of the latter view seems to be accumulating. Bronchitis.— Ritchie2 concludes that acute bronchitis is an infective disease, but is not due to any one specific organism, the most important causal bacteria being the D. pneumonia and streptococci. In every case of acute bronchitis numerous pathogenic bacteria are present in the bronchi, which are usually sterile in health. The commonest organisms are B. pneumonia B. influenza, and M. catarrhal. Spirochetes are present in some forms of tropical bronchitis; m others Castellani has described oidium-like and yeast-like organisms. Chancke, Soft. — An extremely small bacillus, first described by Ducreyi has been found in the ulcers and buboes. It has not been inoculated successfully on animals, but can be inoculated from a chancre, experimentally, from man to i Jnurn. Trop. Med., October 1st, 1907, p. 315. - Jbwn. Path, and Bad,, vol. vii, No. 1, p. 1. :> Comp. Bend. Congres Internal, de Dermatologie (Paris, 1889), p. -~ ■ Summer Diarrhoea 587 man. The bacillus does not stain by Gram's method, and can be cultivated on blood agar, on which it forms shining greyish colonies 1 mm. in diameter, or in guinea-pig blood.1 Conjunctivitis. — Conjunctivitis is of several varieties: (a) Acute contagious conjunctivitis, due to the Koch-Weeks bacillus. This is a slender, non-motile organism, 1-1-5 [x in length, occurring singly ov in pairs, both free and within the pus-cells. It is decolorised by Gram's method, and is difficult to cultivate, growing best on a serum-agar mixture, on which it forms small, pimctiform transparent colonies. It is hardly pathogenic to animals, but in man sets up a typical acute conjunctivitis. (b) Chronic catarrhal conjunctivitis, due to the Morax- Axenfeld diplo-bacillus. This organism is 2 /.i long by 1 /a broad, is not stained by Gram's method, and can be cultivated on blood-serum or serum agar. (c) Gonorrhoea! conjunctivitis. (d) Diphtheritic conjunctivitis. (e) Conjunctivitis of streptococcic origin. (/) Conjunctivitis of pneumococcic origin. — Usualty in children, and accompanied with coryza and scanty muco-puru- lent discharge. (g) Micrococci (aureus and albus) and B. coli may also occasionally cause conjunctivitis. Diarrhcea (Summer) op Infants. — Booker,2 inan elaborate paper, came to the following conclusions : " No single micro- organism is found to be the specific exciter of the summer diarrhoea of infants, but the affection is generally to be attri- buted to the activity of a number of varieties of bacteria, some of which belong to well-known species, and are of ordinary occurrence and wide distribution, the most impor- tant being a streptococcus and the Proteus vulgaris" Lesage obtained a bacillus from the " green diarrhoea " of infants which he believed to be the cause of this complaint. It is a small, motile, non-liquefying bacillus, producing on 1 Einiinel, Ann. de I'Inst. Pasteur, xv, 1901, p. 928. - Johns Unpkim Eosp. lUVs., vol. vi, 1897, p. 159 (Bihlioo-.). 588 Manual of Bacteriology gelatin a whitish expanded growth with ci'enated margins, and giving rise to a green fluorescence in the medium. The B. pyocyaneus may he an occasional cause. In cases with hlood and mucus in the stools, the B. dysenterise (Shiga-Kruse type) has been found to be present in America and in this country. In London, Morgan has isolated in a number of cases a bacillus which in its fermenta- tion reactions is nearly allied to the hog-cholera bacillus (see p. 394). Ealph Vincent ascribes the disease (which he terms " zymotic enteritis ") to the ordinary organisms of putre- faction gaining access to milk and multiplying and causing alterations therein. The stinking motions of the diarrhoea of children have been ascribed to the action of organisms belonging to the Proteus group, particularly B. proteus (P. vulgaris, seep. 658), which occurs in putrefying matter, sewage, and in the intestine. (This organism may also cause abscesses and cystitis, and a form of meat poisoning has been ascribed to its action.) Filtrates of cultures were found by S. Martin to produce a fall of temperature, collapse, and diarrhoea in rabbits. Distemper of Dogs.— According to Galli-Valerio,1 this is caused by a bacillus (B. caniculte) intermediate in character between' the coli-typhoid and lnemorrhagie septicsemic groups of organisms. Evidence has also been brought forward that distemper is due to a filter passer. Probably the term " distemper " may include several different diseases. Dysentery.— Dysentery must be regarded as a term applied to a series of clinical symptoms associated with colitis which is due to different specific agents. There are at least two forms of the disease, one, the so-called tropical or endemic dysentery, met with especially in the East, and characterised by chronicity, a tendency to relapses, amenability to treat- ment with ipecacuanha, and the occurrence of the single liver i Centv f Bald. (Ref.), xli, 1908, p. 563. See also M'Gowan, Jouvn, Pathol, and Bacterial., vol. xv, No. 3, 1911, p. 372 (Bibliog.) Skin Diseases 589 abscess as a sequela ; the other, epidemic dysentery, met with in all parts of the world, particularly in times of war and famine, not amenable to ipecacuanha, and not followed by liver abscess. There are also probably other forms occurring in small outbreaks or sporadically.^Tj^fiitaliUsfintery is due to the Amceba coli, which is found abundantly in the stools, "especnilly in the acute stage, and also in the liver abscesses (see p. 512). In the epidemic dysentery of Japan and other parts of the world a bacillus, or group of bacilli, has been isolated by Shiga, Flexuer, Strong, Kruse, and others. This is the B. dysenteriw described at p. 396. Coli-form bacilli have been isolated from cases of dysentery. Calmette in Tonkin isolated the B. jjyocyaneus, and this organism seems to have been the cause of a small outbreak in New York State investigated by Lartigau.1 In Japan, Ogata isolated a fine G- ram- staining, liquefying bacillus which does not seem to have been met with by subsequent observers. Spirochaetes have been found in large numbers in a form of dysentery occurring in Bordeaux. Vedder and Duval,2 as a result of the study of a number of cases of acute dysentery in the United States, conclude that the disease, whether sporadic, " institutional," or epi- demic, is due to the B. dysenter'ue of Shiga. The B. dysenteric (Shiga type) has been isolated by Eyre, McWeeney, and others from cases of ulcerative colitis or asylums dysentery in the British Isles (see pp. 397-401). The Balantidium coli (p. 536) and certain parasitic worms may also induce a dysenteric condition. Skin Diseases : Acne. — In the acne pustules, the M. pyogenes var. aureus, with or without var. albas, is almost invariably present, and a staphylococcic vaccine generally acts extremely well. In the comedoes a Grrain -positive, Hofmann-like bacillus (B. acnes) is present in considerable numbers, and may be the cause of the comedo. This organism 1 Joum Exper. Med., vol. iii, No. 6, p. 595. 2 Ibid., vol. vi, llJ02, No. 2, p. 181. 590 Manual of Bacteriology was cultivated by Fleming on a neutral agar to which glycerin and oleic acid are added. Siidmersen and Thomp- son1 cultivate on an acid (+ 40) serum-agar. The organism is anaerobic, at least at first, and will grow in glucose- agar stabs. In culture the organism is diphtheroid. A vaccine prepared with it is of service in the comedo stage. Eczema is produced by the action of the pyogenic cocci (ill. pyogenes, var. aureus and albus). Virulent cultures of these organisms, with or freed from their toxins, seem, how - ever, to produce an impetigo rather than eczema. But the filtered cultures, i.e. toxins, are harmful to the skin, and when applied to it for one or two days by means of moist warm pads a typical papular or vesicular eczema ensues. Probably in the human subject in addition to the micro-organisms some peculiarity in the soil is necessary for the disease to develop.2 In so-called seborrhceic eczema, a non-liquefying micrococcus which forms butyric acid has been isolated. Impetigo— The large vesiculo-bullous eruption of impetigo contagiosa is caused by the St reptoccocus pyogenes ; the small pustule in the neighbourhood of hair-follicles, impetigo of Bockhart, is caused by the M. pyogenes var. aureus. The B. diphtherias may also cause an impetigo (p. 285). Pemphigus— A diplococcus has been isolated in acute pem- phigus by Demme, and in the chronic form by Dahnhardt. Bulloch and Eussell Wells, in this country, seem to have isolated an identical organism, and the following description of it is taken from their papers. Cocci 0 8 to 1-5 ji in diameter, mostly arranged as diplococci, and staining by Gram's method, On surface agar the organism forms a thick, white, shining growth. In stab agar the growth has a " nail-shaped ' appear- ance The colonies on agar are at first round, but later, m seven days, they throw out lateral projections and assume a rosette appearance. On gelatin the growth is slow and slight, with some, but not marked, liquefaction. On blood-serum the growth resembles that on agar. On potato a whitish semi-transparent film forms. Milk is curdled. In broth it i Journ. of Pathol, and Bacterial., vol. xiv, 1910, p. 224, - Whitfield, Practitioner, February, 1904, p. 202. Mediterranean Fever causes a general turbidity, with a whitish sediment, and sometimes a pellicle, which soon sinks. Guinea-pigs and mice inoculated or vaccinated with the organism died in four to eight days, fine haemorrhage, occurring in the lungs, and the cocci being obtained from the blood. No bulks appeared on the skin. Th B. pyocyaneus may cause dermatitis and bullous eruptions (see p. 250). The pyogenic cocci or their toxins may produce various bullous eruptions, e. g. pemphigus neonatorum and contagiosus and hydroa gestationis.1 Herpes zoster. — Pfeffer observed bodies in the cells of the vesicles which he believed to be [ rotozoa. Gilchrist, however, regards these merely as altered nuclei. Foot and Mouth Disease.— Various organisms have been described in this disease, but a German commission com- prising Loftier and Abel2 stated that they were unable to prove its ^etiological significance. Loftier and Frosch have determined that the organism must be a very minute one, as it passes through the smallest pored porcelain filter. Malta Fever.3 — Synonyms : Eock, Mediterranean or undu- lant fever. A disease met with especially on the Mediterranean littoral, but also in South Africa, India, China, the Philip- pines, and the subtropical countries of America, and clinically often simulating typhoid fever. A minute micrococcus (M. melitensis), first described by Bruce, is the cause of the disease. Microscopically, the organism from cultures occurs as a coccus, single, in pairs, or in short chains ; it is easily stained by the ordinary anilin dyes, but is Gram-negative. In hansine1- "1 1 • drop cultures it shows decided muvement, which may be only an active Brownian movement, but is perhaps a true motility inasmuch as Gordon has described the presence of flagella (other observers have failed to find them). The organism may be isolated from the spleen of a cadaver. 1 Brit. Med. Journ., 1902, vol. i, p. 73. 2 Centr.f. BaM., xxiii, 1898, March. 3 See Reports of the Mediterranean Fever Commission (Eoyal Society), pts. i-vii, Harrison & Sons, 1904-1907. 592 Manual of Bacteriology On agar it grows as minute transparent colonies, winch first appear when inoculated from the spleen in 90 to 125 hours. In thirty-six hours more the colonies become amber-coloured, and later still, in four to five days, they become opaque, of a slightly orange colour, and round, with granular margins. On gelatin a whitish growth slowly forms without liquefaction, and in broth a diffused cloudiness forms, with a while deposil and without film-formation. Litmus milk becomes alkaline without curdling. Alkali is also produced in glucose media, but galactose, maltose, and saccharose are unchanged (see table, p. 261). The distribution of the M. melitensis in the body corresponds closely with that of the B. typhosus ; thus it is abundant in the spleen, relatively scanty in the blood, and is excreted in the urine. The M. melitensis maintains its vitality outside the body in the dry state in dust or on clothing for two to three months, in tap- or sea- water for a month. The thermal death-point is about 55° C. Inoculated into animals no result usually ensues ; in the monkey, however, a febrile condition is produced, with en- larged spleen, sometimes terminating in death, the course of the temperature resembling that of the disease in man. By intra-cerebral inoculation Durham found that the organism becomes pathogenic for the rabbit and guinea-pig, otherwise it is without effect. For the diagnosis of the disease the agglutination reaction is most valuable. It may be carried out by the microscopic method, a forty-eight-hours' broth culture being employed, the details of the process being the same as described at p. 198. Dilutions of 1 in 30, 1 in 50, and 1 in 100 should be prepared, as well as controls with normal serum, for old laboratory strains sometimes agglu- tinate with normal serum in dilution of 1 in 20 or 30 (see p. 190. Neglect of this precaution led Bentley to ascribe kala-azar to a Malta fever infection). The organism being minute, it is necessary to use the Tyineh oil-immersion, the -i-inch with a high eyepiece and draw-tube extended, or better, a i-inch dry objective. The sedimentation method is pre- ferable. Measles and Meningitis 593 The disease may be conveyed to monkeys by contact, by inhalation of infected dust, and by feeding. Mosquitoes and other insects do not seem to convey it. The investigations of the Mediterranean Fever Commission have shown that the main source of infection of man is by goat's milk. G-oats may be infected (and are largely so in endemic districts, e.g. Malta and South Africa) without showing any symptoms, and excrete the organism in large numbers in their milk. Since the goat's milk has been boiled the incidence of the disease in Malta has fallen from 663 cases in 1905 to 7 cases in 1907 in the Army, and in the Navy there were no cases in 1907 (Bruce). Toxin, vaccine, and serum therapy.— The M. melitensis forms no extra-cellular toxin, but Macfadyen obtained an endotoxin by disintegration. Attempts to prepare an anti-serum have not been successful. A vaccine prepared with cultures killed by heat (see p. 232) has been used in the chronic form of the disease by Bassett-Smith1 and others with some amount of success. Mastoid Disease.— See " Otitis Media." Measles. Doehle and Behla described small flagellated bodies which they believed to be protozoa in this disease. Canon and Pielicke found small bacilli in the blood, which Tchaikovsky confirmed. They are motile, do not stain by Gram s method, and can be cultivated on agar and serum, on which they form delicate colonies. Czajkowski has found a similar organism. Lesage* cultivated a small micrococcus from the nasal mucus and blood, which produced a fatal hemorrhagic septicemia in animals. The influenza bacillus is present in many cases. Meningitis may be caused by D. pneumonia (60 per cent of acute cases), D. intracellulars, Still's dip locoLs B tuherculons gonococcus, and micrococci and streptococci. _ Mumps (Epidemic Paeotitis) . _ Mecray and Walsh isolated from the parotid and blood in some cases of mumps I Journ- °f Hygiene, vol. vii, 1907, p. li; - Comjpt. Bend. Soc. Biol., 1900, p. 203. 38 594 Manual of Bacteriology a coccus resembling that described by LaveraH and Catrin. It occurs chiefly as a diplococcus, but abo in large -roups. The colonies form circular, white, shining points, with slow growth and gradual liquefaction. On potato a white growth occurs; on blood-serum a plentiful cream-coloured growth; and in litmus milk production of acid with coagulation. Noma and Cancrum Oris.— Grawitz in 1890 observed bacilli in the affected tissues in this disease, others fusiform bacilli with or without other organisms; Comba considered that there was probably no specific organism; Durante found the M. pyogenes, var. aureus, with B. protms, ami Eavenna the same micrococcus with the typhoid bacillus. Diphtheroid bacilli have also been isolated. Weaver and Tunnicliff1 in a case of cancrum oris observed the presence of fusiform bacilli and spirilla. Hellesen- isolated a diplo- coccus from a case of noma. The organism is not unlike the pneumococcus, but possesses no capsule, is Gram-posit ive, gives a general turbidity in broth with acidity, forms no gas from glucose, curdles milk with acid production, and forms punctate, whitish-grey, translucent colonies on surface ao-ar. On inoculation into animals a specific necrosis was produced. , Bishop and Evan, in two out of three cases, isolated an organism which culturally and morphologically resembled the diphtheria bacillus, but which only produced some local inflammation on inoculation into guinea-pigs. In the third case the M. pyogenes, Tar. aureus, and the Streptococcus pyogenes were isolated. Guizzetti, and Freymuth and Petmsehkv have isolated the Klebs-Lomer bacillus m noma. Oppler-Boas Bacillus.— Met with in the stomach, parti- cularly in cases of carcinoma, and its detection is suggestive of this condition. The bacilli occur in masses, are long and filiform and non-motile, and frequently 30m one another in angles. They measure usually 6-8 /x m length, but vary between 3 and 10 p. The organism has been cultivated, and is a facultative anaerobe, non-sporing and i Jonm. Infectious Diseases, vol. iv, 1907, p. 8 (Bibliog.). » See Lancet, 1908, vol. i, p. 955. Otitis and Ozaena Gram-positive. It curdles milk and forms lactic acid from various sugars. Otitis Media. — The Diplococcus pneumonise is' perhaps the commonest organism met with ; next in frequency comes the Streptococcus pyogenes, and then the pyogenic cocci. In scarlatinal otitis media, Blaxall found the 8. pyogenes to be always present, and generally accompanied by other organisms, pyogenic cocci, etc. In thirty-seven cases of mastoid disease Blake found the following organisms, and remarks that as a rule the same were found in the middle ear : Streptococcus ........ 12 Staphylococcus ........ 5 Diplococcus (? pnaumonice). . ... . 6 Streptococcus and diplococcus ..... 5 Streptococcus and Bacillus fetidus (? colon bacillus) 3 Streptococcus and Bacillus pyocyanaus ... 1 Streptococcus and diplococcus ..... 1 Streptococcus, micrococcus, and diplococcus . . 2 In two of the cases no organisms could be isolated. Oz^na (Atrophic Ehinitis). — Lowenberg described in this disease encapsuled bacilli somewhat resembling the pneu mo-bacillus morphologically. Some Italian observers found bacilli apparently identical with the diphtheria bacillus. Abel1 described a bacillus somewhat resembling the pneumo- bacillus. It is this organism which produces the atrophy of the mucous membrane, but the fetor is due to the decompo- sition of the secretions produced by other organisms. Perez2 isolated an organism in ozsena (Coceo-bacillus fetidus ozxnte) which has the following characters: it is a short bacillus with rounded ends, non-motile, does not stain by Oram's method, does not liquefy gelatin, does not ferment lactose nor curdle milk, but forms indole and ferments urea. Its cultures are foul-smelling, and it is pathogenic for guiuea- pigs, mice, rabbits, and pigeons. Peritonitis.— Treves gives the following table of the micro-organisms found in peritonitis: J Zeitschr.f. Hy a 5, eft rr" ,Q to 3 g bOT3 .s s br [ a ci I 3 o En 606 Manual of Bacteriology water, in flood time or drought, may be avoided. More- over, storage alone usually markedly diminishes the number of organisms, partly by subsidence, partly by lack" of aeration, and partly probably owing to the struggle for existence going on among them (see also p. 381). (2) Thickness of fine sand in the filter-beds. — Efficient sand filtration removes quite 09 per cent, of the organisms originally present. The hue sand only lias to be taken into account in estimating the removal of organisms and efficiency of a filter bacteriologically. It probably should form a layer not less than 3 ft. to '3 ft. 6 in. in thickness. Moreover, a filter-bed is not efficient at first, but becomes so when the surface film forms, composed of sedimented particulate matter, and of a, zooglceal mass of bacteria and algse. (3) The rate of filtration. — The removal of organisms is less perfect when the rate of filtration is increased; this should not exceed about 1-5 gallons per square foot per hour. (4) ' The renewal of the filter-beds. — New, or recently cleaned, filter-beds allow a greater number of organisms to pass through. The beds must be cleaned from time to time by raking up and clearing away the surface layer of sand, for as time goes on the rate of filtra- tion becomes slower and slower, though the bacterial efficiency of the filter-beds does not appear to be reduced by prolonged use. The normal bacterial efficiency seems to be rapidly regained after cleaning— within two or three days. Besides storage and filtration, sedimentation in the presence of fine particles, either naturally present or artificially added, may also effect a marked removal of micro-organisms from water. Thus, by the addition of alum, an old method of clarifying turbid water, a, large 607 o r. o CO o o ©• To yo 1 O , r-i « o ' m + o jjf- o • o° «k + H ^-2 6° fc- oa CO lit C.C. 11-5 + o " CO •0-5 00To2 1 en's P P-i £73 H w o H - o w w p p i«l CO to CO CM IN o 09 co da CD cd 3 s 3 S3 a to oa CO TO a o Iff »o 25 eg *co CO -a 43 R C C o O s 3 X CO S. a? : V b o CO Ul cj -M 03 a CU B to '3 "3 B ■w JS a 09 u p o c o 3 S CO H St o - - PS o3 si o eS ►J •z} a cs fit ho co a; t .3 <3 CH .3 £ S"5 o a> ell o a g o f CI ^ 2-, <= h> w .3 CO i3 3 a b co ™ 5 g eq o Sh o- co £i CD a o 5) ■ s.>)i 1 1 mis ■_•[ s.-ih.i v \\ MO(i>jorf 'i 1 v Vi \ m.0il unv.'i ; ) a b 1— 1 CS ib rH rH 1*- SO rH r. 10 9 to CM §? CO CD Hi CO O) if rH M 1 © cq IT) Cq p if O CO p cq p cq CO rH p rH O if >H p H cq •(woiiIuun Qggi 1 S-tOIU.U pOAl.lOp . CO a if rH t- ■O as »H CO la OS CO CO oa H CO CO rH .0 CO cq CO p b rH <5 1 00 r—i 05 cq p p CO CO p cq rH O CO p 5j< rH cq te- rn £- cq •(S3[drans izo) . o 1— 1 cq 0 CI CO ?' cq 0 cq CO 0 7-\ O H< CO CM B0 CO CO b p b rH H CM »o -f< 00 Tf rH «i 1 00 ru O co CO t* P p CO CO CO 1— 1 p O if Tf LO cq cq cq Jf b ■(soidums | got) p'nttig . 00 [S rH 10 OS cl H< 1^ H< HH CO CO 00 cq CO b* in p rH 00 if < 1 0 p 1 — 1 p r-l p cq p rH S* O p rH p cq p cq p rH P b D- rH •(S8[dtauB 0X0) U9B[8q.O . CO cb £- 1— 1 co US b ■HI CO X iO IQ CM IO 0 cq jji CO CI CO cq 0 b CI P b cq [.uij uoiduia^i ■ CO b N is CM *H CM C5 CO O TO C5 t> w s CO p C-l b LO 00 p b ■^f if rH rf 00 CO cq Cq b CO c? cs 1 1 p cq CO p b 01 rb c» CI -f if cq O' 1 O CNJ p CM p b 1— 1 p p CO CO ■H 10 cq p rH TO rH ^H H CO rH p rH p b ••saidnnts 8681) . O C6 b rH rH rH 00 'X 0 CO CO 60 00 fa H? l> 10 *H CO CM TO 10 rH cs p b m b rH .taAi^i Aia^j -4 1 O CO O cq p rH p cq US rH p b p b CO p CO rH b p cq p cq 05 cq 1910-11. Month. O CS -O cn qj CD rH be g 1 ID 4 0 CS <5 r-s « a) p4 r^ a hs -^J 3 5) o) CO 1 CD — CD m CO O 0 O CO 1 0 0 rH rl CO 1 QJ 0 © Q a c rd s 3 "co rH CO s 1 ■j. - CO rH ci H 00 < Examination of Water 609 number of the organisms present are carried down in the precipitate. The Clark process of softening water may also reduce the number of organisms present, but is very uncertain (Moor and Hewlett). By the Porter-Clark rapid process, however, Nankivill believes considerable purification is effected. The tables on pp. 607 and 608 illustrate the influence of storage and of sand filtration on the bacterial content of a water. The Bacteriological Examination of Water.1 The bacteriological analysis of water affords valuable indications as to the purity or otherwise of a water, and, if properly carried out, will indicate a pollution so small in amount as to be incapable of detection by chemical methods. The specimen of water should be collected in clean bottles of about 100—200 c.c. capacity, sterilised prefer- ably by heat. If, however, the bottles be thoroughly cleaned and rinsed out with a little strong sulphuric acid, and then thoroughly rinsed several times with the water to be examined before taking the specimen, no error will be introduced. The stopper of the bottle should be tied down with a thin layer of cotton-wool enclosed between two pieces of muslin, and the bottle should not be quite filled. In taking the specimen the following details should be attended to : (1) If taken from a tap, the water should be allowed to flow for at least five minutes before the specimen is collected. i 1 See Savage, Bacteriological Examination of Water Supplies (Lewis, 1906); Thresh, Examination of Water and Water Supplies^ Churchill, 1904) ; Houston, Gordon and others in Reps. Med. Off. LocMov. Board, 1899-1904 ; Houston, Reports to the Metropolitan Water Board. 39 G10 Manual of Bacteriology (2) The water from a cistern is not a representative sample of the water supply; to be so the specimen should be taken dii-ect from the mains. (3) If taken from a stream or pond, the bottle should be held about a foot below the surface and away from the edge before the stopper is removed. (4) If taken from a well the conditions should be noted, e. g. whether the well has been recently dis- turbed or no, whether the pumps have been in opera- tion, etc., for such may markedly influence the number of bacteria found. The specimen should then be examined with as little delay as possible, for if allowed to stand for any time a large increase in the number of bacteria may take place. Frankland, for example, found that in distilled water, even at the ordinary temperature, organisms multiply enormously : Hours Number of organisms in 1 c.c." 0 1,073 6 6,028 24 7,262 48 48,100 In water of good quality the organisms are found to multiply much more rapidly during the first few days, after which time they become less and less numerous ; but in impure water multiplication is slower, and the number more persistent, while in very impure water the number may diminish. It is essential, therefore, if reliable results are to be obtained, for the specimen to be examined at once (within three hours). If this cannot be done the specimen should be packed in ice ; the cold will then inhibit multiplication to any extent. Special double-chambered metal boxes are made for this purpose : the bottle containing the sample (not less than 60 c.c. j the writer prefers to have not less than Examination of Water 611 200 c.c.) is placed in the inner chamber, the outer chamber (which surrounds the inner) being1 filled with a mixture of ice and sawdust, and the whole is packed in a wooden box with felt lining-. According- to Remlingler,1 the addition of 10 per cent, of common salt to the sample preserves the original bacterial content of the water unaltered up to ninety-six hours after taking the sample, without icing. Besides the sample packed in ice, a " Winchester quart " of the water may also be collected for examination for the spores of the B. Welchii (enteritidis sporogenes) . The routine bacteriological examination of the specimen may be carried out according to the scheme (here somewhat modified) drawn up by a committee of the Royal Institute of Public Health.2 PiiOCEDURrcs. — The following procedures should be carried out : (a) Enumeration of the organisms which will develop aerobically in gelatin at 20° C. (b) Enumeration of the organisms which will develop aerobically in agar at 37° C. (Enumeration is carried out by counting the number of colonies which develop in the plates [see p. 80].) (c) Search for Bacillus coli, and identification and enumeration of this organism if present. (d) Search for, and enumeration of, streptococci. As a routine measure it is not necessary to search for the Bacillus Welchii (enteritidis sporogenes), but in special instances it may be desirable to do so. The bottle must be well shaken to mix the sample. Before removing the stopper, it and the neck of the bottle should be swabbed with absolute alcohol, which is then carefully ignited and allowed to burn away. 1 Comp Rend. Soc. Biol., Ixx, p. 64. ; Jouvn. State Med., vol. xii, 1904, p. 471. 612 V Manual of Bacteriology Mkdia, Time of Incuisatfon, etc. — For the gelatin count ordinary nutrient gelatin is employed, the period of incubation being seventy-two hours. In hot weather ifc may be necessary to use 15—20 per cent, gelatin (unless an incubator which can be cooled is available), but the development of the colonies is slower. For the agar count ordinary nutrient agar is used, the period of incubation being forty to forty-eight hours. The media should preferably be recently prepared and be standardised to a reaction of + 10. In addition to the actual numbers of organisms which develop in the gelatin and in the agar, a comparison of the ratio oF the number of organisms developing in gelatin at 20° 0. to those developing in agar at 37°C. also gives useful indications. With a pure water this ratio is generally considerably higher than 10 to 1 ; with a polluted water this ratio is approached, and frequently becomes 10 to 2, 10 to 3, or even less. The actual number of organisms growing at blood-heat ia of considerable value apart from any question of ratio. In certain instances it is true that this ratio may be unreliable. Thus with surface waters, especially in the tropics (as pointed out by Horrochs) varieties of the B. fluorescpiis Liquefaciens and nun-liquefacieus and B. liquefaciens may be abundant and grow well at blood- heat. Distilled water gelatin and agar have also been recommended, but since the organisms of polluted water develop better in the ordinary nutrient media, the latter are preferable for routine use. Amounts to be Plated, Size of Dishes, etc.— Gelatin —For an ordinary water amounts of 0"!, 0'2 and 0-3 c.c. may be plated in Petri dishes of about 10 cm. diameter, preferably done in duplicate. Examination of Water <;|:; Agar.— Two plates may be made with (H and 0'2- 0-3 c.c., and arc preferably duplicated. The desired volume of water should be run into the sterile Petri dish by means of a sterile 1 c.c. pipette graduated m hundredths. The tubes of gelatin should be melted in a water- bath at a Low temperature (4lT to 45° C). A tube is taken out of the water-bath, wiped dry to prevent the adherent water running down into the Petri dish, its mouth well singed in the Bunsen flame to sterilise it, and the contents are (hen quickly poured into the dish and mixed with the water by tilting the dish several times. ' The agar tubes must first be boiled, then cooled to about 45° C, and similarly treated, or surface plates may be made. If waters are constantly being examined, it saves trouble to have the gelatin and agar in small flasks, 30-60 c.c. of the former and 20-40 c c. of the latter ; a flask of each will then be used for an examination . In dealing with an unknown water, and in all cases of doubt, additional plates should be prepared with a dilution of the water (made with sterilised tap-water) of ten or hundred fold, according to circumstances. The amount of the medium in a plate should be 10 c.c. The counting is done with the naked eye, preferably in daylight, any doubtful colony being determined with the aid of a lens or low- power objective. The number of liquefying colonies in the gelatin plates should also be noted. The plates should be inspected daily, in oi*der that the count may be made earlier should liquefaction rentier this necessary. In examining an ordinary drinking-water there is no need ever to dilute. As 1000 or 1500 colonies can be counted in a plate, and if the number on a plate should be, owing to crowding, uncountable, ipso facto this would be sufficient to (ill. Manual of Bacteriology V. condemn without an actual count. Dilution is necessary when dealing with river or other water known to be polluted, and of which an estimate of the number of organisms present is desired. In order to count the colonies if very numerous, ink lines may be drawn across the bottom of the Petri dishes so as to divide them into sectors. Ruled paper discs (Pakes's discs) upon which the dishes are placed can also be obtained. The colonies in the sectors are then much more easily counted: or if the colonies be very numerous and evenly distributed, the number in two or three of the sectors may be counted, and the total number on the plate estimated by calculation. SKARCH FOU BACILLUS com, etc. — Various media may be employed for the detection, isolation, and enumeration of jB. colt. The writer generally employs as a pre- liminary, glucose bile-salt peptone-water, but many other media may be employed, e. g. formate or neutral- red broth, or if the organism is abundant, neutral-red bile-salt agar. As a routine, 50 c.c. should be the minimal quantity examined lor the presence of the Bacillus cult, quan- tities from a minimum of 0"1 c.c. to a maximum of 50 c.c. being1 added to the tubes of culture media. It is preferable to add the water directly to the tubes of culture medium, even with the larger amounts, and not to concentrate the bacteria by any method. The culture media may be diluted with at least an equal volume of the water without interfering with their cultural properties, and large tubes or small flasks are used for the larger amounts. In the case of glucose or lactose bile-salt peptone- water, the medium may for the larger amounts be pre- pared of double strength. The glucose or lactose bile-salt peptone water should be incubated at 42° C. for not less than forty-eight hours. Examination of Water 615 \ For composition of glucose formate broth, glucose and lactose bile-salt media, and neutral-red broth, see p. 623, et seq. While a lactose medium has the advantage, of excluding a number or forms which, though fermenting glucose, do not ferment lactose, and are therefore not typical B. coll, Houston has found that a glucose medium is more delicate jTulS^nactose one^ For general purposes, quantities ot troni ~cFl to 25'0 c.c. may be added to tubes of the medium selected. For the examination of an ordinary drinking-water, the writer usually employs five tubes with 1 c.c. of the water in each, two tubes (double strength) with 10 c.c. in each, and one tube (double strength) with 25 c.c. For the larger amounts large test-tubes and boiling tubes must be employed. If the medium shows changes (acid + gas) suggestive of the presence of B. coli, it is only presibmptive evidence of the presence of this organism. Occasionally other organisms produce a similar change, e. g. B. lactis asroqeites^, clnarn^ Hence the necessity for the isolation and identification of the organism, as recommended in the next section. Isolation op Bacillus coli, if pkesent. — If indica- tions of the presence of the Bacillus coli be obtained in the preliminary cultivations (acid + gas), the organism must be isolated and identified. If several tubes show acid + gas, one or two of the tubes with, the smallest quantities of the water should be used for this purpose. This may be done by making' surface cultures on plates (sloping tubes generally suffice) of either (a) litmus lactose agar, reaction + 10 ; (b) litmus lactose bile-salt_agur ; (c) (Jonradi and Drigalski agar, which the writer generally employs ; or (d) ordinary nutrient gelatin. Agar media, incubated at 37° C, have the advantage of saving time. (For composition of media, see p. 624, et, seq.) Identification op, and Tests por, the Bacillus 616 Manual of Bacteriology coli. — Having obtained coli-like colonies on fche plates made from the preliminary cultivations of the water, various tests must be used for identification. The organism should conform in morphology, motility and staining reactions with the characters of the typical B. coli as given at pp. 401—405, and must be subjected to various cultural tests, e.g. the " llaginac " reactions of Houston (p. 405). The writer generally employs these, with the addition of the fermentation reactions given by dulcitol, mannitol, and adonit litmus peptone water, and gelatin for absence of licpxefaction. If atypical Bacilli coli (see pp. 401 and 409) are met with, the fact should be noted, but their significance is not yet fully determined. Stueptococci. — It is a distinct advantage to search for streptococci. They may be looked for by making hanging-drop preparations of the fluid media employed for the preliminary cultivation of the B. coli (glucose or lactose bile-salt peptone water, etc.) The presence or absence of streptococci in these tubes gives also a quantitative value to the examination, just as in the case of B. coli, and the result obtained should be stated. The streptococci can be readily isolated on Conradi-agar plates. According to Houston (loc. cit.), faeces contain at least 100,000 streptococci per gram. The type of streptococcus generally present is one forming short chains, producing a, uniform turbidity in broth, acid and clot in litmus milk within five days at 37° C., and non-pathogenic for mice. (See table, p. 248.) Bacillus WEr.CHil. — As already stated, it is not essential as a routine procedure to search for fche Bacillus Welchii (enteritidis sporogenes), though in cer- tain instances it may be of advantage to do so. A Examination of Water 617 negative result in such cases is probably of more value than a posit ive one. For the isolation of B. Welchii, 500 c.c. of the water may be filtered through a Pasteur-Charnberlaiid filter, the deposit is suspended in 5 to 6 c.c. of sterile water, and 1 c.c. of the j suspension added to each of five to six tubes of sterile milk, which are then heated to 80° C. for ten minutes in a water-bath, &fl0tf\JL& and inciibated anacrobically at 37^C. for forty-eight hours u (filter-brushing method). A better method' is to employ large boiling tubes or small Erlenmeyer flasks, each containing 25 to 50 c.c. of sterile milk. To each tube a quantity of water equal to that of the milk isadded, the tubes are then heated in a water- bath to 80° C. for fifteen to twenty minutes, A^^}/ some sterilised oil or melted vaseline is poured on the surface to exclude air, the tubes are cooled in water to 37° C. or WwJa* thereabouts, and incubated for forty-eight hours at 37° C. Not less than 200 c.c. of the water ^should be used. The typical change m the mine (see p. 45'2)inclicates the probable presence of the organism. To make sure that the change is due to the J. Welchii and not to the C. hutyricum, 1 c.c. ojthejvhe^pei- 100 grm of body weight should kill a guinea- RigjiyiQxlyJm^^ subcutaneously. The virulence qfcTpepfone-icater culture has been suggested as an index of contamination, but in the writer's hands has not given reliable results. If sufficient peptone and salt be added to a measured volume of the water to form a 1 per cent, solution of the former and a i per cent, solution of the latter, the mixture incubated at 37° C. for twenty-four hours and injected intra-peritoneally into a guinea-pig, a bad water is stated to kill, whereas a good one does not. The amount to be injected is 2 c.c and death should ensue within forty - eight hours. Interpretation of Results— The interpretation of the results of the bacterioscopic examination of water is a difficult matter, for which experience is necessary. Just 1 B. T. Hewlett, Trans. Path. Sue. Lond., vol lv, 1904, p. 123. 018 Manual of Bacteriology as in chemical analysis, if. is not possible to lay down an absolute standard, a knowledge <>lr the source and surrounding- conditions being of the greatest importance in forming an opinion. The ultimate aim is, of course, the detection of sewage or fseeal pollution j the bac- terioscopic analysis does not give any information as to the suitability of the water for household, trade, or factory purposes. O H*. iTT'h Number of colonies on the gelatin plates. — The number of colonies represents approximately the number ^ — of organisms in the original sample capable of develop- ment aerobically at 20° C. in gelatin. This number in a good water rarely exceeds 100 or 150; in pure waters, particularly those coming from deep chalk-wells, there may be only a few — 5 to 10 per c.c. (the results are always expressed in numbers per cubic centimetre of the original water). In waters of poorer quality the number may approach 500 per c.c. Anything over OAAk/fJ^this casts suspicion on the water, and 1000 per c.c. or ^ywJr*™ ^rnore should probably condemn the sample, always .-_^0 supposing, of course, that multiplication in vitro can be (/V* . excluded by the proper storage of the sample bottle in ice. As a rule in water of good quality liquifying organisms are scanty, while in a polluted water they are numerous. Number of colonies on the agar plates.-r-As men- tioned before (see p. 612), it is the ratio of the number of organisms developing on the agar plates to those developing on the gelatin plates that is of importance. Number of B. coll.— The detection and enumeration of B. coli are regarded by all as perhaps the most im- portant part of water examination. The number of B. coli is estimated from the amounts of water that have been added to the tubes of media, which, however, assumes that the organism is regularly distributed Examination of Water Gil) throughout the sample, and must so far as possible be ensured by thorough mixing-. The results generally come out fairly concordantly, though irregularities exceptionally occur which can only be obviated by making duplicate sets of cultures. It is better to state the result as " B. coli present in c.c. of water" rather than to say that so many B. coli are present, thouo-h as a matter of fact the latter statement is approximately correct. Adopting the writer's methed for B. coli. (p. 615), if none of the tubes contains B. coli, we say that " B. coli is absent from 50 c.c"; if the 25 c.c. tube contains B. coli, but not the remainder, " B. coli is present in 25 c.c. but not in less," and so on. If nothing is known about the water, the following standards may be adopted : (a) Waters of good quality . — B. coli absent in 50 c.c. of the water. 3^ *" (b) Waters of medium quality. — B. coli. present in 50 c.c. but absent m 25 c.c. (c) Waters of poor quality. — B. coli present in 50 c.c. and 25 c.c, but absent m 10 c.c. (d) Waters of suspicious quality.- — B. coli present in 50 Co., 25 c.c, and 10 c.c, but absent in 1 c.c (e) Waters -ml fib jor drinking. — B. coli present in 1 c.c. or less.-"* Waters which show uo B. coli in 50 c.c. are of a high degree of purity, and therefore the proved absence of this organism in this amount, and still better in larger quantities, is of great value. B. coli should be absent from at least 50 c.c. of spring or deep well water, possibly from greater amounts. Iu upland surface waters the presence of B. coli in 40, 10, or even 2 or 1 c.c. means contamination, but not necessarily a contamination which it is essential to prevent, It may be 620 Manual of Bacteriology Erom contamination with tlie excreta of animals grazing on the gathering areas, and is by no means necessarily from sewage or other material containing specific organisms of infection. If B. coli are present in numbers greater than, say, 500 per litre (or even in that amount), such a water is suspicious, as it is rare to get so many B. coli in a water from the kind of animal contamination indicated, and further investigation is desirable. In filtered samples the number of B. roll is, as a rule, considerably reduced. In surface wells B. coli in large numbers indicate surface or other contamination, generally very undesirable if not actually dangerous. It must clearly be understood that the presence of the B. coli in water is used as an index of pollution, just as the organic ammonia is in a chemical analysis. This organism is not necessarily harmful in itself ; it is what it indicates, viz. pollution, probably with human excremental matters, which mav contain the organisms of specific disease, e. g. typhoid, dysentery, and cholera. As a routine, the typhoid bacillus is never looked for, and the statement sometimes seen in the report on the bacteriological examination of a sample of water that " no typhoid bacilli have been detected " is of little value. It is on the general results of the examination, as detailed in preceding pages, that a conclusion is arrived at respecting the purity or otherwise of a water. Bacillus Welchii. — This organism being- abundantly present in faeces and sewage, its presence in water has been suggested as an indication of pollution. Its spores, however, are very resistant, and it might, therefore, gam access to the water in ways ot her than by direct pollution e. g. in dust — and for this reason the committee did not recommend the search tor this organism as a routine procedure. On the other hand, Thresh1 lays a good deal of stress on it, and the following are standards 1 Public Health, 19 4. Examination of Water 621 suggested by him, based on an examination for, and detection of," B. cult and B. Welchii : 1 Water showing the absence of organisms capable of fermenting glucose, and of the B. Welchii. These we regard as being free from any evidence of pollution. 2. Waters showing the absence of organisms capable of fermenting glucose, but containing the B. Welchii, or its near ally. In the few cases of this kind which have come under our observation we have inferred the absence of sewage pollution, but the possible presence of water derived from fertile soil. This inference has been verified on more than one occasion. 3. Waters containing organisms capable of fermenting glucose, but not lactose, but free from the spores of the B. Welchii. These are regarded as unpolluted. 4. Waters differing from No. 3 only in containing spores of the B. Welchii. These we regard as free from sewage pollution, but as probably containing soil washings. 5. Waters containing lactose fermenters, none of which belongs to the Bacillus coli group, and free from the spores of the B. Welchii. These we do not regard as being sewage- polluted, but as containing surface water or subsoil washings. 6. Waters resembling No. 5, but containing the spores of the B. Welchii. These waters are usually from a source requiring careful watching, manurial matter probably being used on the collecting area. 7. Waters containing organisms of the colon group other than the B. coli, but no spores of the B. Welchii. These we do not regard as dangerously polluted, bit as probably coming from a source such as that referred to under No. 6. 8. Waters containing organisms of the colon group other than the B. coli, and also spores of the B. Welchii. Pollution indicated, but possibly from a source not close at hand. The necessity for frequent examination is essential, especially after heavy rains, as such waters usually sooner or later show more serious signs of pollution. 9. Waters containing the true B. coli, but no spores of the 622 Manual of Bacteriology B. Welchii. Such waters are occasionally met with. No opinion can be expressed without an intimate knowledge of the source. We have had such water from a source absolutely free from the possibilities of contamination, but usually subsequent examination has revealed the presence of the spores of the B. Welchii. The proximity of manured soil is strongly indicated. 10. Waters containing the true B. coli and spores of the B. Welchii. These we regard as being decidedly contaminated with faecal matter of recent origin. Streptococci. — Streptococci are abundant in fteces and sewage, but are extremely rare, if ever present, in unpolluted natural waters ; hence the value of their detection. Streptococci as a class are delicate organisms, and it was supposed that their presence indicates recent pollution.1 Horrocks, on the other hand, believes that they maintain their vitality longer even than B. coli, and this is rather the opinion at present. We need further data before we can exactly estimate the value of streptococci as indicators of pollution. There can be no question, however, that the detection of many streptococci, together with B. coli, indicates serious pollution. There can be no doubt of the value of the bacteriological examination of water, but it cannot entirely supplant chemical analysis, which on account of its rapidity and the valuable data it yields will probably always remain an integral part of the examination of potable waters. If the water be pure and uncontamiuated, the bacteriological examination will occupy three days ; but if contamination be present, though it may be presumed in the same time, ten days or a fortnight may be required to convert this presumption into a certainty, owing to the length of time necessary for determining the characters of the organism present. 1 Houston, Rep. Med. Off. Loc. Gov. Board for 1898-99. Isolation of Bacillus Coli U23 Media Employed for the Isolation of B. Coli. (1) Carbolised gelatin— Ordinary nutrient gelatin with the addition of 0 05 per cent, of phenol. (Hardly used now.) (2) Bile-salt peptone water (MacConkey and Hill). — The composition of this medium is as follows : Sodium taurocho- late 0-5 grm., glucose or lactose 1-0 grm., peptone 2'0 grin., water 100 c.c. The constituents are dissolved by heating ; the mixture is filtered, and after filtration sufficient neutral litmus solution is added to give a distinct colour. The medium is then distributed into Durham's fermentation-tubes and sterilised by steaming for twenty minutes on three successive days. The medium may be put up in various sized tubes, a measured volume in each — e. g. 10 c.c, 20 c.c, 25 c.c, etc., according to the quantity of water which is to be added. For the larger quantities the medium may be made double the above strength. The inoculated tubes are incubated at 42° C. for forty-eight hours. The B. coli reddens and ferments both the glucose and lactose media, so that gas collects in the fermentation tube. (3) RfeutraLzed broth (Hunter, Makgill, Savage). — The dye known as neutral-red (G-riibler's) is reduced by the action of the B. coli, the colour changing to a canary yellow, accom- panied by a green fluoi-escence. The B. enteritidis (G-iirtner) also reduces neutral-red, but the B. typhosus doesnpt do so, rim- do stTfipfrpponci. B. pvomia)ieus. and Spirillum choleras. Some anaerobes also possess a reducing action. G-lucose agar or broth (0-5 per cent, of glucose) is employed, and to every 10 c.c. of the medium 0-1 c.c. of a 0'5 per cent, aqueous solu- tion of neutral-red is added. Savage recommends the follow- ing procedure : 10 c.c of the water are added to a 10 c.c. tube of neutral-red broth ; also to 40 c.c. of the water contained in a bottle or flask a 10 c.c. tube of the broth of quadruple strength is added. Both arejincubated at 37° C, and examined daily up to eight days. If reduction occurs, B. coli is almost certainly present in the water ; if reduction does not occur its presence is highly improbable. (4) Glucose formate broth (Pakes).— To ordinary meat 024 Manual of Bacteriology infusion 1 per mil., peptone, 0-5 per cent, sodium chloride, 2 per rent, glucose, and 0;4 per cent, sodium formate are added. When these have been dissolved by heating, the medium is neutralised (indicator, litmus), and after neutrali- sation 2 c.c. of normal caustic soda solution per litre are added ; the broth is then steamed for twenty minutes, filtered, and distributed into test-tubes, 10 c.c. in each, which are steamed for twenty minutes on each of three successive days. These tubes are inoculated with the water, and incubated anaerobically at 42J C. for twenty-four to seventy-two hours. Tubes showing any growth at the end of twenty-four, forty- eight, or seventy-two hours are removed and examined micro- scopically^and by plating. (5) Bile-salt lactose agar (MacConkey) . — This medium is prepared by adding to 1000 c.c'. of tap-water in a flask 2 per cent, of peptone, 0-5 per cent, of sodium taurocholate, and T5 per cent, of agar. The mixture is autoclaved at 105° to 110° C. for H hours, cleared with a small addition of white of egg, and filtered. To the filtrate 1 per cent, of lactose is added. The medium is then distributed into test- tubes, 10 c.c. in each, and sterilised by fifteen minutes' steam- ing on three successive days. Plates are made and incubated at 42° C. for forty-eight hours. The colonies of organisms which ferment lactose with the formation of acid are sur- rounded with a cloudiness or haze owing to the precipitation of the taurocholate. Neutral -red or krystal violet may be added (proportions, see Nos. 3 and 6). (6) Conraa U-DrigaUki agar.— Mixture A. — To 1 litre of acid beef broth (p. 55) add : Witte's peptone 10 grm. Nutrose . . . • . 10 „ Sodium chloride . . . . . 5 ,, Steam for one hour, and add 25 grm. of powdered agar. Steam for three hours, bring to a reaction of + 10, and filter through " papier Chard in." Mi -dure B.— Boil for a few minutes 100 c.c. of Kubel- Tiemann's litmus solution, add 15 grm. of pure powdered lactose, and boil again for a few minutes. Specific Organisms in Water G25 Add B to A, and to this mixture add 2 c.c. of a hot 10 per rent, solution of anhydrous sodium carbonate and 10 c.c. of a 0-1 per cent, solution of krystal violet. The medium is y$\Ls b-jir^" sterilised . In using the medium it should he employed as surface plates. then tubed, 10 c.c. being placed in each test-tube, and The required number of tubes are melted in a water-bath, and / i t heir contents poured out into sterile Petrie dishes and allowed J^JI to set. These sterile plates are then placed in the warm - — / incubator for an hour or so with the lids slightly tilted at one ^fsj^i edge, so that the surface of the medium may dry somewhat. ^y\/^Jl^ The matter to be plated is sufficiently diluted, and from a few drops to 0-5 c.c. are run on to the surface and spread by means of a glass rod bent into a flattened hook, and sterilised \ by boiling. ^Qn this medium in forty-eight hours -^ggZL. fornis large^recl(^h2niesA^. typhosus and B. dysenteric small "jlue .colonies, and streptococci small delicate red colonies. tier organisms are to a large extent inhibited from developing. (7) S.D.8. rebipelagar (Houston). — "Eebipelagar" has been much used by Houston1 for the isolation of B. coli. It lias the following composition : Agar 20 grin., taurocholate of soda 5 grin., lactose 10 grm. neutral-red 4 c.c. of a 1 per cent, solution, peptone 20 grm., water 1 litre. The S.D.S. rebi- pelagar has the following composition : Agar 20 grm., tauro- cholate of soda 5 grm., lactose 2-5 grm., neutral-red 4 c c, of a 1 per cent, solution, peptone 20 grm., saccharose 2-5 grm., dulcitol 2-5 grm., salicin 2-5 grm. The Isolation of Specific Organisms from Water. The principal disease-p inducing organisms conveyed by water are the B. typhosus, B. paratyphosvs, B. dysenteric, and Spirillum cholera'. The Isolation of B. typhosus, B. paratyphosus, and B. dysenteric prom Water.— There is great difficulty in isolating the B. typhosus from water that lias been very 1 First Rep. on Research Work, Met. "Water Board, 1908. 40 626 Manual of Bacteriology copiously contaminated with specifically polluted sewage, there is, therefore, far greater difficulty when the specific pollution has been small in amount. The earlier records of the isolation of the B. typhosus must be accepted with much scepticism, as the methods of identification were formerly incomplete and unsatisfactory. It is necessary to bear in mind that usually, when drinking-water has suffered sewage- pollution, the amount of the pollution is relatively very minute when compared with the great bulk of the water supply. Moreover, allowing ten days as the average incubation period of typhoid fever, another week before the disease comes under notice, and another week before the fact that an epidemic is in progress is recognised, at least a month will have elapsed between the date of infection of the water supply (supposing this to have occurred on one occasion only, as may be the case) and the taking of the samples for examination, a period during which all the typhoid bacilli may have died out. The contamination of water may, however, be of an intermittent nature. Numerous methods have been devised for the isolation of the typhoid bacillus from an infected water. With rare exceptions, it is impossible to detect the organism by direct plating ; it is too scanty and too mixed with other organisms to admit of this, and therefore concentration of the bacterial content of the water must be attempted. The following are some of the methods which have been suggested for this purpose ; they serve equally well for B. paratyi)lw*n< and B. dysenterise. 1. Filtration through a porcelain filter.— By passing one to two litres of the water through a sterile Pasteur-Chamber- land filter, the whole of the organisms present may theoreti- cally be collected in a few c.c.'s. Practically, however, a large proportion of the organisms are lost in the process : perhaps they get carried into and remain in the superficial layers of the filter-candle, and for this reason, though some- times employed, this method has been largely given up. i See H. S.' Wilson, Journal of Hygiene, vol. v, 1903, p. 429 ; McWeeney, Brit. Med. Journ., 1909, vol. ii, p. 866. Isolation of Bacillus Typhosus G27 2. Concentration. — W. J. Wilson1 has devised the following method : The water is placed in one or two Winchester quart bottles, and 10 c.c. of nutrient broth are added for every litre. The bottles are placed in a water-bath' main- tained at 37°-40°C., and are connected by rubber corks and tubing with a condenser (at a lower level) through which cold water continuously passes, and the tube of the condenser is connected to a large bottle (at a still lower level) . This bottle is kept partially exhausted by means of a filter-pump. The water evaporates and is thus concentrated, the evaporated water being condensed and collected in the exhausted bottle. It requires twenty-one to twenty-two hours to evaporate a litre of water. The water remaining in the bottles, now concentrated to a few c.c.'s., is then plated on Conradi- Drigalski or malachite-green agar. 3. Chemical precipitation. — These methods depend on the formation in the water of a fine, inert precipitate, which entangles and carries down with it a large proportion of the bacteria present. Thus, in the Vallet-Schuder2 method, to 2 litres of the water are added 20 c.c. of a 775 per cent, solution of sodium hyposulphite and 20 c.c. of a 10 per cent, solution of lead nitrate. The precipitate is allowed to settle or is centrifugalised off, is dissolved in a small volume of a saturated solution of the hyposulphite, from which plates are made in suitable media. Ficker3 uses ferrous sulphate after making the water faintly alkaline with caustic soda; the ferrous hydrate formed carries clown the micro-organisms (this must be a risky procedure, as the typhoid bacillus is very sensitive to caustic alkalies). Iron oxycbloricle may also be used as the precipitant. H. S. Willson {Ion. cit.) employs alum. A stock solution of alum is prepared, containing 10 grm. per 100 c.c, and of this sufficient is added to the water to obtain 0"5 grm. to the litre. After the precipitate of aluminium hydrate has formed, the vessel is well shaken to mix its contents, and the mixture is centrifugalised for fifteen 1 Brit. Med. Journ., 1907, vol. i, p. 1176. 2 Zeitschr.f. Hyg., xlii, No. 2, p. 317. 3 Hyg. Rundschau, xiv, No. 1, 1904, p. 7. 628 Manual of Bacteriology minutes at 2000 revolutions per minute. The clear, super- natant fluid is then syphoned or poured carefully off from the precipitate, and the mass of precipitate in the conical extre- mity of the tube stirred up with the little fluid (0"5 to 1 c.c.) remaining. The suspension is then plated out on Conradi- Drigalski, malachite-green or brilliant-green, agar. This seems to be a very promising method. 4. Serum agglutination. — An anti-typhoid serum— the serum of an animal which has been inoculated several times with the typhoid bacillus, having the power of agglutinating typhoid bacilli — if added to a water would presumably agglu- tinate any typhoid bacilli into masses which will sediment or may be centrif ugalised off. The method has been used by Scliepilewsky,1 who adds 10 to 20 c.c. of the water to flasks containing 50 c.c. of nutrient broth, to which after three or four days' incubation at 37° C. an addition of the typhoid serum is made, and after standing for some hours andcentri- f ugalising, the deposit is plated out, 5. Method of enrichment.— The principle of this method is to devise a medium which shall allow of the multiplica- tion of the typhoid bacillus, and at the same time prevent, or at least retard, the growth of B. coli and allied forms. Almost all the methods which have been introduced for this purpose fail, inasmuch as, though they inhibit the growth of a great many organisms, they do not inhibit the growth of the B. coli, or, if they do, inhibit the B. typhosus to a°still greater degree. Roth* found that caffeine in broth would retard B. coli, but allow B. typhosus to multiply. The method has been further elaborated by Hoffmann and Fieker,3 who convert the water itself into a nutrient medium by the addition of 1 per cent, of nutrose, 0'5 per cent, caffeine, and 0-001 per cent, of krystal violet. The mixture is incubated at 37° C for not more than twelve to thirteen hours, at the end of which time the typhoid bacilli should have multiplied to such an extent as to permit of direct isolation by plating, the i Centr.f. Bald., Oriij., xxiii, No. 5, 1903. - Hyg. Rundschau, xiii, 1903, p. 489. » Ibid., xiv, 1904, p. 1. Isolation of Bacillus Typhosus 629 B. coli being inhibited. Many observers have shown, how- ever, thai while caffeine may materially help, it cannot be entirelv relied on to eliminate B. coli and allied form. 6. Process of Cambier — Cambier1 has devised a process ba sed on the idea that an actively motile organism will find its way through the pores of a porcelain filter more quickly than feebly or non-motile forms. His procedure is to make use of a special alkaline peptone medium, which is placed in a glass jar. In this is immersed a Pasteur-Chamberland filter- candle half filled with the same solution, to which is added a little of the fluid to he examined, and the Avhole is incubated at 37° C. Sooner or later growth appears in the fluid outside the candle, and Cambier states that if typhoid bacilli be present they will make their appearance before B. coli. In hands other than those of Cambier, however, the method has not proved successful. 7. Fuchsin Agar (Endo). — One litre of 3 per cent, nutrient agar is made alkaline with 10 c.c. of 10 per cent. NaOH solution after neutralisation. Pure lactose 10 grin, and saturated alcoholic fuchsin solution 5 c.c. are added, and after mixing, 25 c.c. of fresh 10 per cent, solution of sodium sulphite are added. The medium when cold should be colourless. The medium is used as surface plates, and on it typhoid and para- typhoid colonies arj colourless, coli colonies are red. 8. Malachite-green Media. — Loffler has found that mala- chite green (No. 120 Hoechst) in the proportion of about 1 in 5000 in media inhibits the growth of B. coli while still per- mitting the growth of B. typhosus. The dye may be Lidded either to liquid or to solid media. The medium recommended by Loffler2 is composed of 3 per cent, agar made with meat infusion, with 1 per cent, nutrose, and containing in every 100 c.c. 2-25 c.c. of a 1 per cent, solution of malachite green. On this medium the B. typhosus grows in twenty-four hours as delicate, slightly crinkled colonies, surrounded by a colourless zone (due to alkali formed by the bacilli). Thus 1 Rev. d'Hyg., 1902, p. 64. a Deutsch. med. Woch., 19U6, No. s. 630 Manual of Bacteriology it is possible to detect one colony of It. typhoms among 300 to GOO colonies of other bacteria. As a medium for "enriching" — i. e. for specially advancing the growth of the B. typhosus — Loftier recommends a 15 per cent, gelatin, prepared with beef- juice and peptone, and containing per 100 c.c. 3 c.c. of doubly normal phosphoric acid and 2 c.c. of 2 per cent, malachite-green solution. With the suspected matter, firstly, one series of malachite-gelatin plates is prepared and incubated at 25° C. for twenty to twenty-four hours ; secondly, a tube of malachite gelatin is inoculated and incubated at 37° C. for twelve to twenty-four hours ; from this a second tube is inoculated and incubated at 37° C, and then plated out on malachite gelatin and incubated at 25° C. The colonies of B. typhosus are well marked after twenty to twenty-four hours, as large as a pin's head, transparent, highly refractile, light grey and granular. Their shape is circular or oval, and they show characteristic offshoots resembling a bone- corpuscle or the body of anacarus. By using this 15 per cent, gelatin, which can be incubated at 25° C, there is the double advantage of speedy growth and formation of very characteristic colonies. Houston recommends S.D.S. rebipelagar (p. 625) with the addition of malachite-green to the extent of 1 in 5000 (0-2 grm. to the litre). On this medium B. typhosus forms colourless colonies ; most other bacteria do not grow, or appear as blue- black colonies. 9. WerbitzkVs China green agar, — Fur this 3 per cent, nutrient agar (reaction +13) is used, and to every 100 c.c. of the agar 1-4-1-5 c.c. of a 0-2 per cent, aqueous solution of china green (G-riibler's) are added. 10. Brilliant green agar. — Conradi devised an agar containing brilliant green and picric acid, and this has been modified by Fawcus1 as follows : To 900 c.c. of tap- water are added sodium taurocholate, 5 grm.; powdered agar, 30 grm.; peptone, 20 grm. ; and sodium chloride, 5 grm. Dissolve the constituents by steaming for three hours, filter through wool, and bring to a reaction of + 15 (by means of lactic acid or NaOH, as the case may be). In 100 c.c. of distilled water dissolve 10 grm. i Journ. Boy. Army. Med, Corps, February, 1906, p. 147. Isolation of Bacillus Typhosus (3:31 lactose and add this to the former, filter, distribute m Masks (100 c.c. in each), and sterilise. At time of using, melt and add to each 100 c.c, 2 c.c. of a 1-1000 aqueous solution of brilliant green and 2 c.c. of a 1-100 aqueous picric acid (extra-pure, Griibler's). Typhoid forms round, transparent retractile colonies of a light pale green colour by transmitted light, B. coll dark green colonies with an opaque spot at the centre. Conclusion.— The writer would suggest for the isola- tion of B. typhosus from water: (1) Concentration of the organism by precipitation with alum (Wilson's method") or iron oxychloride, followed by plating of the precipitate on Conradi-Drigalski agar, or, better, on malachite-green agar (Loffler's or Houston's, No. 8 above), or brilliant-green agar (No. 10 above) ; (2) enrich- ment by LOffler's method and subsequent plating. In all cases the organism isolated must be examined as to its morphological, cultural, and biological characters, and should have its agglutination and Pfeiffer reactions tested with a high-grade typhoid serum. Two organisms which are likely to be mistaken for the B. typhosus, unless all tests are applied to them, are the B. (J)]^caUs)-udJadigenes and_-B. (aquatilis) sidcatus. Both occur in the dejecta and in polluted water, and are very like the B. typhosus in morphology, motility, staining, and cultural reactions, but neither aggluti- nates with typhoid serum. The B. alfcaLigenes some-- times produces a brownish growth on potato, it renders litmus milk alkaline and produces alkali, but no gas, in glucose, lactose, dulcitol, mannitol, sacchai*ose, and salicin. The B. sidcatus hardly grows at 37° C. and is almost a strict aerobe, little growth occurring in the depth of a stab. Some varieties of typical and of atypical B. coll agglutinate with typhoid serum, so Manual of Bacteriology that a positive agglutination reaction does not. neces- sarily prove that an organism is B. typhosus. The Isolation of the Cholera BacillttsfromWateb. — The detection of Koch's comma bacillus (Spirillum choleree) in water, as in the case of the typhoid bacillus, is a matter of some difficulty, as this organism is rapidly overgrown by the ordinary water bacteria. In the examination of suspected water supplies, the best method to employ for the detection of this organism is to take advantage of the fact, first noted by Dunham, that the cholera spirillum multiplies with great __rapidity in alkaline saline peptone "solution. The suspected water is examined as follows : To 300-500 c.c. of the water are added 1 per cent, each of pure peptone and of common salt ; the mixture is made faintly alkaline with sodium carbonate, distributed in a dozen small Erlenmeyer flasks having a layer not more than an inch deep in each, the flasks are loosely capped with caps of filter-paper, and incubated at 37° C. At intervals of ten, fifteen and twenty hours respec- tively, hanging-drop and cover-glass preparations are made from the top of the liquid, on which there is often a, surfacefilm, and care must be taken not to disturb this; these are then examined microscopically for vibrios and spirilla. At the same time agar, or, better, blood alkali agar (p. 471) plates are prepared and incubated at- blood-heat, Any colonies that appear which resemble the cholera spirillum are examined microscopically ; if the organisms are comma-shaped, they are at once subcultured into peptone water and other media. The original peptone water culturesare tested for the indole reaction with piu-e~hydrochloric~acid, withdrawing some of the contents of the flasks with a sterile pipette. Any likely spirillum isolated must have its cultural and biological reactions investigated and be tested for H^ii«»-o-l|itinn,tirm and Pfeiffer reactions with a high-grade cholera serujn. ' On the survival of the typhoid and cholera organisms in water, see pp. 381 and 461 respectively. Ice and ice -creams may be examined by methods similar to those used for water, the material being first melted at a- low Sterilisation of Water 633 temperature. Some of the fluid should also be cemfcrif ugalised and the deposit examined microscopically for gross con- tamination. The infection in typhoid fever and cholera, and perhaps also in bacillary dysentery, is perhaps more frequently water-borne than conveyed in any other way. It might be supposed that the acid gastric juice would prevent this, and it may do so in many instances. Experiments by Macfadyen1 showed that, whereas in fasting animals, to which suspensions in water of the cholera spirillum were administered, living spirilla pass into the intestine, when the vehicle is milk none could be detected in the intestines. The inference is that when there is no food there is no gastric juice secreted and the organisms are able to pass on into the intestine, but when food is present the gastric juice is secreted and the organisms are destroyed. Sterilisation or Water.- -This maybe done on the small scale by heat, by the use of germicidal agents, or by filtration through a filter (see p. 635). Heat may be applied by simple boiling, or by the use of apparatus in which the water is heated to 65°-90° C, and the outgoing hot water is cooled by the ingoing cold water, which itself is thus warmed, thereby effecting economy in fuel (Griffiths' and other sterilisers). The chemical germicides that have been employed are (1) sodium bisulphate, 15 grams to the pint ; (2) Potassium permanganate, sufficient to tinge the water deeplv for at least half an hour ; (3) chloriue gas or iodine tablets,2 in both cases the taste of the agent being destroyed by the addition of sodium sulphite; (4) copper and copper sulphate. Sufficient metal is dissolved from bright copper in twenty-four hours to destroy typhoid and cholera. Copper sulphate 1 in 100,000 or less is similarly germicidal, and in still smaller quantities (1 in 1,000,000) destroys algae, and has been used for the purifica- 1 Journ, of A mi I. and Physiol., vol. xxi. 2 Nesfield, Journ. Prev. Med., vol. xiii, 1905, p. 623. 634 Manual of Bacteriology tiou of reservoirs overgrown with algae. On the large (also small) scale, chlorine derived from hypochlorites is one of the simplest and most efficient agents. Moor and Hewlett1 showed that 0'25 part of chlorine (equivalent to about 0- 75 part of good chloride of lime) per million parts of chalk water is sufficient to kill B. co!i in half an hour. The taste disappears quickly in bright sunlight and on standing, or may be removed by an addition of sodium sulphite. If the water is organically polluted, more chlorine must be used. Ozone produced by high-tension electric discharge is also employed on the large scale for the sterilisation of water supplies, e. g. at Chartres. Examination op Shell-Fish. — Shell-fish may come from sewage-polluted layings (see p. 382). The following method may be employed for their examination (after Houston) : The outside of the shells are. cleansed by thorough scrubbing and rinsing in tap-water, and a final rinse in sterile water. The fish after cleansing are laid on a sterile towel. The operator then cleanses his hands and opens the shells aseptic-ally with a sterile oyster-knife, care being taken to avoid loss of their contained liquor. The liquor as each fish is opened is poured into a sterile litre cylinder, and the fish is cut up with sterile scissors and added to the liquor in the cylinder. Ten fish should be treated , the volume of fish + liquor noted, and sterile water is then added to make up to 1 litre; 100 c.c. liquid therefore corresponds to one fish. In addition, four dilutions of the liquid are prepared— 1 in 10, 1 in 100, 1 in 1000, and 1 in 10,000. With the liquid and dilutions gelatin and agar plate cultivations are prepared for the enumerations of the organisms present. Cultures are also made in litmus lactose bile-salt peptone water and in milk for the enumeration and isolation of B. coli and B. Welchii respectively, taking 100 c.c, 10 c.c, and 1 c.c. of the liquid, and 1 c.c. of each of the four dilutions ; in this way the contents of the fish, ranging from one fish to T,ro0ooo of a fish, are examined. The process and principles involved 1 Rep. Mad. Off. hoc. Gov. Board for 1909-10, p. 559. Filters correspond to those described for water. Houston Las suggested for oysters as a lenient standard less than 1000, and as a stringent standard less than 100, B. call per oyster. Even ten B. eoli per fish should be viewed with suspicion, for Hewlett and others have shown that oysters from pure layings contain no B. cub. Watercress, etc., may be examined in a similar manner, 100 arm. being weighed out and transferred bit by bit with sterilised forceps and scissors to a flask containing 900 c.c. of sterile water. The flask is shaken vigorously, and the washings examined in a manner similar to that employed for shell-fish. Filters. — Keference has already been made to the removal of organisms in water by sand filtration. With regard to filters for domestic use, few of those in the market are capable of doing more than removing particles of suspended matter, while they allow from 5 to 50 per cent., or even more, of the bacteria present in the water to be filtered, to pass through. Such filters are, of course, useless for the prevention of disease — in fact, rather favour it, by engendering a false sense of security ; and when in use for some time without cleaning, the water after filtration may be worse, bacteriologically and chemically, than before filtration. Woodhead and Wood1 found that the only filters which were capable oE completely removing organisms were the Pasteur-Chamberlancl, Berkefield, and Porce- laine d' Andante. The Berkefeld, while more rapid in action than the other two, after being in use for a few days may nilow some organisms to appear in the filtrate. This, perhaps, is due rather to a growth of organisms through the pores of the filter-candle than to 1 Brit. Med. Journ., 1894, vol. ii, p. 1053 el seg_. Manual of Bacteriology a direct passage. Lunt] Pound that while the ordinary water bacteria,, such as the B. fluoresceins liqiiefaciens, appeared in the filtrate from a Berkefeld filter within a lew days of the infection of the sample, the typhoid bacillus and the comma bacillus similarly introduced had not passed through the filter four or five weeks after infection. Horrocks,3 however, does not confirm this, and 1ms found that when sterile water is inoculated with typhoid bacilli and run daily through a Berkefeld filter, the bacilli appear in the filtrate in one to two weeks, whereas this is not the case with the Pasteur-Chamber- land. The writer has made some similar experiments, which partially, but not entirely, support Horrocks's conclusions. Much evidently depends upon the chemical composition of the water. Messrs. Doulton have constructed a porcelain filter which seems to be perfectly efficient, like the Pasteur- Chamberland. All porcelain filters should be cleaned weekly by well scrubbing with a, nail-brush and boiling in water containing some sodium carbonate. The Bacteriological Examination of Water- Filters. The large majority of water-filters at- present in use are incapable of preventing organisms from being washed through into the filtrate. In order to ascertain whether this is the case with any particular filter, it should be sterilised in the steam steriliser, and water containing organisms of known species (B. prodigiosus, B. violaceus, and M. agilis are very suitable) should be passed through it for twenty -four hours. This water and the filter should during this period of Hie examination be maintained, if conveniently possible, at a temperature below 5° C. This will almost invariably prevent J Trans. Brit, hid o/Prev. Med., vol i, 1897. - Brit. Med. Journ., 1901, vol. i, p. 1471. Protozoa and Algae in Water 637 any growth or multiplication of the organisms. Samples should be taken immediately after the filtration has begun, and at intervals during the day, and again at the end of twenty-four hours. If they are all sterile, the filter is capable of preventing organisms from being directly washed through, In the case of niters of very great density or depth of filtering medium, it may be necessary to prolong the period of examination beyond the first day ; but most ordinary filters which permit organisms to be washed through do so within the first few hours. Protozoa and Algae in Water. The examination of water for the minute forms of life other than bacteria, and their enumeration, can be carried out by the Sedgwick-Eafter method.1 A 6-inc!i glass funnel is plugged at the bottom of the stem with a perforated rubber cork, over the upper end of which a disc of fine silk bolting cloth, cut by a wad-cutter, is laid. Sharp, clean, dry quartz sand is then poured into the stern of the funnel to the depth of half an inch above the plug. The sand should be of such a size that the grains will pass through a sieve of 60 meshes to the inch, but not through one of 120 meshes. The sand is washed into place and well moistened with a little distilled water free from organisms. The water to be examined is thoroughly shaken and 500 c.c. are poured into the funnel ; it runs through the sand, which detains any organisms it may contain. After the water has all passed through, the rubber plug is carefully removed and the sand washed clown into a test-tube with 5 c.c. of distilled water. The contents of the test-tube are agitated and the tube is allowed to rest until the sand has deposited. Immediately this is the case the supernatant fluid is decanted into a second test-tube, carrying with it the organisms. One cubic centimetre of this is withdrawn by a pipette from mid- way between the top and bottom and transferred to the 1 Calkin, Twenty-third Ann. Rep. Slate Board of Health, Massa- chmetts; 1891. f>38 Manual of Bacteriology counting plate. This consists of an ordinary glass slide on which a rectangular brass cell (20 x 50 mm.) is cemented, so enclosing exactly 1000 square mm. The brass cell is 1 mm. thick, so that the cell contains exactly 1 c.c. The preparation is covered with a cover-class and examined with a low power.1 The Bacteriology of Air. Just as in water, the bacteria in the air vary con- siderably at different times and seasons, under different conditions, and in various localities. The species met with are mostly saprophytes, consisting largely of chromogenic forms. A number of moulds occur fas spores), and, in fact, ordinarily are in lai'ge excess, together with yeasts and torulas. It is not easy for micro-organisms to become diffused through the atmosphere ; they are incapable of a voluntary rising, and cannot be torn from a fluid or moist solid medium by a strong current of air. The medium on which they are growing must dry up completely and crumble into fine dust before they can be distributed through the agency of air-currents (but see p. 385). The number of organisms in the air varies with the season, with rain, with altitude, with movement, etc. At Montsouris, Miquel found in one cubic metre of air 49 organisms in winter, 85 in spring, 105 in summer, and 142 in autumn. After heavy rain the air is largely freed from organisms. Frankland found at Norwich Cathedral at an altitude of 300 feet 7 organisms in two gallons, while on the ground 18 were found; at the Golden Gallery at St. Paul's two gallons of air contained 11 organisms; in St. Paul's church- 1 On the microscopy of water, see Whipple, Microscopy of Drinking Water. Bacteriology of Air 639 yard the number was 70. On high mountains organisms are nearly absent from the air, and the same is the case at sea at a distance from land exceeding about 100 miles. Organisms are much fewer in the air of the country than in that of towns. At the entrance-hall, Natural History Museum, South Kensington, Franklancl found in the morning 30 organisms ; in the afternoon, when many visitors were present, the number had risen to 292, showing the influence of movement. By keeping a volume of air absolutely still, enclosed in a box the walls of which were smeared with glycerin, Tyndall was able to free it completely from particles and organisms. The writer found from 43 to 150 organisms per 10 litres of air in some of the principal streets of London during the daytime. Gordon,1 by exposing dishes of neutral-red broth to the aii', or by aspirating air through neutral-red broth (p. 623), has been able to detect the presence of the 8. salivarius, M. epidermidis , and scurf micrococcus (p. 241) in air subjected to human contamination. By these tests and by the use of B. prod/giostis as an indicator he concludes that particles of saliva are disseminated as far as 40 feet in the act of loud, speaking, indicating the possibility of the wide distribution of such pathogenic organisms as the tubercle, plague, and influenza bacilli and the pneumo- coccus by speaking, and still more so by coughing. The number of dust particles in the air may be enormous. In London Macfadyen and Lunt observed as extremes from 20,000 to nearly 600,000 per c.c. The ratio of micro-organisms to dust particles is therefore a very small one. 1 Reps. Med. Off. Lor. Gov. Board for 1902-1904. 640 Manual of Bacteriology Bacteriological Examination of Air. A number of methods have been devised for the estimation of the number of micro-organisms in the air, of which the following are the principal ones : (1) Plate method — Melted sterile nutrient gelatin is poured into a sterilised Petri dish, and allowed to set. The plate is then exposed to the air, by removing the lid, for a given time —one, live, ten, or fifteen minutes, etc— the lid is replaced, and the plate incubated at 22° C. for some da vs. The number of colonies of moulds, bacteria, yeasts, etc., is counted, and, having estimated the area of the gelatin plate,1 the result is expressed as the number of organisms falling per square foot per minute. The results obtained by this method are roughly comparative, but no estimate can be formed from it of the number of organisms contained in a, given volume of the air. (2) Hesse's method. — This is a quantitative method for estimating the number of organisms contained in a given volume of air. The apparatus consists of a glass tube 30 in. long by 1 \ to 2 in. in diameter. One end of this tube is plugged with a rubber cork through which a glass tube passes, the other end is covered with a piece of sheet rubber perforated with a hole ] to | in. in diameter; over this is placed another sheet of rubber, imperforated. The small tube being plugged with cotton-wool, the whole is sterilised for an hour in the steam steriliser. Just before use 40 to 50 c.c, of melted sterile nutrient gelatin are poured into the tube, and ils walls coated with the medium. The tube is then strapped horizontally on to a tripod stand, and the small tube con- nected by means of a piece of rubber tubing to an aspirator consisting of two flasks arranged so as to form a reversible syphon. A litre of water is poured into the flask connected with the tube, and the outer sheet of rubber having been removed from the end of the tube, the Avater is syphoned over to the second flask, placed at a lower level, and an equal volume of air is thus aspirated through the tube. The second 1 The area of a circular dish is calculated by multiplying the square of the diameter by 0-785. Frankland's Method 641 flask is then connected with the tube, and the position of the flasks being reversed the water is again syphoned over and a second litre of air passes through the tube, and this process is repeated until 5, 10, 15, or 20 litres of air have been drawn through the tube. The rate of flow is controlled by a screw- clamp ou the rubber connecting-tube; it should not exceed half a litre per minute. With this rate of flow all the organisms are deposited on the gelatin-coated tube. The aspiration being completed the rubber tube is disconnected and the sheet of rubber replaced over the end of the tube, which is then incubated, and the colonies are counted when they have developed. (3) Petri's method. — Petri aspirates the air through a glass tube containing sterilised sand, kept in place by fine wire- gauze wads. When the sample has been taken, the sand is a B c Fig. 67. — Frankland's tube for air analysis. distributed in Petri dishes, and melted sterile gelatin is poured over it and allowed to solidify, plate cultures being thus prepared. The objection to this method is the presence of the opacpie particles of sand in the culture medium. (4) FranJcland's method.— The air to be examined is aspirated through a tube 5 in. in length and \ in. in diameter (Fig. 67). One end of the tube is open, the other (c) is plugged with cotton-wool. At a distance of 1 in. from the open end the tube is slightly constricted to support a plug of glass wool (a). At a distance of 2^ in. from this plug the tube is again constricted to support a second plug (B), con- sisting of glass-wool and finely powdered, cane-sugar, supported in front and behind by plugs of glass-wool. Several such tubes having been prepared, they are placed in a tin box and sterilised at 130° C. for three hours, and can then be easily transported without risk of contamination. When required for use, a tube is quickly removed from the box, being handled 41 642 Manual of Bacteriology by the plugged end, which is connected by stoul rubber tubing 1o aspirating flasks such as are used in Hesse's apparatus. The tube is clamped horizontally to a retorl stand, and by attaching the second flask to a, small hand exhaust-pump, the water can lie syphoned over from the first flask, a, corresponding volume of air passing through the tube. When the desired volume of air has been aspirated '«rv^ through the tube, it. is disconnected ami placed in another sterile tin box. As many tubes as desired can be employed to con- trol one another or to examine the air in different localities and under different con- ditions. All the samples having been taken, the tubes are manipulated on re- turning to the laboratory. The tubes, as before, beinsj; handled by the ends only, a file-mark is made across the centre of each tube, which is then broken in half and the plugs of glass-wool and sugar are. shaken, or pushed by means of a, sterile wire, into a sterile flask of about 250 c.c. capacity. Into this 10 or 15 c.c. of liquefied sterile nutrient gelatin are then introduced; the sugar dissolves, the glass-wool becomes disintegrated, and a roll-culture is made on the walls of the flask, which is incu- bated at 22° C, and the colonies are counted Fm. 68 — Sedgwick when they have developed, and Tucker's tube (5) Sedgivich and Tucher's method.— for air analysis. 0m q£ f]w best all<1 most convenient methods for the bacteriological examination of air. A o-lass tube of special form is employed (Fig. 68); this consists of an expanded portion (a) about 15 cm. long and 4-5 cm. in diameter; one end of this is contracted so as to form a neck 2"5 cm. in diameter and m length ; to the other end is fused a glass tube (b c) 15 cm. long and 0"5 cm. in diameter. The neck of the tube is plugged with cotton- wool, and two cotton-wool-or, better, glass-wool-plugs are Bacteriology of Soil 643 inserted in the narrow tube, one at its open end, the other (c) about 6 to 8 cm. horn the wide part. The whole is then sterilised. When cool, the narrow part of the tube, from its origin at the wide part down to the first plug (c), is filled with powdered cane-sugar (No. 50, B.P. gauge) which has beea carefully dried and sterilised at 120o-130° C. The tube is again sterilised at 120°-130° for two or three hours, the greatest care being taken not to melt the sugar. After sterilisation the tube is ready for use. The wool plug is removed from the mouth and a measured volume of air is aspirated through the layer of powdered sugar by means of a small hand air-pump, the volume of air being measured by the displacement of water in a flask. Having taken the sample (5 to 20 litres), the wool plug is replaced in the neck. The powdered sugar is then shaken down into the wide part of the tube (a), and 15 c.c. of melted sterile nutrient gelatin are poured in. The powdered sugar readily dissolves in the melted gelatin, and when solution is complete a roll-culture is made in the tube, just as in Esmarch's method (p. 83). The tube is then placed in an incubator at 20° C, and the colonies are allowed to develop. In both Frankland's and Sedgwick and Tucker's methods the sugar, after powdering and sifting and before introducing into the tubes, should be thoroughly dried by keeping in the warm incubator for several days with occasional stirring. Unless this be done, the sugar is apt to cake and discolour during sterilisation. Soil. The upper layers of soil contain large numbers of organisms, chiefly bacilli. The species are very varied; among patho- genic ones may be named the bacillus of tetanus and of malignant oedema. The B. mycoicb's is very abundant, and the varieties of Proteus, the hay and potato bacilli, are common, while the nitrifying forms are of course present, but do not develop on ordinary media. Below five or six feet aerobic organisms become scanty, but (1 14 Manual of Bacteriology the anaerobic and thermophilic ones are still met with. The number of organisms present in soil is variable, from 200,000 to 45,000,000 in ordinary earth, while in dirty and busy streets there may be as many as 1,000,000,000 per gnu. According to Houston, uncultivated sandy soil averages 100,000, garden soil 1,500,000, and sewage polluted 115,000,000 per grin. Houston1 found that ia virgin soils the B. coli, B. Welchii, and streptococci are practically absent, but that in soils polluted with animal excrement by manuring or otherwise the spores of B. Welchii a,re present in great abundance, also B. coli and streptococci if the pollution be of recent date. The length of time pathogenic bacteria retain their vitality in buried corpses has been the subject of experiment by Losener,2 who injected cultures into the bodies of pigs, which were then wrapped in linen, placed in wooden coffins, and buried. The conclusions he arrived at were that, provided the soil has good filtering properties, there is practically no chance of the dissemination of a virus. Klein/ experimenting with the bacilli of diphtheria, cholera, plague, typhoid fever, etc., also found that the vitality and infective power of these organisms passed away in a compara- tively short time, in most cases within a month. On the survival of the typhoid and cholera organisms in soil see also pp. 383 and 461 respectively. Examination of Soil. The bacteria in the soil may be examined by adding traces of the soil to sterile nutrient broth, thoroughly crushing and soaking it, and then mating plate or roll cultures, aerobic and anaerobic. To make anything like an accurate quantitative examina- tion ie almost' impossible. Weighed amounts of the soil, 1 Rep. Med. Off. Loc. Gov. Board for 1889-1900. 2 Centr. f. Bald. (lle Abt.), xx, 1896, p. 454. 3 Rep. Med Off. Loc. Gov. Board for 1898- 99, p. 344. Bacteriology of Sewage after thorough pulverisation is an agate mortar, may be introduced into sterile test-tubes and thoroughly exhausted by repeated washing with sterile water or broth, plate cultiva- tions being made with the washings. Various boring apparatus have been devised for withdrawing soil from different depths. Sewage.1 Sewage is exceptionally rich in organisms, but the numbers present are variable. Jordan in Massachusetts found an average of 708,000 per cubic centimetre. Laws and Andrewes found from 905,000 to 11,216,000, the latter being the highest number obtained. The number of organisms naturally varies at different seasons and with the amount of dilution. The organisms present are very varied, but moulds, yeasts, and sarcinse only occasionally occur. A few micrococci are met with and streptococci are present in considerable numbers, at least 1000 per c.c, but bacilli, especially licpaefying forms, largely predominate. The commonest species are the B. fluorescens liquefaciens and varieties, several varieties of Proteus, the B. filamentosus, vai'ieties of the B. mesentericu?, B. mycoides, B. subtilis, B. cloacte, and the colon bacillus. The latter numbers from 20,000 to 2,000,000 per c.c, and the other bacilli mentioned number 200,000 to 2,500,000 per c.c. Many anaerobic sporing bacilli are also found, especially the B. Welchk, the spores of which number from 30 to 2000 per c.c, averaging 500-600. Foreign bacteria introduced into sewage are probably soon suppressed by the predominant species of the sewage. The air of well- ventilated sewers differs but little from that of the external air, and the organisms in it contrast with those of sewage by the abundance of moulds. Specific organisms may, however, gain access to it (p. 340). The powerful liquefying and solvent actions of the bacteria 1 See various Reports to the London County Council by Clowes, Houston, Laws and Andrews ; Klein, Houston, Reps. Med. Off. hoc. (lor. Board I'm' L897-1904; Rep. of the Sewage Commission. 646 Manual of Bacteriology present iu sewage have suggested a means of dealing with sewage bo as to make use of these properties, and many bacteria] systems of sewage disposal have been devised. The principle most widely adopted is to run the sewage into large covered reservoirs (septic tanks), where it remains at rest for twenty -four to forty-eight hours. Here it is under practically anaerobic conditions, and anaerobic bacteria exert their action on the solids, partly dissolving them, partly disintegrating them, with the formation of a sludge which has to he cleared out from time to time. From the septic tanks the sewage passes on to beds composed of broken brick, coke, or some similar material, through which it slowly percolates, and here it is subjected to the action of aerobic organisms, which complete the decomposition to such an extent that the effluent does not affect fish life nor putrefy, so that it may be run into a stream Avithout causing a nuisance. Four sets of these aerobic bacterial beds are usually provided, each set being worked in turn for six hours and resting for eighteen hours during the twenty-four hours. The effluent from such bacterial beds may contain as many bacterias as, or more than, the sewage itself. Pathogenic organisms may be present in it, for Houston found that the B. pyocyaneus added to the beds soon appeared in the effluent. On the survival of the typhoid and cholera organisms in sewage see pp. 383 and 461 respectively. Examination of Sewage and Sewage Effluents. To ensure a fair average sample, the sewage or effluent should be collected in small portions at intervals. The portions are mixed, strained through muslin, and dilutions of 1 iu 10, 1 in 100, 1 in 1000, and 1 in 10,000 made with sterile tap-water. These are then examined according to the following scheme : Bacteriology of Milk Tests. 1. Total number of bacteria 2. Number of spores of aerobes 3. Number of spores of anaerobes Amount of sewage in c.c. 4. Number of orga- nisms liquefying- gelatin 5. Spores of B. Wel- chii (enterit idis sporogenes) 6. Number of B. coli 7. Number of strep- tococci Gelatin and agar plate cultivations Gelatin plate cultures with material pre- viously heated to 80° C. for ten minutes ; Agar plate cultures with material pre- viously heated to 80° C. for ten minutes and incubated anae- robically Surface gelatin plates 0- ooi, o-oooi, o-ooooi 1- 0, 0-1, o-oi i-o, o-i, o-oi Milk cultures heated to 80° C. for ten minutes and incu- bated anaerobically Surface plates of Con- radi - Drigalski, or bile-salt media, etc., as described for water (p. 623) Sur /'ace-plates of Con- radi - Drigalski me- dium (p. 625) 0-001,0-0001,0 00001 0-1, 0-01, 0 001 o-ooi, o-oooi, o-ooooi o-oi, o-ooi, o-oooi Effluents only. 8. Incubate some of the effluent in beakers at 22° C. and 37° C. for some days. A good effluent should yield little or no unpleasant odour (an unpleasant odour indicates the presence of decom- posable organic matter, and such an effluent might give rise to a nuisance). 9. Place a gold-fish or two in a bowl of the effluent. The fish will live in, and be unaffected by, a satisfactory effluent. (This may be done only by a licensee under the Vivisection Act.) Milk.1 Milk is an admirable nutrient soil for the develop- 1 See Houston, Rep. to the London County Council, No. 933, 1905 ; MacConkey, Journ. of Hygiene, vol. v, 1905, p. 333; Hewlett and Barton, ibid., vol. vii, 1907, p. 22; Savage, Rep. Med. Off. Loc. Gov. Board for 1909-10, p. 474; Swithenbank and Newman, Bacteriology of Milk. 648 Manual of Bacteriology ment and multiplication of micro-organisms, and, though sterile in the udder/ as delivered to the consumer may contain an appalling number of bacteria. In milk as ordinarily supplied there are from one to five million bacteria per c.c, and it frequently contains ten to fifteen millions, with an average of about three to four millions. Hewlett and Barton found an average bacterial content of about 1,500,000 in London milk as delivered at the railway termini (the range was from a minimum of 20,000 to a maximum of 8,390,000), but this does not represent the condition of the milk as delivered to the consumer, for the bacteria present rapidly multiply in warm weather. Eyre2 in the middle of summer found the following rate of multiplication : Microbes per c.c. Initial content . . . 56,000 After 12 hours . . . 526,000 After 24 hours . . . 20,306,000 After 30 hours . . . clotted A similar specimen in the middle of winter gave the following results : Microbes per c.c. Initial content . . 20,000 After 12 hours . . . 24,000 After 24 hours . . . 43,000 After 30 hours . ' . . . 280,000 - In New York, Park estimated the average bacterial content of milk as supplied to the consumer at 1,000,000 per c.c. in winter and 5,000,000 per c.c. during the hot months. Byre (loc. cit.) states that, as the result of his observations, the numbers are in London about 3,000,000 1 The " fore " milk may contain organisms which have lodged in the milk-ducts, and it is extremely difficult to obtain completely sterile milk. 2 Journal of State Medicine, vol. xii, 1904, p. 728. Bacteriology of Milk 049 to 5,000,000 in December, January, and February, and 20,000,000 to 30,000,000 in June to September, smaller numbers than these always being associated with the presence of boric acid or formaldehyde. Even in so-called sterilised milks bacteria are rarely com- pletely absent. Cream is even richer in bacteria than milk, and averages about 8,000,000, and may contain as many as 30,000,000 organisms per c.c.1 Although all the ordinary species may be met with, milk has a bacterial flox-a largely its own, comprising many forms producing lactic and butyric acid fermentations. Organisms also occur having more or less specific effects, and giving rise to bitter milk, viscid milk, etc. The lactic ferments are mostly non-sporing, the butyric chiefly sporing, species. The commonest of the lactic ferments is the B. acidi lactici, which has some similarity to the colon bacillus (see table, p. 400). Another common lactic organism is the O'idium lactix, a mycelial form, the colonies of which appear as little fluffy tufts. In addition to the organisms named, pathogenic species may be met with — viz. the tubercle, diphtheria, typhoid, paratyphoid, Gartner, dysentery, and comma bacilli, the M. melitensis, M. 'pyogenes, and the Streptococcus pyogenes (lactic-acid-forming streptococci are also common). The B. coli and B. Welchii are generally present in milk, and the B. lactis arrogates is sometimes found (p. 410). Scarlatina (see " Scarlatina ") and foot-and-mouth disease may likewise be conveyed by milk, and the diarrhoea of infants is largely due to the use of milk swarming with microbes, some of which in themselves may be harmful, and which also by the products they form tend to set up gastro-enteritis. The percentage of samples infected with tubercle bacilli 1 Russell, CenLr.f. Bald. (2'e Abt.), i, 1895, p. 741. 650 Manual of Bacteriology varies much : Barton and Hewlett found only one out of 20 .samples taken at London railway termini, The supply of the large dairy linns is also comparatively free from tuberculous infection, as considerable pre- cautions are taken to exclude tuberculous animals. For the quarter ending March 31st, 1911, of 760 samples examined for the London County Council, 106, or 13:9 per cent,, were found to be tuberculous, and since 1007 of 5698 samples, 640, or 1T2 per cent,, proved tuber- culous (see also p. 337). A poisonous body, tyrotoxi- con (p. 39) has been isolated from milk and milk products. Sources of contamination and infection are derived from the insanitary conditions of many farms and dairies and the dirty methods of those handling the milk. In order to render milk wholesome for infants and free from infective organisms under the present conditions of supply, two methods may be adopted — sterilisation and pasteurisation. To ensure sterilisation it is necessary to heat the milk to boiling-point for six- hours, or to expose it for a shorter period to steam under pressure. Such treatment, however, markedly alters the flavour of the milk, and is said to diminish its nutritive value. If the milk be heated to a temperature not exceeding 70° C, the flavour and nutritive qualities are far less altered, while the patho- genic species are all destroyed. This method is termed '•'pasteurisation," and consists in heating the milk to about 68° C. for twenty to thirty minutes. Pasteurisa- tion destroys 92-99 per cent, of the total organisms present. The objections to pasteurised milk are that t he natural enzymes present in fresh milk are destroyed, the lactic-acid-forming organisms are killed, and if the treated milk be kept, the resfduum of resistant putrefac- tive, etc., bacteria multiply enormously, without obvious change in the milk. Behring has advocated the Bacteriology of Milk 651 addition of formaldehyde to all milk used for the feed- ing- of children. Another method for sterilising milk is the Budde process,1 in which the milk, after the addition of hydrogen peroxide, is heated for three hours to 52-53° C. All non-sporing organisms are destroyed, and the added hydrogen peroxide is decomposed into 11,0 and 0. All milk should be distributed in closed bottles, and pasteurised milk should be consumed within thirty-six hours of treatment. The thermal death-point of pathogenic organisms in milk is as follows Organism. Temperature. Period of Exposure. B. tuberculosis 60" C. 20 min. B. typhosus 60° C. 2 min. B. diphtherix 60° C. 1 min. Spir. cholerse 60° C. 1 min. B. dysenterix 60° C. 10 min. M. melitensis 60° C. 20 min. The thermal death-point of tubercle bacillus, especially in milk, has been the subject of some controversy (see also p. 325). De Man found that an exposure of fifteen minutes at 65° C. was necessary to destroy the infective properties of tuberculous milk. Bang, of Copenhagen, considers that pasteurisation cannot always be relied upon, and recommends that milk should be heated to 85° C. The writer found that the vitality of the ordinary non-virulent laboratory cultures was destroyed by a temperature of 60° C. acting for ten minutes, and that the infective properties of tuberculous sputum, tested on guinea-pigs, were destroyed by a temperature of 65° C. acting for fifteen minutes in five out of six instances. Wood- head's experiments (First Eoyal Commission on Tuberculosis) gave irregular results which seem to be explained by 1 Hewlett, Lancet, 11106, vol. i, January 27th. 2 Rosenau, Hygienic Lab., Washington, Bull, 4-2, 1908. 652 Manual of Bacteriology Theobald Smith's careful work.1 This showed that tuber- culous milk was rendered non-infective by heating to 60° C. for ten to fifteen minutes, provided there was no forma/ion of a surface scum; the latter seems to protect the bacilli. Russell and Hastings2 confirmed Smith's experiments, and assert that it is sufficient to heat milk to 60° C. (140° F.) in a closed receptacle for a period of not less than twenty minutes in order to destroy the tubercle bacillus. The surface scum forms on milk only when it is heated in contact with air ; all pasteurisers, therefore, should be closed vessels. The writer lias devised a simple form of domestic pasteuriser, which is made by Messrs. Allen & Hanbury. The occurrence of so-called leucocytes and pus-cells in milk must be considered. A certain number of cells resembling polymorphonuclear leucocytesare always present in milk, more numerous during the first week of lactation and then accompanied by colostrum cor- puscles. An excess of these cells may indicate some local inflammatory affection of the udder, or, if streptococci and blood are present in addition, suppura- tion, but not necessarily, for Russell and Hoffman, and Revis have shown that a very large cell count (500,000— 1,000,000, or even 10,000,000, per c.c.) may often be obtained from quite healthy cows. The nature of these cells has been the subject of an extended investigation by Hewlett, Villar and Revis.3 Their conclusion is that the majority of these cells are not leucocytes, but are germinal cells of the secreting epithelium of the udder. Blood may also be present transitorily in health (Revis). The presence of squamous epithelial cells indicates desquamation from the teat or udder or from the hand of the milker — i. e. want of cleanliness. There is no doubt that micro-organisms are far more 1 Joum. Exper. Med , vol. iv, 1899, p. 217. 2 l~th Ann. Rep. Wisconsin Agricult Exp. Station. 3 Joum. of Hygiene, vols, ix, x, .and xi, Sour Milk 653 abundant in milk as supplied to the consumer than should be. This arises from the ignorance and care- lessness of those charged with the duty of providing and distributing this important article of diet. The udder and teats of the cow and the hands of the milker (who should wear a special dress) should be wiped before milking, and all vessels should be clean and steamed or scalded before use. The milk should be cooled at once, some more efficiently closed vessel than the present form of milk churn adopted, and the milk not stored, but forwarded without delay by the railway companies in special refrigerator vans. Distribution in bottles would be a great improvement. The following might be suggested as a bacteriological standard for milk : 1 (a) Number of organisms not to exceed 1,000,000 per c.c. ; (b) absence of excess of leucocytes or of pus-cells ; (c) B. coli, B. Walchii, and streptococci should not be pi'esent in 1 c.c. or less ; ('/) the sediment after centrifue-alisino- should be less than 100 parts per million ; («) the milk as delivered should not have a temperature above 10° C. ; (/) absence of pathogenic organisms. Sour milk. — Sour milk is used as au article of diet iu many parts of the world, e. (j. Bulgaria. In these sour milks a particular micro-organism or a variety of it, the B. bulgaricus or "bacillus of Massol," is generally present in association with lactic streptococci. It is a large, pleomorphic, Gram-positive bacillus, non-motile, non-sporing, growing best at about 40° C, but only iu milk or in culture media made with milk or whey. It lias been much employed for the preparation of a soured milk which is of considerable service in the treatment of certain disorders.3 1 See "Eep. of a Committee on Milk Supply," Philad. Med. Journ., October, 1900, p. 758 j Park, Journ. of Hygiene, vol. i, 1901, p. 391 ; Houston, loc. ext. 3 See Hewlett and others, Brit. Med. Journ., 1910, vol. ii. (Bibliog.) 6 VI Manual of Bacteriology Examination of Milk. 'Number of organisms per <■.<■. — This is curried out by diluting the milk to 1 in 1000—1 in 1,000,000 with sterile water, or preferably nutrient broth, as a better mixture is obtained. Plates are then made either in gelatin or in distilled water agar (1| grin, powdered agar, distilled water 1 litre, Eastes), or preferably in both media. B. coli, B. Welchii, and streptococci. — These are searched for quantitatively by the methods detailed for "Water" (pp. 609-617). Amounts of milk in decreasing decimal order from 100 c.c. to O'OOOOOl c.c. should be examined. Pathogenic organisms. — The detection of these, with the exception of the tubercle bacillus, is difficult and uncertain. In all cases the milk should be centrif ugalised and the deposit examined. 1. For the detection of the tubercle bacillus1 staining methods are almost useless (except in cases of advanced tuberculosis of the udder) and inoculation must be performed. At least 250 c.c. of the milk should be centrifugalised at 2000 to 2500 revolutions per minute for an hour. As many organisms become entangled in the cream, it is advisable to stop the machine after half-an-hour, stir in the cream, and again centrifuge. The fluid is poured or pipetted off carefully, so as not to disturb the sediment, leaving about 3 c.c. in the tube. The sediment and the remaining fluid are then well mixed and about 1 c.c. is inoculated subcutaneously and intra- peritoneally into two guinea-pigs respectively (see also p. 30 1) . For staining, a process of solution of the milk may be em- ployed, 20 c.c, of the milk being mixed with 1 c.c. of a 50 per cent, potash solution, and heated in a water-bath until the solution turns brownish ; 20 c.c. of acetic acid are then added. The mixture is shaken, heated in a water-bath for three minutes, and centrifugalised for ten minutes. The fluid is poured off, 30 c.c. of hot water are added to the sediment, and the mixture is again centrifugalised. Films are then i See Delepine, Rep. Med. Off. Loc. Guv. Board for 1908-09, p. 134. Examination of Milk 655 prepared from tlie sediment, and stained for the tubercle bacillus (see also p. 343). Non-pathogenic acid-fast bacilli occur in milk (p 358). •_>. The diphtheria, bacillus is searched for by making serum cultures from, and inoculating guinea-pigs with, the sediment. It' a, diphtheroid organism is detected it must be isolated and examined by culture tests and animal inoculation. In milk and cheese a bacillus is frequently met with closely resembling the diphtheria bacillus in its morphological and cultural characters; it is, however, cpaite non-pathogenic.1 3. The typhoid, paratyphoid, Gartner, dysentery, and comma bacilli may be searched for by the methods given for " Water." (4) The M. pyogenes and the Streptococcus pyogenes may be searched for by means of plate cultures on glycerin agar. (5) Examination of sediment. — Houston and Savage (loc. cit.) have devised methods for the cpuantitative estimation id' the sediment by centrifuging in special graduated tubes. For the microscopical examination of the sediment the milk is centrifugalised for twenty minutes at 1500 revolutions per minute, and the upper fluid is pipetted or syphoned off. Some of the sediment should be examined with the § in. and ! in. objectives for the presence of " dirt," e. g. hairs, straw, etc. Three smear preparations are then made, each with four drops of the sediment, which are spread evenly over three fourths of the slide. The slides are air-dried, and may be treated with a mixture of absolute alcohol and ether for ten minutes. One slide is stained with Loffler's blue, another by Gram's method for streptococci, and a third by the tubercle method. The Loffler's blue specimen gives a general idea of the number of bacteria present, and of the presence of cells. Prom Avhat has been said above (p. 652), considerable cauti> m must be exercised in stating the presence of pus-cells. Streptococci present are not necessarily pathogenic, as non- pathogenic lactic-acid-forming streptococci are common. For counting the number of cells present, Eevis2 employs a 1 See Scientific. Bull. No. 2, Health Dept., City of New York, 1895.,p. 10. " Jount. of Hygiene, vol. x, 1910, p. 5S. 656 Manual of Bacteriology centrifuge tube of 10 c.c. capacity, the lower third of which is contracted to 0-8 cm. in diameter, and contains 1 c.c. The procedure is as follows: In the tube are placed 5 c.c. of the well-mixed milk, diluted to the 10 c.c. mark with 0"8 per cent,, salt solution. After inserting a rubber stopper the contents are well mixed. The tube is then centrifuged at about 2000 revolutions per minute for two minutes, the cream is broken up by violently shaking the upper part of the tube, and the rotation continued for four minutes longer. A glass rod, fitting roughly the narrow neck of the tube, is inserted, and the major part of the milk poured off, and the upper part of the tube well rinsed with water to remove cream, etc. ; the contents of the narrow end down to within £ in. of the deposit are sucked out with a fine glass pipette, the upper part of the tube is wiped clean, and the tube is then filled to the 10 c.c. mark with salt solution. The tube, having been violently shaken till all the deposit is distributed through the liquid, is then rotated for four minutes, and the liquid down to within } in. of the deposit again removed. In the case of small deposits, two to three drops of saturated aqueous solution of methylene-blue are added, and the deposit is stirred up by blowing through a fine glass capillary pipette (which is afterwards used for filling the counting chamber) . After fifteen minutes, water is added to the 1 c.c. mark, and counting done in the usual way with a Thoma-Zeiss blood counter. Counting should not be restricted to the ruled spaces, but the field so arranged that a definite number of squares is included, and fields are counted all over the chamber. At least two different prepara- tions should be made of the same deposit for counting. Pood Poisoning. — Apart from the presence of the ordinary poisons, food may be poisonous on eating — (a) naturally, e. g. certain fish, (b) from the results of the activity of micro- organisms with the formation of toxic products, the ordinary " ptomine poisoning," (c) from infection with certain organisms, particularly B. enteritidis, which generally induce gastro-enteritis. In the last named, symptoms do not usually Meat, Bread, and Butter 657 ensue until a lapse of twelve to forty-eight hours after the consumption of the food. Meat is not likely to convey any infective disease with the exception of tuberculosis and anthrax. It may be examined by cultures and plate cultivations, and by inoculation and feeding experiments. Tinned meats, etc., frequently contain sporing organisms of the B. subtilis and mesenteric as groups. They may be examined by aerobic and anaerobic cultures, and by feeding mice. Poisonous ptomines are occasionally present. The B. enteritidis occurs in meat, and causes a form of poisoning (see p. 392). 1 In certain intoxications due to bad meat, known as " botulism," Van Ermengem isolated the B. hotidinus (see p. 450). Bread — Troitzki states that new bread contains no micro- organisms, but Waldo and Walsh found that such organisms as the comma bacillus are not destroyed by passing through the ordeal of the baker's oven. Cut bread forms a good nidus for the development of pathogenic organisms. The Bacillus prodicfiosus may grow upon various food-stuffs, and give rise to suspicion of foul play. L. Parkes- describes cases of diarrhoea which he suggests were caused by this organism. Blotter contains from two to forty-seven millions of micro- organisms per gramme. Tubercle bacilli have been found in butter, and the comma bacillus artificially introduced survives for over a month. "Acid-fast" non-pathogenic forms also occur (p. 358). For the isolation of the tubercle bacillus from butter and cheese the only certain method is by inoculation. Butter may be melted and allowed to stand in the incubator at 37° C. for some days, and the sediment inoculated. As this involves the multiplication of septic organisms, it is preferable to centri- fugalise the melted butter, keeping it melted during the process, and to inoculate the sediment immecliatelv. Clothing, etc.— Attempts have been made to examine clothing, bedding, Hock, etc., by bacteriological methods for filth contamination, but without much success. 1 See Savage, Rep. Med. Off. Luc. Gov. Board for 1909-10, p. 440 2 Brit. Med. Joiidi., 1905, vol. ii, 1330. 42 a 41 = 0 St . 3 » o ^ o = HO P o an- t- t, C G) S S ^ T H c Z = = 2 ' « io 2 i ■. B QJ :a- : a: ; P ■ § a' £ Q> B '£_- S.% of .3 c - o •g a! 0 *^ : K S ,0 E C K fl 6 . , B o'-§ 6 - = s 3 « s .£ & £ B O — S C C*E as V ~- ES. «! — ^ u _ a iff*0 W ana a c'~ o >! io *^ t- ^. 2 ft C ja oa W x B - Eh Eh ' C b '- i ii ■? « 5 B - e 5 a « .2 be - = p 4< . o T o s •- o O c a s g 02 o 13 CO © S o a ;j= III III 1 ^ H £. h - bf.-"^ St, -if. .-c= ,"C-r->c — - hj — ■- i - ^ ' ^ t : - - +3 if ■= s •= s £ £ 5 0 ^ ^ i «-5 So S3 P.H-T P l2 i < < x aj p P X03 •asoonifl 1 I <; ) + + + + + + +1 II •uor4IMII.ini -9JO(lS 1 1 + + + + + 1 II •A'tH^OK !+ II + + + + + + + i 0 COCCUS, (lipid- coccus, tetra- cocc-us, chains and masses Cocci A nthrax-like Somewhat anthrax-like Large rods and filaments Slender rods and filaments Slender rods and filaments Slender rods, filaments, and threads Short rod, al- most coecoid Slender ri >d £ s P DO 5i ; ■ , — y. at it « *-o - i.i' . « a = = S s 0 s j s 1 4! i "? i S^f = § a -2 S'S 8 -0 Spl'f '5 1 %A S -5.1 * - o - = ■ o't ■ CD 2 * r = fq a c - aj a u o C > X ^ 'Z r S 0 B.S i. Disinfection 659 CHAPTER XXII. Disinfection.1 HEAT — STEAM DISINFECTION — OH EM1CAI DISINFECTANTS THEORY OF DISINFECTION METHODS OF DETERMINING DISINFECTANT POWER, Natural agencies restrain the multiplication of disease organisms, but enough survive to determine the persistence of infective diseases, and to call for measures by which communities attempt to cope with them. These measures are broadly isolation, prophylactic inoculation, general improvement in sanitation and nutrition, and disinfection. In the present chapter the methods by which the fourth means of protection may be applied are considered. Disinfection implies tin- removal or the destruction of infective properties, but, for practical purposes, should be understood to mean the killing of the infective organisms to which those properties arc due. For this purpose, the two agencies ordinarily used are heat and chemical action, though, in addition, other methods can occasionally be employed for destroying or excluding micro-organisms. Such are light, desiccation, and filtration. Hea t. — Fire is the simplest and most efficient agent for destroying infective matter. Burning should always 1 See Hewlett, " Milroy Lectures," Lancet, l'JO'J, vol. i. 660 Manual of Bacteriology be employed where possible, as for rags, old clothing or bedding, native huts, etc. For surfaces which would not be unduly injured, such as stables, pens, yards, etc., a torch-fire generated by means of the cyclone burner described by Forbush and Femald has been favourably reported on by Stiles. The apparatus consists of a portable tank, from which paraffin gas oil is driven by a pump through a hose (such as is used for the delivery of oil) to which is attached a pole, consisting of an iron pipe 12 ft. long, which is protected by a covering of wood, and to the end of which is attached a cyclone nozzle. • The fine spray from the nozzle is ignited, and the resulting fierce flame passed over the surfaces to be disinfected. The thorough wetting with water of all such surfaces would practically abolish danger from fire, and by proper adjustment of the power of the flame, and experience on the part of the operator, the method is an efficient one. Dry heat, may also be used, and forms the basis of some disinfectors (Ransome's), but is not nearly such an efficient means as moist heat. The objections to dry heat are, that to ensure the destruction of bacteria and spores the temperature must be high and the heating prolonged. Koch and Wolfhugel found that two hours at 150° C. did not always ensure sterilisation, and Gaff'ky and Loffler state that the spores of some organisms are killed only by exposure to hot air at 140° C. for three hours. Moreover, dry heat has little power of penetration, and it recpiires many hours for the centre of a mass of bedding, or the like, to attain the temperature recpiisite for sterilisation, while some articles and fabrics are distinctly injured by the pro- longed heating. The highest temperature which can be safely adopted for a dry-heat disinfector is about Steam Disinfection 061 190° 0., and then if large masses have to be treated the heating has to be continued for from eight to ten hours. A rise of 5° C. above this temperature is sufficient to damage many woollen goods, which en- hances the objections to a dry-heat disinfector, as it is difficult to keep the temperature of a large chamber constant. For the reasons given above, disinfection by dry heat is often impractible ; on the other hand, moist heat is more effective, is found to work well in practice, and is now generally adopted. In the household, for articles which cannot be burnt, brisk boiling for an hour or so will suffice. Steam disinfection. — For public disinfectors, steam under pressure — i. e. at a pressure greater than that of the atmosphere— is employed. Steam under pressure has not such a deleterious action on articles, with the exception of leather, as dry heat, while its penetrating- powers are far greater. By "saturated steam " is meant steam at the temperature at which it can condense, and the temperature of the condensation point rises as the pressure increases. By "superheated steam" is meant steam at a temperature higher than that at which it can condense; therefore superheated steam has to be cooled down into the state of saturated steam before con- densation ensues. If superheated steam is used for disinfection, it loses heat by conduction, and the rise in temperature of the articles treated approximately corresponds to the fall in temperature of the steam. With saturated steam, on the other hand, immediately it is cooled an enormous amount of latent heat is set free by the change in state from the gaseous to the liquid condition, therefore saturated steam is a far more efficient disinfectant than superheated steam. These considerations should always influence the choice 662 Manual of Eacteriology of a steam disinfecting apparatus for efficient work- ing. The Equifex disinfector is worked with saturated steam at 10 11,. pressure (239° K). The chamber consists of a cylinder of mild steel, made without steam jacket, so as to avoid risk of superheating. The cylinder is lagged with non-conducting composition and wood, to reduce loss of heat by radiation, and, as usually supplied, is furnished with separate doors In,' infected and disinfected articles respectively. An arrangement can be supplied to prevent both doors being opened simultaneously. The Washington-Lyons apparatus, or its modifications, is an elongated cvlin- drical boiler with double walls, forming a jacket, and a door at each end. The chamber is of sufficient size to admit bedding, and is built into the partition wall between two rooms, so that each door opens into a different room. Into one of the rooms the infected articles are conveyed, and are placed in the disinfector as lightly packed as possible ; when disinfected they are removed by the opposite door into the other room, thei-eby avoiding all chance of reinfection. Steam at a- pressure of about 20 lb. is admitted into the jacket and then passes to the inner chamber, the object of the jacket being to warm the chamber, and so prevent condensation. For the same purpose hot air is some- times injected beforehand to warm the chamber and articles, and after the steam disinfection, can again lie injected for drying. The length of time required for disinfection does not exceed a half to one hour. In Thresh's disinfector the steam is generated from a saline solution (calcium chloride), which has a boiling- point (105° 0.) higher than that of water. The thermal death-point of a number of organisms in pure Light and Desiccation ^ culture has been determined by many investigators. Eyre suggests the following as "standard conditions" for deter- mining thermal death-points ; 1. Length of " time exposure " to be ten minutes. 2. Emulsion to be prepared from "optimum cultivation.'' 3. The vehicle in which culture is suspended to be sterile salt solution or sterile distilled water. 4. Strength of emulsion to correspond to about 1 milli- gramme of culture per cubic centimetre. 5. Bulk of emulsion to be not less than 3 c.c. 6. Emulsion to be contained in test-tube of 1-5 cm. dia- meter with walls 1 mm. thick. 7. Emulsion to be exposed to moist heat in a water-bath regulated by a delicate and accurate thermo-regulator. 8. Broth cultivations and agar plates both to be used in determining the death of the bacteria, and the period of observation of these cultures to be extended, when necessary, to seven or fourteen days. The experiments to be repeated at least once. 9. Thermal death-point to be first roughly determined to within 5° C. 10. Thermal death-point to be finally determined to within 1° C, and to be defined as that temperature which causes the death of all micro-organisms exposed to it, within the ten minutes in these standard conditions. Light is not used directly for disinfection, but in- directly in nature and in our homes may not be an unimportant factor. It has previously been referred to at p. 23. Sunlight, and artificial light rich in violet and ultra-violet radiations, such as that emitted by a quartz mercury vapour lamp, are efficient germicides. The latter lias been tested by Barnard and the writer with excellent results, but, unfortunately, the germi- cidal rays have practically no power of penetration and are stopped even by thin glass. Desiccation, although one of Nature's methods of 6G4 Manual of Bacteriology disinfection, is not made use of to any extent by man except as an inhibitory agent for the preservation of many articles of food. FILTRATION is a method of disinfection by exclusion, and in the form of sand filtration and filtration through porous porcelain, as in the Berk ef eld and Pasteur- Chamberland filters, is made use of for the sterilisation of water and other fluids. Chemical Disinfectants. — A large number of chem- ical substances variously known as germicides, anti- septics, disinfectants, deodorants, etc., have the power of interfering with, or masking the results of, the vital activities of micro-organisms. Germicides are sub- stances which kill bacteria or germs; antiseptics, by inhibiting bacterial development, prevent sepsis or putrefaction ; and by " disinfectant " is meant a substance which prevents the action of, or destroys, infective matters, while deodorants destroy or absorb foul-smelling gases the result of putrefactive and similar processes. All germicides are disinfectant and antiseptic, but many antiseptics, though preventing or inhibiting the development of bacteria, are not neces- sarily germicidal. Many deodorants act largely mechanically, and although often not germicidal, and hence not ideal disinfectants, are of some value in preventing the deleterious and depressing effects of the emanations from decomposing organic matter. Such are charcoal, ashes, dry mould, and peat (peat has also a germicidal action). Other deodorants, such as quicklime and chloride of lime, act chemically. The germicides and antiseptics may be considered together, for although many antiseptics are not germi- cidal, all the germicides in small amounts act as anti- septics. The principal germicides and antiseptics are Theory of Disinfection 665 the halogen elements, the mineral acids, a large number of metallic salts, phenol and many coal-tar derivatives, and various organic bodies and essential oils. Theory of chemical disinfection. — The theory of chemical disinfection is not yet fully understood. It is probable, as suggested by Paul and Kronig, that the degree of ionisation of a solution may have an impor- tant bearing on its disinfecting efficiency. Paul and Kronig1 made a 'number of experiments on the M. -pyogenes, and spores of anthrax, with a view of determining the effects of various acids, bases, oxidising agents, and metallic salts on bacteria. The salts of mercury, gold and silver exert a marked germicidal action, strongest in the case of mercury, while the platinum salts are almost inactive. The efficiency of mercuric chloride is markedly lessened by the presence of sodium chloride or other chlorides. Of the oxidising agents, nitric, chromic, chloric, and permanganic acids act in the order stated; chlorine has the most powerful action of the halogens. Phenol acts better in a 5 per cent, solution than in higher concentrations, and the efficiency is increased by the addition of sodium chloride, but diminished by the presence of alcohol, and under the most favourable conditions it is not such a powerful germicide as mercuric chloride. Mercuric chloride dis- solved in absolute alcohol has little or no efficiency, and the addition of sodium chloride reduces its activity. Organisms in masses are less readily acted upon by antiseptics than when they are isolated. The efficiency of a germicidal salt in solution seems to vary with its dissociation. It is believed that the molecules of a salt in solution are more or less dis- sociated into constituent electrified atoms or " ions," and the greater the dissociation the more active will the 1 Zeitschv.f. physical. Chem., 1896, xxi, p. 414. 666 Manual of Bacteriology substance bo as a germicide. Taking mercuric chloride, bromide and cyanide, it, is found that the ionisation of the chloride is greater Chan that of the bromide-), and this is more ionised than the cyanide, and the following results show that the germicidal power of the three is in I his order :! Number of colonics which developed. After After Solution. 20 minutes' 85 minutes1 treatment. treatment. 1 mole HgCl2 in 64 litres . 7 0 1 „ HgBr2 „ „ . 34 0 1 „ Hg(CN), in 16 litres 8 33 Since the amount of tins dissociation may be greatly influenced by the presence of other substances, much oiiilion should lie exercised in adding salts, etc., to increase solubility or prevent precipitation, as the addition may seriously impair germicidal or antiseptic power (see p. 072). The disinfection process is a, gradual one. In the early stages of disinfection large numbers of organisms are killed, lint I he rale of killing becomes slower and slower as time elapses. Madsen and Nyman and Miss ('hick" have found that if the results be plotted, ordi- nates representing the numbers of surviving bacteria, and abscissa' the corresponding times, the points lie on a logarithmic curve. The curve so obtained, in fact, appears to be similar in form to thatof a " unimolecular reaction," and may be expressed by the formula 1 91 lo<>- 1 = K, where n, and n.-, are the numbers of k-h e™2 ■ bacteria surviving after times /L and tz respectively, and K is a constant. In the case of disinfection of anthrax spores with phenol, Miss Chick found the mean 1 Findlay, Physical Chemistry, 1905. 2 Jowm. of Hygiene, vol. viii. 1908, p. 92, (Summary and Bibliog.) Factors Modifying Disinfection 667 value of 1\ to be 0'44. In the case of B. paratyphosus, however; the course of the disinfection is different unless the culture is very young, and Miss Chick concluded that the older individuals are less resistant than the younger. The progress of heat disinfection apparently follows the same course. Miss Chick asserts that the act of disinfection is a unimolecular reaction, but it is difficult to accept this view. Disinfectants in emulsion tend to be more efficient than when in solution. Factors modifying disinfectant action.1 — The efficiency of a disinfectant liquid partly depends on its concen- tration. The rate of penetration into bacterial cells decreases as the concentration increases above a certain limit. Most disinfectants yield, therefore, a greater amount of disinfectant energy per gramme-hour in dilute than in strong solutions. In oil, glycerin, or alcohol, disinfectants lose some or most of their activit^y. Of fats, lanolin alone seems compatible with disinfectant efficiency. Some disinfectants form an emulsion on the addition of water, and their efficiency for a given amount of active material may vary within wide limits according to the manner in which they are emulsified. The temperatui'e at which the organism is exposed to the disinfectant has a considerable influence on the extent or rate of disinfection. Up to the optimum temperature at which the organism to be disinfected gi'ows on the medium in which it is exposed the activity of a disinfectant may fall off as the temperature rises, owing to the increased vigour which the organism derives from the improvement in its conditions in respect of temperature. A relatively small difference of temperature — two or three degrees — may make an appreciable difference in the activity of the disinfectant, ' 'VU]A section is largely taken from Applied Bacteriology, Moor and Eewlett, 1907. 668 Manual of Bacteriology and in the examination of disinfectants the failure to remember this fact has led to serious error. Above the optimum a rise of temperature increases the activity of the disinfectant, sometimes to an enormous extent. The same is sometimes the case even at temperatures below the optimum, when the organism is in unfavour- able conditions for growth. A mixture of disinfectants in many cases has a more powerful effect than can be produced by either separately (Chamberland). The resistance of bacteria to disinfection by chemical agencies is extremely vai-iable and is also selective. Bacteria of one class may be many times more sensitive to one disinfectant than to another when both sub- stances exert an equal effect on bacteria of another class. The presence of organic matter may profoundly modify the action of chemical disinfectants, particularly those acting by oxidation, considei'ably reducing their efficiency. Requirements for an efficient disinfectant. — The con- ditions which should be satisfied by an efficient disin- fectant for general use are simple, but not eas}^ to obtain. Because a disinfectant effect depends on the strength of the solution, the substance should have an approximately definite efficiency for particular organisms in given conditions, and for the same reason it should be permanently homogeneous. In practice disinfectants must be used with water or in an aqueous solvent ; it- should, therefore, yield a stable solution or uniform emulsion in all proportions. Because bacteria as pre- sented for practical disinfection usually have some organic coating, it should be stable in the presence of organic matter ; and as this coating is often of a greasy character, it should, especially if intended for use on dirty or greasy surfaces, have high solvent power for grease. For use when heat can also be applied, whereby Acids and Sulphur 669 iis activity is enhanced, unless it breaks up, it should be stable at all reasonable temperatures. These con- ditions may be considered to be indispensable It is further desirable that it should have a sufficiently high specific efficiency to allow of its being used in a readily diffusible dilution ; that it should yield a cheap solution or emulsion, not act on metals, and be neither caustic nor toxic. Some disinfectant substances may now be considered more in detail. Acids. — All acids have disinfectant action, and their relative values are interesting in the respect that for them a general law has been fairly well established by Yon Lingelsheim, and confirmed by Boer— namely, that the efficiency varies with the degree of acidity. Solu- tions of acids not of equal percentage concentration, but of equal acidity, have approximately the same disinfectant efficiency, whatever may be the acid, and whether it be inorganic or organic. The acids have no great practical application in dis- infection. That which has been most commonly used is sulphurous acid, applied either direct from burning of sulphur (in which case it will also contain S03 if there is sufficient moisture to hold the sulphur dioxide in solution) or by the use of the liquefied gas. It produces a slow superficial disinfection of a Aveak and uncertain character even under laboratory conditions. Such experiments avoid, however, to a far greater extent than is possible in practice the difficulty of diffusion, and the unequal diffusion of sulphurous acid in air and its small power of penetration make it less efficient in practice. To obtain even the poor efficiency which is its maximum possible it is necessary for the air to be damp and the room most cai-efully sealed, and in these conditions it is often more injurious to the objects under treatment than to the bacteria against which it is directed. 117,1 Manual of Bacteriology One of the most efficient methods of applying sulphurous acid disinfection is by means of the Clayton apparatus. The gas is generated by burning sulphur in a current of air at a high temperature, and contains, in addition to S02, traces of higher oxides of sulphur. It is also a very efficient vermin-killer, destroying rats, cockroaches, bugs, fleas, flies, etc. Alkalies and soaps.— The degree of alkalinity of a solution affects, but does not by itself altogether determine, its germicidal power, which is also depen- dent on the nature of its metal. The hydrates of thallium, lithium, barium, calcium, potassium, sodium, and ammonia have widely different efficiencies, roughly in the order named. For practical purposes only those of potassium, sodium, and calcium need be considered.1 They exhibit notably the characteristic of all disinfec- tants that they work much more vigorously in hot than in cold solution. It is to the hydrates or alkaline carbonates of potassium and sodium that the soaps owe such power as they possess against naked organisms. The relative efficiency of soaps in practical disinfection may be understated by the results of comparative experiment on laboratory cultures because the resist- ance of the microbe itself to disinfection by chemical substances, and, indeed, by other agencies, may be small compared with the resistance offered by the envelope of grease or greasy dirt, derived from perspira- tion, pus, fat, and the oily grime which pervades cities and is everywhere caused by handling. A disinfectant of greater efficiency than soap on a laboratory culture may, therefore, be of much less efficiency on an infection in actual practice. Soaps are incompatible with most disinfectant substances, but not with all. Biniodide of 1 See Forrest and Hewlett, Journ. Roy. Army Med. Corps, February, 1904. Lime and Halogens 673 mercury can be prepared with soap, ami Eor surgical purpose's is a disinfectant of high value. The " carbolic soaps " of commerce are, for the most part, worthless. Caustic lime, used generally as a 20 per cent, milk, has considerable disinfectant power, and has been applied to the disinfection of fasces. For this purpose care has to be taken to break up any lumps of excreta, and whenever practicable a heat process, of which the efficiency and rapidity may be greatly increased by an alkaline disinfectant, is much to be preferred. Lime is inefficient against the more resistant organisms, and lime-washing cannot be considered a sufficient precau- tion against them or against infections, such as those of scarlet fever and smallpox, of which the exciting orga- nism is unknown. Halogens. — The disinfectant values of dry chlorine, iodine, and bromine are low. Both in a dry and a damp state chlorine is inconvenient, and the others are costly; and the use of halogens is therefore practically confined to solutions, notably " chloride of lime " (a mixture of calcium hypochlorite, hydrate, and chloride) and hypo- chlorite of soda (chloros). These have a powerful effect on laboratory cultures, but in practice need to be used in excess proportionate to the amount of orgauic matter which may be present. Thus, for instance, a 1 per cent, solution of hypochlorite of soda mixed with an equal volume of urine loses the whole of its available chlorine almost immediately, and becomes inert as a germicide. Where the amount of organic matter is small, and the objects are not likely to be injured, the hypochlorites are among the best of known disinfectants, provided they are used fresh. The slow addition of hydrochloric acid, yielding nascent chlorine, increases the activity of a hypochlorite considerably. A solution of iodine is now used for skin disinfection in surgical 672 Manual of Bacteriology practice. Iodine trichloride is a powerful disinfectant, of which the use has been suggested, among other purposes, for the sterilisation of water. Nessfield has suggested the use of chlorine for sterilising water on the large scale, and iodine for the same purpose on the small scale (p. 033). Chloride of lime or other hypo- chlorite may be used for sterilising water on the large scale (p. 634). Other inorganic substances. — Solutions' of salts of mercury exercise a powerful disinfectant action in pro- portion to the amount of dissolved metal which they contain. The most commonly used is the perchloride (corrosive sublimate). Apart from its extremely poisonous character, it has the disadvantage of forming with albuminoid substances both insoluble and soluble com- pounds of little or no germicidial value, sulphuretted hydrogen converts it into the insoluble and inert sulpiride, and it acts on some metals. The addition of acids or salts (e. g. hydrochloric or tartaric acid or sodium or ammonium chloride) prevents or largely reduces the formation of insoluble compounds ; but it does not prevent the reactions resulting in soluble substances, it may reduce the germicidal power, and the action of jierchloride in the presence of albuminoids is therefore very variable. The reduction in germicidal power by addition of sodium chloride is well seen from the following results (Finlay, loc. cit.) : Number of colonies lb' litres of solution contained developing- after treat- ment for 6 minutes. 1 mole HgCl3 8 1 „ HgClo + 1 mole NaCl . . 32 1 „ HgClg + 2 moles NaOl . .124 1 „ HgClo + 4 „ NaCl . . 382 1 „ Hg > 1'04 3; -I II „ (with 50 per cent, urine) Walker ft ('upper sulphate Cyllin*. t> (i ( )4' 33 1 W 1 1 \ ) \ i I Fowler 11 0o 33 ,, (with 50 per cent, urine) 5-06 33 110 Cyllin . Klein 505 iLT. pyo(j cues 9' 3 jj . . . Simpson and Hewlett 6 06 O'i.' \J Formalin Fowler 3 05 B. t ypliosus 07 Hydrochloric acid Walker 2-05 11*0 Izal* . Fowler 3 06 33 11*0 Kerol* . 3 3 9 -06 S3 120 ,, (with 50 per cent, urine) 33 806 ' J 8-5 Little's phenyle . 33 5 04 >9 2-0 Lysol . 33 206 33 25 Mercuric chloride 8-05 33 looo-o >> >t Walker 8-05 >9 400-0 Potass, permangau ate Fowler 8-05 „ 42-0 » „ (with 3 per cent, organic matter) Walker 1-07 3i l-o Zinc chloride 3) 1 06 " 0-15 * The germicidal efficiency of these substances has been increased since the date of the experiments recorded, and they now have a carbolic-acid co-efficient of from 10 to 20-22. proportion, but probably when in contact with living cells a decomposition is effected, free iodine being liberated, hence its value. The essential oils,. peppermint, mustard, cloves, thymol, and menthol, are powerfully antiseptic. Disinfectant powders at best exert but a super fi cial 1 Fowler. Ju am. Roy. Army Mad. Corps, July, 1907. Antiseptic Treatment G79 action. They act chiefly as deodorants, but may be useful iu preventing the breeding of flies in garbage, etc. It is useless to add a small quantity of disinfectant to a large volume of fluid or solid ; the disinfectant must be added iii sufficient amount so that the mixture contains the minimum percentage which has been found by experiment to be efficient. For this reason the attempt to disinfect sewers, sewage, streets, etc., by relatively small quantities of disinfectants is useless, and the money so wasted would be far better em- ployed in providing more water for flushing purposes. In medical practice, while antiseptics can be applied locally with success and, to some extent, for disinfecting the alimen- tary tract, no substance has yet been discovered which can be administered with safety to such a degree as to saturate the body, and so exert a general germicidal action in infective diseases. In surgical practice no unbiased observer can doubt the efficacy of antiseptic treatment, but many so-called " anti- septic operations " are marred by faults of omission and commission which render them far from being perfectly anti- septic. There has been some controversy between the advo- cates of " antiseptic " and of " aseptic " surgery. Undoubtedly antiseptics do diminish the vitality, and therefore the re- parative power of the tissues and aseptic methods should so far as possible replace antiseptic ones. The skin of the patient and the hands of the operator having been disinfected as far as possible, no antiseptic should be permitted to come into contact with the wound, which may be irrigated with warm sterile physiological salt solution. A dry wound is an important element to success, and a dry, sterile, unirritating dressing should be employed. Instruments, sponges, etc., may be kept in sterile salt solution after the preliminary disinfection — by heat (not sponges) or chemicals. But the aseptic system requires more care to ensure success than the antiseptic one, and unless the assistants can be trusted and the details rigorously carried out, the latter seems preferable. (38U Manual of Bacteriology The Determination of the Germicidal Power. For determining germicidal power oh spuria- organisms anthrax spores are generally used, on non-sporing organisms cultures of the B. typhosus are usually employed. (1) Thread method.— Sterilised silk threads are impreg- nated with sporing and non-sporing organisms, lightly dried, and then exposed to the action of the antiseptic solution of a known strength for a given time. After treatment the threads are thoroughly washed with distilled water to remove the antiseptic, and sown on the surface of agar or other suitable culture medium. If no growth occurs the organisms are assumed to have been destroyed. As a matter of fact, however, it is extremely difficult to get rid of the last traces of the antiseptic, which may inhibit growth although the organisms may yet be alive, a fallacy which caused an exaggerated value to be assigned to many substances— for example, corrosive sublimate. The thread method may still he employed, but after treatment the threads should be sown in broth, or, better still, if pathogenic organisms be the subject of experiment, inoculated into a susceptible animal. The writer finds that in disinfection experiments with anthrax spores, surface agar is a much better medium than broth. /// experiments with corrosive sublimate, by whatever method, the titsf I nice* <>/ the substance must be converted into the inert sulphide by treatment with hydrogen or ammonium sulphide. (2) Garnet method. -Small garnets the size of a pea are sterilised, soaked in a suspension or a broth culture of the organism, removed and dried. The garnets with the organisms attached are then soaked in solutions of the disinfectant of known strengths for various periods of time ; they are then removed from the solution, well washed with sterile water, and finally placed in tubes of broth. (3) mdeal-Wallcer or drop-method.— Moov first suggested that the germicidal efficiency of a disinfectant might be compared with that of a standard solution of carbolic acid, which has a definite composition, is stable, and can lie accurately standardised, and Kidea.1 and Walker devised an <2fU I, 1 u u u Rideal-Walker Method 681 ingenious and simple method for carrying tins out. A special test-tube rack is very convenient (Fig. 69), iu which the lower tier has five holes which hold three or four tubes containing the solutions of decreasing strengths of the disinfectant to be tested, and two tubes or one tube containing standard carbolic acid solution of known strength for comparison. The upper tier has thirty holes in two rows, and spaced into six sets of five holes each. These hold tubes of sterile nutrient broth which are numbered from 1 to 30. The test is usually made with a broth culture of B. typhosus, Fig. 69. — Test-tube rack with test-tabes arranged for the Rideal-Walker method of testing disinfectants. but other organisms may be employed. The process is as follows : The five tubes in the lower tier each contain 3 c.c. of the disinfectant and carbolic solutions. Into each in succession, at intervals of half a minute, three drops of the typhoid broth culture are added with a pipette. Half a minute after the last tube has been inseminated, a loopful is taken from the first tube and inseminated into the first broth tube, and this process is repeated at half-minute intervals until all the broth tubes have^been inoculated. The inocu- lated broth tubes are then incubated at 37° C. for three days, and the occurrence or not of growth is taken as indicating the killing or non-killing of the organism respectively. Obviously the lirst set of five broth tubes inoculated arc subcultures in Gy2 Manual of Bacteriology which the organism lias been acted upon by the disinfectant and carbolic solutions for two and a half minutes, the second set for five minutes, and so on. The results (taken from an actual test) may be charted as follows : B. typhosus, 24-hour broth culture at 37° C. Boom-temperature 60° F. Time culture exposed to action Sub-cultures. Disinfec- of disinfectant (in minutes). tant. Dilution. Period of Tempera- -a 5 J 7£ 10 15 incubation. ture. X 1-1400 + # # # # 3 clays 37° C. X 1-1500 + + * # # # X 1-1600 + + + * # # x 1-1700 + + + + * # Carbolic 1-100 + + + # # # + = growth in the sub-cultures. * = no growth in the sub-cultures. From this it will be seen that the disinfectant X in a solu- tion of 1 in 1600 kills in the same time (7h minutes) as carbolic 1 in 100. This result is expressed as a coefficient obtained by dividing the strength of disinfectant by the strength of carbolic which kills each in the same time ; in the present instance the co-efficient is lT8^0 — 16'0, and this figure is known as the " carbolic acid coefficient." If nothing is known about the strength of the disinfectant, some preliminary experiments should be performed with dilu- tions at wide intervals as regards strength (e. — symptomatic, 155 — vaccine, 274 Anti-bodies, L56 Anti-endotoxic sei-a, 42 Anti-ferments, 202 Antigen, 157 — test (syphilis), 530 Antiseptic action, conditions modifying, 667 — power, determination of, 680 — treatment, 679 Antiseptics, 664-679 Anti-sera, 179 Anti-serum, anthrax, 274 — cholera, 468 — colon, 408 — dysentery, 399 — hydrophobia, 570 — plague, 42 L — pneumonia, 434 — streptococcus, 246 — tubercle, 339 — typhoid, 386 Antitoxic constituent, 175 — treatment, 1H7 Antitoxin, cholera, 468 — diphtheria., 291 — tetanus, 447 Antitoxins, 157 — in normal blood, 286 Anti-venin, 171 Appendicitis, 585 Archebiosis,6 Area of dish, 640 Arthritis, 25S, 434, 5! 17 — deformans, 598 Arthus phenomenon, 177 Ascitic fluid culture medium, 61 Ascococcus, 16 Ascospores of penicillium, 501 — of yeast, 498 — of yeast, staining. 497 Aseptic treatment, (579 Asiatic cholera, 457 Aspergillus nig Br, 501 — fumigatus, 502 Atrepsy, 216 Autoclave, 47 Azotobacter, 34 Bacteriology Babesia, 557 Bacilli, capsulated, 27 1 Bacilli carriers, cholera, 462 diphtheria, 286, 299 — dysentery, 399 — typhoid, 379 Bacillus, definition of, 17 — uriili lactic!, 400, 649 — acidophilus, 601 — acnes, 589 — aerogenes eapsultitiix, 4-51 — aertryck, 393, 395 — albus va rioltn, 579 — anthrncis, 265 — aquatilis sulcatus, 631 — bifidus, 601 — bottle, 507 — botulinus, 450 — buccalis, 4K6 — bulgaricws, 658 — butyricu*, 36, 456 — cadavsris sporogenes, 455 — caniculie, 58H — capsulatus, 437 — — 7iomi in is, 2 7 1 — ravicida, 410 — chativsei, 453, 455 Bacillus cloacx, 41(1 — coli, 401 — — communis, 401 — — communior, 404 — — immobilis, 271 — coryzte, 312 — diphtheria;, 279 — — columbarum, 313 — diphtheroid, 286, 300, 312, 594 — dysenteriai, 370, 397 — enteritidis, 371, 391, 400, 649, 657 — — sporogenes, 451, 453 — feecalis alkaligenes, 7, 574, 631 — filamentosns, 645, 658 — fetidus, 599 — fluorescens liquefaciens, 30, 37, 212, 250, 383, 612, 645, 658 — fluorescens non - liquefaciens, 61 2, 658 — fluorescens slercolalis, 283 — fusiformis, 311 — glanders, 361 — grass, 359 — icleroides, 394, 576 — infantilis, 602 Index 689 Bacillus infiuenzse, 138 — isosarcinus, 371 — lactis aerogenes, 410, 601, 649 — lepra', 352 — mallei, 361 — megaterium, 658 — mesentc ricits, 6 15, 658 — mist, 359 — mucosas capsulatus, 271 — muriseptiais, 427 — mycoides, 30, 643, 645, 658 — neapolitanus, 410 — of Achaline, 451, 597 — of black quarter, 455 — of chicken cholera, 426 — of Danysz, 37 1 , 394 — of Ducrey, 586 — of Friedliinder, 429, 435 — of gastro-enteritis, 392 — of Hof maun, 300 — of hog cholera, 393 — of Johne, 350 — of Koch and "Weeks, 587 — of Laser, 410 — of Lustgarten, 357, 525 — of malignant cedema, 449 — of Morax and Axenfeld, 587 — of mouse septicEemia, 427 — of ozsena, 595 — of rabbit septicaemia, 427 — of rheumatoid arthritis, 598 — of rhinoscleroma, 598 — of Massol, 653 — of swine fever, 393 — of swine plague, 394, 427 — of symptomatic anthrax, 455 — of syphilis, 525 — of xerosis, 311 — Oppler-bSoas, 594 — paracoli, 37], 395 — paratyphosus, 371, 391, 395 — pcrfringens, 451 — pertussis, 441 — pestis, 413 — pneumonise, 271, 429, 435 — prodigiosus, 37, 263, 657, 658 — proteus, 24, 30, 251, 587, 588, 643, 645, 658 — pseudo-diphtherise, 300 — pseiido-dysenterise, 399, 571 — psexido-tuberculosis, 350, 416 — psittacosis, 371, 391, 394 — putrificiis coli, 443, 601 Bacillus pyocyaneus, 37, 219, 570, 585, 588, 589 — pyogenes fetidus, 408 — smeijmatis, 356 — sublilis, 15, 520, 658 — suicholerw, 393 — suipestifcr, 371, 393, 395 — sulcatus, 574 — tetani, 443 — timothy grass, 359 — tuberculosis, 317 — typhosus, 371 — typhi/murium, 37J, 392, 391 — typhosimilis, 371 — vagina1, 602 — violaccus, 37, 658 — Welchii, 451, 620, 644, 645, 649 — X, 576 — xerosis, 311 Bacteria, action on artificial sugars, 22 — classification of, 15 — conditions of life of, 19 — effect of electricity on, 24 — effect of light on, 23 — effect of pressure on, 23 — influence of chemical agents on, 21 — influence of oxygen on, 20 — influence of radium on, 24 — influence of temperature on, 19 — nutrition of, 19 — selective action of, 22 — structure of, 9-14 — study of, 66, 123 et suq. — thermophilic, 20, 644 — variation of, 6 — vitality of buried, 644 Bacterial poisons, 153 — products, 38 Bacteriological diagnosis. See Examinations — microscope, 138 Bacteriolysis, 180 Bacteriotropines, 219 Bacterium, definition of, 17 — species of. See Bacillus — termo, 30, 658 Bacteroids, 33 Balantidium coli, 536, 589 Beer, 492-495 44 690 Manual of Bacteriology Bell-jars, is Beri-Beri, 585 Berkefeld filter, 19, 635 Bird-pox, 148, 582 Bismarck bro-w n, LOS, ;JU8 Black leg, 455 lll.u k quarter, 455 Black water fever, 552 Blastomyeetes, 487 — classification, 487 — examination, 191, 196 — fermentative, 4! 12 — identification, 493 — pathogenic, 488 — spores of, -111:; Blastomycetic dermatitis, 490 Bleeding animals, 131 Blood films, 98, 553 Blood, germicidal action of, 208 — serum, 6U — to obtain, 61, 131, 533 Blood-agar, 63, 4-67 Blue pus, 2 10 Boils, 238 Borax-methylene blue, 554 Bordet- Durham reaction, 195, 201 Bordet-Gengou phenomenon, 191 Boric acid, — diagnosis of, — lesions in, 258 < i nun's met hod, 105 » Slaudius's modification, 108 — Giinther's modification, 107 — thionin, 109 — Weigert, 109, 115 Gram -negative cocci, 261 Granuloma, ulcerating, 525 Grass bacillus, 359 Gvegarincs, 559 Griffith's steriliser, Grinding machine, 42 Grouse disease, 427 Guarnieri bodies, 580, 581 Gum for freezing, 90 Hjemamosba, 541, 556 Hamiatin stain, 345 Hematoxylin, 103, 514 Hsemoflagellates, 516 Ha-moglobin agar, 63 Hsemogregarines, 559 Hemolysins, 188 Haemolysis, 1S7 — test, 1S9 Hemolytic serum, 192 — system, 191 Henioproteus, 556 Hemosporidia, 541 Halogens, 671 Halteridium, 523, 556 Hanging-drop cultivations, 13 I — anaerobic, 137 Haptines, 162 Haptophore gronp, 161 Heat as a disinfector, 659 Heat, dry, 660 — moist, 6(il Keidenhain's iron-h a- matoxy lin, 514 Herpes zoster, 591 Herpetomonas, 516 Hesse's method for air analysis 640 Hofmann bacillus, 300 Hog-cholera, 39$ Hot-air steriliser, 46 Hyd roa gestation is, 59] 1 1 ycbvocele-fluid culture medium 61 I [ydrochloric acid, 678 1 [ydrogcn peroxide, (>7 1 I [ydrophobia, 564 I I ypersensitai ion, 1 7(i HyphsB, 498 I Hyphomycetes, 498 — examination, 502 ' — pathogenic, 501 Ice, org-anisms in, 604, 632 — creams, 632 Identification of organisms, 125 Illumination, 140 — dark ground, 1 16 Immersion lenses, theory of, 142 Immune body, 181 Immunity, 20 t — acqiured, 205, 21 1 — atreptic, 216 — natural, 205, 206 — active, 215 — passive, 215 — humoral, 211 — phagocytic, 211 — transmission of, 215 Impetigo, 239. 285, 590 Impression specimens, 100 Incubator, 66 Index, opsonic, 221, 229 — determination of, 224 Indian ink method, 82 — for syphilis, 528 Indole, 25 — influence of culture medium, 26 Infantile paralysis, 572 Infection, 151 — modes of, 155 Infective process, 154 Influenza, 438 — cold, 260, 312, 440 Infusoria, 536 Inoculating tubes, method of, 7(i Inoculation, intra-venous, 128 — of animals, 128 Interlamellar films, 137 Intestine, organisms of, 601 Intoxication, 152 Intracellular substances, 153 tntra-venous inoculation, 128 Invertase, 37 Manual of Investigation of micro-organisms, Iodine, 671 — Gram's, 106 — trichloride, 07 L Iodoform, 677 Irrigation, 133 Isolation of micro-organisms, 124 12G Izal, 677 Jenner's blood stain, 104 Johne's disease, 350 Kala-Azar, 6 KeroL 614, 677 Klebs-Loffler bacillus, 278 Koch's " comma" bacillus, 457 — postulates, 154 Koch-Weeks bacillus, 587 Kraus's test, 460 Landry's paralysis, 565, 573 Lankesterella, 560 La rerania, malaria;, 550 Leguminosae, fixation of nitrogen by, 32 Leishman-Donovan body, 521 Leishman stain, 104 Leishmaniosis, 521 Lenses, microscopical, 1 1 1 — immersion, 142 Leprosy, 351 — diagnosis of, 355 Leprosy-like disease of vats, 355 Leptothrix bxiccalis, 486 Leucocytes, migration of, 212 — in milk, 652, 655 Leucocytozoa, 523, 559 Leucocytozoon run is, 55!) Leuconostoe, 17 Levaditi's stain, 529 Life-history, studying, 124 Life without bacteria, 3 Light as a disinfector, 663 — effect of, on bacteria, 23 Litmus media, 59 Local Government Board disin- fecting solution, 673 Loffler's methylene bhie, 101 — serum, 61 Loop, standard, 683 Lustgarten's bacillus; 525 Bacteriology Lymphadenitis, ovine, 35] Lymphangitis, 244 — epizootic. 367. 49] Lysins, 180, 188, 192 Lysol, 677 Macrogamete, 509 Macrophages, 212 Madura disease, 482 Mai de Caderas, 519 Malachite green media, 370, 621 1 Malaria, 541 — diagnosis of, 553 — parasites, 542-552 mosquito phase, 545 species, 549 Malignant disease, 488, 583 — oedema, 449 clinical examination, 454 — pustule, 271 Mallein, 366 — in diagnosis, 368 Malta fever, 591 Marasmus, 250 Mastigophora, 516 Mastoid disease, 595 McConkey stain, 116 — media, 623, 624 McDougall's fluid, 677 Measles, 593 Measurement, microscopical, 149 Measures and weights, 6S5 Meat, 392, 657 Media, culture, 54. See Culture media Medical antiseptics, 679 Mediterranean fever, 59] Meiostagmin reaction, 202 Membranous rhinitis, 286 Meningitis, 434, 593 — cerebro-spinal, 253 — posterior basic, 255 Mercaptan, 24, 38, 39 Mercuric chloride, 672 — iodide, 673 Mercury pyogenic, 235 ■ — vapour lamp, 140, 663 Merismopedia, definition of, 16 Metchnikoff's spirillum, 472 Methylated spirit, 88 Methylene blue, Loffler's, 101 ■ — borax, 554 — carbol, 101 Index 697 Micrococci, Gram-negative, 261 Micrococcus, definition of, 16 — agiUs, 658 — bombycis, 56] — ■ ccmdicans, 658 — catarrhalis, 260, 440 — cereus albns, 240 jiavus, 37, 241 — cinereus, 256 — epidermidis albns, 240 — flavescens, 240 — gonorrhoea, 256, 599 — laneeolatus, 430 — Melitensis, 196, 591, 649 — meninffitidis, 253 — neoformans, 242, 583 — Pasteuri, 430 — pyogenes, aureus, 237 albus, 240 — — citreus, 240 — — tenuis, 430 — salivarius, 241, 601 — scurf, 241, 639 — tetragenus, 260 — «re«, 30, 37 — zymogenes, 241 Microgamete, 509 Micrometer, 149 Micro-millimetre, 150 Micron, 150, 685 Microphages, 212 Microscope, bacteriological, 138 Microsporidia, 560 Microsporon Audouini, 503 — furfur, 507 — minutissimum, 507 Microtomes, 90, 94 Miescher's corpuscles, 561 Milk, 647 — diphtheria-like bacillus in, 288, 655 — examination of, 654 — leucocytes in, 652, 655 — organisms in, 648 — Pasteurisation of, 650 — pathogenic organisms in, 649 — examination for pathogenic organisms, 654 — sour, 653 — standard for, 653 — sterilisation of, 650 — curdling of, 36, 649 — culture media, 59 Moeller's spore stain, lis MolluscTini bodies, 584 Morax-Axenfeld bacillus, 587 Mosquitoes, 548 — and malaria, 545, 54S — and yellow fever, 576 Motility of organisms, 11, 136 Moulds, 498 Mounting sections, 111 Mouse plague, 394 — septicaemia, 427 Mouth, organisms of, 486, 600 Movement, Brownian, 136 Mucor mucedo, 498 Mucous membranes, organisms of, 599 Mumps, 593 Mustard oil, 678 Mycelium, 498 Mycetoma, 482 Mycetozoa, 515 Mycoses, 501 Mycosis tonsillaris, 485 Mytilotoxin, 39 Myxomycetes, 515 Myxosporidia, 560 Nagana, 518 Nasal mucus, germicidal action of, 600 Nasgar, 254 Nastin, 353 Necrosis, 37 Needles, 50, 51 Negri bodies, 565 Neisser's stain, 308 Neuritis, diphtheritic, 285 Nitragin, 33 Nitrification, 28 — stages in, 30 — solutions for, 31 Nitrifying organisms, isolation of, 31 Nitrobacter, 30 Nitrogen, fixation of, 32 Noctiluca, 516 Noma, 594 Nomenclature, 18 Normal solutions, 65 Nosema bombycis, 560 Nose, organisms of, 600 Nose-piece, 149 Nncl,eins, 21 () 6,98 Manual of Bacteriology < >BJECTIVES, I M ( >bjeuts, measuremenl of, I 19 (Edema, malignant . 449 O'idiwm albicans, I'.ll — birlis, 649 ( HI immersion Lenses, 142 ( His, essential, as antisepl ics, 678 ( )ld age, 60] I >okinet, 5 L-6 Ophthalmia* 258, 587, 599 ( >phthalmitis, 240 Ophthalmoreaction, in glanders 365 — in tuberculosis, 349 — in typhoid, 390 Oppler-Boas bacillus, 594 < >psonic index, 221, 229 — — determination of, 224 < >psonins, 220 I >range-rubin, 103 Organisms and disease, 123, L51, 153 ( >rganisms, cultivation of, 66 — enumeration of, 77. 230 — identification of, 12.", — influence of a mixture of, 21 — isolal ion of, I t, 77. 1 2 t — of air, water, and soil, 658 — Of air-passages, 62 — ultra-microscopic, 1 t8 — variation of, (i Osmic acid fixation, 100, 537 Osteomyelitis. 237, 238, 244, 374 Otitis. 249, 59o (3 veil, hot-air, Hi Oztena. 595 Ozone, 634, 674 Pakes' discs, 614 Pappataci, 578 Pappenheim's solution, 358 Para-colon bacillus, 371, 395 Para-dysentery bacillus, 40] Para-typhoid fever, 395 Paraffin, embedding in, 9] — sections, 94 Paraffin seel ions, mounting, 1 13 Paralysis, diphtheritic, 285, 287 29!) — general, 286, 535 — infantile, 572 — Landry's, 565, 573 Paramecium coli, 536 Parasites, 152 Parotitis, 593 Parthenogenesis, 509 : Pasteurisation of milk, 650 Pasteur's fluid, Peat, germicidal action of, 383, 461 Pebrine, 560 Pemphigus, 590 Penicillium glaucum, 500 Peppermint oil, 678 Peptone water, 57 Pericarditis, 258, 434 Peritonitis, 431, 595 Permanganates, 674 Pertussis, 441 Petri dishes, 79 Petri's method for air analysis, (ill Petruschky's litmus whey, 406 Pfeiffer's reaction. ISO, 183 Phagocytes, 2 1 1 Phagocytosis, 2 1 1 — estimation of, 220 Phenol, 665, (inn Phlebitis, 23ii Phlebotomus fever, 578 Phlogosin, 239 Phosphorescence, 37, 462, 516 Physiological salt solution, 97 Picro-carmine, 103 PictOU cattle disease. 408 Piedra, 507 Pigment, formation of, 37 Pink torula, 658 Pinta, 507 Pipettes, 51, 53, 227 Piroplasmata, 557 Pitfleld's flagella stain, 120 Pityriasis, 507 Plague antiserum, 421 — bacillus of, 413 — diagnosis, 424 — epidemiology, 422 — pathogenesis, 117 — vaccines, -119 Index 699 Plasmodiophora brassiest, 5 1 5 Plasmodium, 5 11 — Kocli ii, 552 — mala Has, 5 19 — prtecox, 556 — I'ii'd.r, 550 Plate bottles, 80 — cultures, 77 — — agar, SI — — anaerobic, 83 gelatin, 7S silica jelly. 31 Platinum needles, 50 Plant's method, 4S2 Pleomorphism, 15 Pleuropneumonia, 148, 430 Primmer bodies, 583 Pneumobaeillus of Friedliinder, 435 Pneumococcus, Frankel's, 430 Pneumomycosis, 501 Pneumonia, 429 — diagnosis, 437 Poisons, bacterial, 38, 153 — tolerance to, 2.>4 Poliomyelitis, 572 Porcelain filters, 49, 635 Porges' reaction, 536 Post-mortems, 129 Postulates, Koch's, 154 Potassium permanganate, 074, 678 Potato, 59. See Culture media. Powders, disinfectant, 678 Precipitins, 203 Pressure, effect of, on bacteria. 23 Products of bacteria, 38 Pn.skauer-Capaldi media, 405 Proteins, bacterial, pyogenic, 235 — germicidal, 209 — toxic, 40 Proteosoma, 556 Proteus capsttlatus homini*, 271 — mirahilix, 658 — vulgaris, 658. See B. proteits. — Zenke-ei, 658 — in putrefaction, 24, 30 Protophyta, S Protozoa, 508 — in water, 637 Pseudo-diphtheria bacillus, 300 Pseudo-diphtheria, relation to B. diphtherise, 305 Pseudo-tuberculosis, 350 Pseudomonas, is, 30 Psilosis, 596 Psittacosis, 304 Psorospermosis, 540 Ptomines, 38 Puerperal fever, 596 Pugh's stain, 308 Pump, exhaust, 48 Purpura, 596 Pus, blue, 249 — in milk, 652 Putrefaction. 24 Pyaemia, 2:16 Pyle-phlebitis, 236 Pyoctanin, 677 Pyocyanase, 250 Pyocyaneus infection, 249, 570 Pyocyanin, 249 Pyogenic organisms, 237. 202 Pyorrhoea, 597 Pyrogallic acid, 73 Pyrosoma, 557 Quartan fever. 549 Quarter evil, 455 Quinine and malaria. 552 — and tetanus, 445 Eabbit septicemia, 427 Rabies, 564 — diagnosis, 571 Radium, effect of, on bacteria, 24 Rag-sorter's disease, 271 Rat virus, 39 I Rauschbrand, 455 Ray fungus, 478 Reaction, Bordet-Durham, 105, 201 — cholera-red, 27, 459 — indol, 25 — meiostagmin, 202 — Pfeiffer's, 180, 1 S3 — Porges', 536 — Voges- Proskauer, 110 Rebipelagar, 625 Receptors, 161 — chemo-, 204, 216 Relapses, theory of, 388 Manual of Bacteriology Relapsing fever, spirillum of, 524 Resolving power, 147 Retention theory, 217 Rheumatism, 597 Rheumatoid arthritis, 598 Rhinitis, membranous, 2Kb' — atrophic, 595 Rhinosoleroma, 598 Rinderpest, 598 Ringworm, 502 — cultivation, 504 — examination, 506 Rocking microtome, 94 Roll cultures, 83 Romanowski stain, 104 Roup, diphtheritic, 313 Rubin, 103 Ruffer bodies, 583 Russell's corpuscles, 583 Saccharimeter, 85 Saccharomyces anomalus, 490 — cerevisix, 492, 495 ■ — ellipsoideus, 495 — litor/enes, 488 — pastorianus, 495 Saccharomycetes, 487 Saliva, germicidal action of, 601 Salt solution, physiological. «)? Sand-fly fever, 578 Saprsemia, 235 Saprophytes, 20 Sarcina, definition of, 16 — liitea, 37, 658 — ventrieuli, 2(i2 Sarcoma, 489 Sarcosporidia, 56] Sarkodina, 510 Saturation test, 201 Scarlet fever, 562, 649 Schizomycetes, 8 Schizophycea?, 8 Sulerotium, 498 Sections, frozen, 89 — paraffin, 91 — fixing to slide, 95 — staining, 11 1 — to mount, 113 Sedgwick and Tucker's method for air analysis, (542 Sedimentation test, 200 Septic diseases. 233 Septic tank process, 646 Septictemia, L55, 235 — a, 155 Sera, anti-microbic, 179 — antitoxic, 157 Serum, culture medium of, 60, (il — germicidal action of, 208 Serum disease, 1 76 Seven-days' fever, 578 Sewage, 645 Sewers, air of, (545 Shake culture, 84 Shell-fish, examination of, 034 ■ — pathogenic organisms in, 382 Side-chain theory, 159 Silica jelly, 31 Silkworms, disease of, 5G0 Silver salts, 673 — pyogenic, 235 Skatole, 28 Skatole-carboxylic acid, 27, 244 264 Skin diseases, 502. 507 — organisms of, 589, 599 Sleeping-sickness, 517 Slides, cleaning, 119 — hollow-ground, 134 Smallpox, 579 Smear preparations, its Smegma bacillus, 356 — staining, 346, 357 Sodium bisulphate, 633 Soil. 643 — nitrification in, 1's Solubilities, 685 Solutions, normal, 65 Sour milk, 653 Species of bacteria, 7, 18 Specimens, preserving patholo- gical, 121 Spirilla, 17, 457. See also Vibrio Spirillum, definition of, 17 — cholera' A s in I 'icsB, 457 varieties, 463, 467 — of cholera, isolation from water, 632 — of Pinkler and Prior, 473 — Metehnikori, 472 — Obermeieri, 524 — rubrum, 37, 474 — tyrogenum, 474 S2)irochaeta, 16, 522 — Diiltinii, 524 Index 701 Sjrirochaeta Obcrmeieri, 524 — pallida, 525 — pertenuis, 524 — recurrentis, 52-4 — refringehs, 52(5 — Vincenti, 31 1 — in bronchitis, 586 — in cancer, 533 — in dysentery, 589 — in ulcerating granuloma, 525 — in ulcers, 525 — in yaws, 524 Spirochaetosis, 522 Spironema pallidum, 525 Spleen, germicidal substance from, 209 — in immunity, 214 Sporangium, 499 Spore formation, 13 Spore staining, 117, 4U7 Spores, resistance to heat, 20, 060 Sporidium vaccinate, 581 Sporotrichosis, 490 Sporozoa, 538 Spotted fever, 253 — - of Rocky Mountains, 575 Sprue, 596 Stage, microscopical, 138 Staining methods, 101. See under respective names — cover-glass specimens, 109 — capsides, 115 — flagella,118 — Gram, 105 — sections, 114 — spores, 117, 497 Stains, 101. See under respective names Standard loop, 683 Standardisation of antitoxin, 293, 448 — of media, 64 Staphylococcus, 17 — species of. See Microccocus. Steam as a disinfector, 661 — steriliser, 46 Stegomyia, 548, 576 Sterilisation, 45 — discontinuoiis, 5 — of cotton-wool, 52 — of glass vessels, 53 — of milk, 650 Steriliser, hot air, 46 Steriliser, G ninth's, 633 — writer's, for milk, 652 — steam, 46 Still's diplococcus, 255 Stimulins, 219 Stomach, organisms of, 601 Strangles, 246 Streptococcus, definition of, 16 — diagnostic table, 248 — anginosus, 248 — bye vis, 246 — conglomerate, 245, 247, 583 — equinns, 248 ■ — erysipelatis, 244, 248 — ftecalis, 248 — longns, 247 — medius, 248 — pyogenes, 243 anti-serum, 246 — — in milk, 649, 655 — rhetimaticus, 248, 597, 598 — salivarius, 248, 601, 639 — scarlatina' , 247, 562 Streptothrix infections, 475 Streptothrix, acid-fast, 315 — actinomyces, 477 — Eppingeri, 475 — Freeri, 484 — leproides, 353 — madurse, 484 — Nocardii, 481 Streptotricheffi, 475 Sub-stage condenser, 138 Sub-tertian fever, 550 Sugars, resolution of, 20 Sulphurous acid, 671 Supersensitation, 176 Suppuration, 233 — clinical examination, 250 — conditions modifying, 236 — due to chemical agents, 234 — influence of dose, 237 embolism, 236 injury, 234 Surgical antiseptics, 679 Surra, 519 Swine erysipelas, 427 — fever, 393 — plague, 394 Symbiosis, 21 Symptomatic anthrax, 455 Syphilis, 525 Syringes, 128 ADLARD AND SON, 1MPE., LONDON AND DOBKINCr. o No 2. ) ) ) J.&A. CHURCHILL Recent Works for Students and Practitioners of Medicine. LONDON ) 7, Great Marlborough Street. JULY, 1910. INDEX. PAGE 2 Anatomy. Physiology. 3 Materia Med ica. Phar= macy. 4 Hygiene. Bacteriology. 5 Pathology. Psychol = ogy. Dictionaries. 6 Medicine. 7 Medicine. 8 Surgery. 9 Surgery. Anaesthetics. 10 Neurology. Urinary Disorders. 11 Midwifery. Gynaeco= logy. Medical Juris= prudence. 12 Ophthalmology. 13 Otology. Paediatrics. Dentistry. 14 Tropical Diseases. Dermatology. 15 Chemistry. Physics. 16 Microscopy. Miscel- laneous. FREE ON . . . APPLICATION. 1. Complete Illustrated Catalogue. 2. Catalogue of Science Books. Anatomy a Physiology A Treatise on Human Anatomy. Bv various Authors Edited by Sir Henry Morhjs, Bart., M.A., F.B.C.S., President of the Royal College of Surgeons, England ; and J. Playfair McMurrich, A.M. w-;r'JA\r^essor °f -Anntnmy, University of Toronto. Fourth Edition! With 102o Engravings, of which 319 are printed in 9 or 4 colour*. 30*. net Also issued in 5 parts. Parts I, II, and III, 8s. net each ; and Parts IV and V, o*. net each. Anatomical Terminology, with Special Reference to the B.N. A. By Llewfllys F. Barker, M.D., Professor of Medicine Johns Hopkins University, Baltimore. With Illustrations and 2 Coloured Plates. 5*. net. A Manual of Practical Anatomy. By the late Professor Alfred W. IIcgites, M.B., M.C.Edin., Professor of Anatomy King's College, London. Edited and completed by Arthur Keith, M. D., Lecturer on Anatomy, London Hospital Medical College. In three parts. -20*. 6rf. Heath's Practical Anatomy : a Manual of Dissec- tions. Edited by J. Ernest Lane, F.R.C.S., Surgeon and Lecturer on Anatomy at St. Mary's Hospital. Ninth Edition. 321 Engravings, of which 32 are coloured. 12*. Gd. Clinical Applied Anatomy ; or, The Anatomy of Medicine and Surgery. By Charles R. Box, M.D., F.R.C.P.Lond., Physician to Out-patients, St. Thomas's Hospital, and W. McAdam Eccles, M.S. Loud., F.R.C.S.Eng., Assistant Surgeon, St. Bartholomew's Hospital. Illustrated by 45 Hates. 12*. Gd. net. Essentials of Surface Anatomy. By Charles E. Whittaker, F.R.C.S., L.R.C.P., etc., Demonstrator of Anatomy, Sur- geons'Hall, Edinburgh. 2*. Gd. net. Text = Book of Anatomy for Nurses. By Elizabeth R. Bcndy, M.D. With a Glossary and 191 Illustrations. 7*. Gd. net. Human Osteology. By Luther Holden. Eighth Edition. Edited by Charles Stewart, F.R.S., Conservator of the Museum R.C.S., and Robert W. Rkid, M.D., F.R.C.S., Regius Professor of Anatomy in the University of Aberdeen. 59 Lithographic Plates and 74 Engravings. 1G*. Landmarks, Medical and Surgical. Fourth edition. 3*. Gd. Elements of Human Physiology. By Ernest H. Starlino, M.D., F.R.C.P., F.R.S., Jodrell Professor of Physiology in University College, London. Eighth Edition. 323 Illustrations. 12*. Gd. net. A Course of Experimental Physiology. With an Introduction by Prof. E. H. Starling, F.R.S. By N. H. Alcock, M.D., and F. O'Brien Ellison. M.D.. B.A.O.Dub., Physiological Department, St. Mary's Hospital. With 30 Illustrations. 5*. net. Practical Physiological Chemistry. By Philip B. Hawk, M.S., Ph.D., Professor of Physiological Chemistry in the Uni- versity of Illinois. Second Edition. With Coloured Plates and 126 Figures. 16*. net. The Cell as the Unit of Life, and other Lectures, Delivered at the Royal Institution, London, 1899-1802. An Introduction to Biology. By the late Allan Macfadyen, M.D., B.Sc, I 'u lenan Professor of Physiology, Roval Institution, London. Edited by K. Tanner Hewlett, M.D.', F.R.C.P. 7*. Gd, net. . The Functional Inertia of Living Matter: a Contn= bution to the Physiological Theory of Life. By David Frakfr Hareis, M.D., C M., B.Sc.(Lond.),F.R.S.E., Physiological Department, University of Birmingham. 12 illustrations. 5*. net. J. & A. CHURCHILL 2 Materia Medica * Pharmacy A Text = Book of Materia Medica. By C. E. Marshall, j M D. Professor of Materia Medica and Therapeutics in the University of st. Andrews. 127 Illustrations. 10s. 6d. net. A Manual of Pre- scribing for Students and Practitioners of Medicine. 5s. net. Materia Medica, Pharmacy, Pharmacology, and Therapeutics. By W. Halt: White, M.D., F.R.C.P., Physician to, and Lecturer on Medicine at, Guy's Hospital. Eleventh Edition. 6s. 6rf. net Southall's Organic Materia Medica. By J. Barclay, ) B.Sc.Lond. Seventh Edition revised by Ernest W. Mann. 7s. Crf. net. A Text=Book of Materia Medica. By Henry G-. Greenish, P.I.O., F.L.S., Professor of Pharmaceutics to the Pharma- ceutical Society. Second Edition. 269 Illustrations. 15s. net. The Microscopical Examination of Foods and Drugs. 168 Illustrations. ]0s. 6rf. net. An Anatomical Atlas of Vegetable Powders. 138 Illustrations. 12s. 6rf. net. Practical Pharmacy. By E. W. Lucas, F.I.C., F.C.S. Second edition. 221. Illustrations. 12s.6if.net. By the same Author. The Book of Prescriptions (Beasley), with an Index of Diseases and Remedies. Beivritten by E. W. Lucas. F.I.C., F.C.S. With an Introduction by A uthub Latham, M.D., F.R.C.S. ) Ninth Edition. 6s. net. ) The Book of Receipts : containing a Veterinary Materia ) Medica. A Pharmaceutical Formulary. A Photographic Formulary. A Synopsis of Practical Methods employed in the Examination of Urine, ( Milk, Potable Waters. Sputum, etc., together with numerous Chemical and other Tables. With 10 Plates. 7s.6ri.net. ) First "Lines in Dispensing. With 93 Illustrations. 3s. Gd. net. The National Standard Dispensatory. By H. A. Hake, P. Sc. , M.D., and others. 178 Illustrations. 31s. Gd. net. Medical and Pharmaceutical Latin for Students of Pharmacy and Medicine By Reginald E. Bennett, Pharmacist and Teacher of Pharmacy at University College Hospital, London. 6s. net. A Companion to the British Pharmacopoeia. By Peter Wyatt Squire, F.L.S., F.C.S. Eighteenth Edition. 14s. net. ) Pocket Edition, 7s. Gd. net. The Pharmacopoeias of Thirty of the ) London Hospitals. Arranged in Groups for comparison. Eighth Edition. 5s net. ^ The Pharmaceutical Formulary : a Synopsis of the British and Foreign Pharmacopoeias. By Henry Beasi.et. Twelfth ) Edition by J. Oi.dhasi Braithwaite. 6s. Gd. \ Tuson's Veterinary Pharmacopoeia, including the Outlines of Materia Medica and Therapeutics. Sixth Edition. Edited by James Payne, F.C.S. 7s.0rf.net. ) Year=Book of Pharmacy: containing the Trans = actions of the British Pharmaceutical Conference. Annually, 10s. net \ ' ) O J. & A. CHURCHILL ~-^p Q Hygiene a Bacteriology TheI^°pKaSi Practice <>f Hygiene. (Notter and (some ot «-l„ch are in colours) and 200 other Illustrations. 21». net By the same Author. Military Hygiene. A Manual of Sanitation for soldiers. With 40 Illustrations. 3s . Crf. net. Manual of Hygiene. By W. H. Hamer, M.D., D.P.H. A^'Ct'P7 ^ecturer oix Public Health, St. Bartholomew's Hospital, Assistant Medical Officer of Health of the Administrative County of London. 93 Illustrations. 12s. 6rf. net. A Handbook of Hygiene and Sanitary Science. By Geo Wilson M.A., M.D., LL.D., D.P.H.Camb. Medical Officer of Health tor Mid-TK arwickshire. Eighth Edition. Illustrated. 12s. Grf. Lessons on Elementary Hygiene and Sanitation, With Special Reference to the Tropics. Bv W. T. Puout 0 M G MB U.M.(Ediu.), Hon. Lecturer School of Tropical Medicine, Liverpool"! Second Edition. With GO Illustrations. 2». Grf. net. The Effects of Borax and Boric Acid on the Human System. Third Treatise. With Diagrams. By Dr. Oscar Liebkeich. 08. net. A Simple Method of Water Analysis. By John C. Tiikesh, M.I). Vic, D.Sc.Lond., D.P.H.Camb. Sixth Edition. 2s.0rf.net. By the same Author. The Examination of Waters and Water Supplies. 19 Plates and 11 Figures in the Text. 14s. net. Also, with Arthur E. Porter, M.D., M. A. Cantab. Preservatives in Food and Food Examination. 8 Plates. 14s. net. Foods and their Adulteration. By Harvey W. Wiley, M.D., Ph.D. With 11 Coloured Plates and'86 other Illust-ations. 21s. net. A Manual of Bacteriology, Clinical and Applied. By R. Tanner Hewlett, M.D., Professor of General Pathology and Bacteriology in King's College, London. Third Edition. 24 Plates and 72 Figures in the Text. 10s. Grf. net. By the same Author. Serum Therapy, Bacterial Therapeutics, and Vaccines. 20 Figures. 5s. net. Clinical Diagnostic Bacteriology, including Serum - and Cyto-diagnosis. By Alfred 0. Coles, M.D., D.Sc, F.R.S.E. 2 Coloured Plates. 8s. net. Lessons in Disinfection and Sterilisation. By F. W. Andeewes, M.D., F.R.C.P., Lecturer on Pathology, St. Bartholomew's Hospital. Second Edition. 31 Illustrations. 3s.0rf.net. J. &. A. CHURCHILL Pathology * Psychology Dictionaries ) Pathology, General and Special, for Students of Medicine By R. Tanner Hewlett, M.D., F.R.C.P., D.P.H , Prolessor o ten nvl Patoology and Bacteriology in King's College, London. 31 Plates and 13 Illustrations in Text. Second Edition. 10s.6d.net. A Manual of General or Experimental Pathology. By W S. Lazarus-Bari.ow, M.D., F.R.C.P., Director of the Cancer Research Laboratories, Middlesex Hospital. Second Edition. 21.. net. By the same Author. The Elements of Pathological Anatomy and Histo- logy for Students. 7 Coloured Plates and 171 Figures in the Text. 24s. net. Surgical Pathology and Morbid Anatomy. See p. 8. Post= Mortem Manual. A Handbook of Morbid Anatomy and Post-mortem Technique. By 0. R. Box M£ F.R UP F.R.C.S., Lecturer on Applied Anatomy, St. Thomas s Hospital. 19 Illustrations. 6s. net. The Pathologist's Handbook: a Manual for the Post-mortem Room. By T. N. Kelynack, M.D. 126 Illustrations. Pegamoid, 4s. 6d. Psychological Medicine: a Manual of Mental Diseases. By Maurice Craig, M.A., M.D., F.R.C.P., Physicmn and Lecturer on Mental Diseases, Guy's Hospital. 22 Plates. 12s. 6d. net. Mental Diseases: Clinical Lectures. By T. S. Clouston M.D., F.R.CP.Edin., Lecturer on Mental Diseases m the University of Edinburgh. Sixth Edition. 30 Plates. 14s.net. The Force of Mind; or, the Mental Factor in Medicine. By Alfred T. Schofield, M.D., Hon. Physician to Frieden- heim Hospital. Third Edition. 5s.net. Unconscious Therapeutics; or, The Personality of the Physician. Second Edition. 5s. net. I he Management of a Nerve Patient. 5s. net. The Journal of Mental Science. Published Quarterly, by Authority of the Medico-Psychological Association. 5s. net. Dictionary of Medical Terms: English, French, German. Edited by Paul Blaschke. 8s. net. Dictionary of Medical Conversation; English=German, 4s. net. German-English, Is. net. A German- English Dictionary of Terms used in Medicine and the Allied Sciences. By Hugo Lang, B.A., and Bertram Abrahams, M.B., B.Sc, F.R.CP. 15s. net. Dunglison's Dictionary of Medical Science. By Thomas L. Stedman, A.M., M.D. Twenty-third Edition. 577 Illustra- tions, including 81 page-plates. 34s. net. A Medical Vocabulary. By B. G. Mayne, M.D., LL.D. Seventh Edition, by W. W. Wagstaffe, B.A., F.R.C.S., and G. D. Parker, M.B. 12s. M. J. & A. CHURCHILL f Medicine o Lectures on Medicine to Nurses. By Herbert E. Cuff, M.D., F.R.C.S., late Medical Superintendent, North-Eastern Fever Hospital, London. Fifth Edition. au Illustrations, 3s. urf. net. How to Examine the Chest: a Practical Guide for the use of Students. By Samuel W est, M.D., F.K.C.P., Physician lo St. Bartholomew's Hospital. Third Edition. 40 Engravings. 5s. - J. & A. CHURCHILL O ( A Text- Book of Medicine. Be^uu by the lale C ,/p. h. pS-ti Manual of the Practice of Medicine. By Frederick l AYLoit, M.D F.K.C.P., Consulting Physician to, 'and Lecturer on ( KuX^lom^B^^ ^Kf^on. 8 Skiagraui S'a 4b Illustrations, lbs. net. Some Disorders of the Spleen. 3s. net. ( A Short Practice of Medicine. By Eobert A Fleuikg, M A, M.D., F.R.C.P.E , F.K.S.E., Lecturer on Practice of { Medicine, School of the Royal Colleges, Edinburgh ; Assistant Physician Royal Infirmary, Edinburgh. 48 Illustrations (10 Coloured) . 10s. Orf. net. ) The Practice of Medicine. By M. Charteris, M.D., Professor of Therapeutics "hud Materia Medica in the University of' ) Glasgow. Ninth Edition. Edited by F. J. Chautekis, M.D., Ch B \ Illustrated. 9s. 6rf. net. ' ' ) A Medical Vademecum, in German and English. By B. Lewis. With a Preface by Prof. Dr. A. Polmzek. 15s. net. ) Student's Guide to Medical Diagnosis. By Samuel Fenwick, M.D., F.K.C.P., and W. Soltau Fenwick, M.D., B.S. Ninth ) / Edition. 13i) Engravings. Us. } A Dictionary of Medical Treatment for Students and > Junior Practitioners. By AttTHUii Latha.ii, M.D.Oxon., F.R.C.P.Lond., ) Physician and Lecturer on Medicine at St. George's Hospital. 0s. Orf. net! ) Text=Book of Medical Treatment (Diseases and ( . Symptoms). By Nestou I. C. Tikakd, M.D., F.R.C.P., Professor of the ( Principles and Practice of Medicine, King's College, London. 15j. A Manual of Family Medicine and Hygiene for India. Published uuder the Authority of the Government of India. By Sir WiLi.usi J. Mooue, K.C.I.K., M.D. Seventh Edition revised by Major ( J. H. Tull Walsh, I. M.S. 70 Engravings. 0s.net. Waring's Bazaar Medicines of India. By Lt.-Col. \ 0. P. Lukis, I. M.S., Principal of the Medical College, Calcutta. Sixth Edition. 0s. net. ) The Blood : how to Examine and Diagnose its Diseases. By Alfked C. Coles, . M.D., D.Sc, F.R.S.Edin. Third ( ( Edition. 7 Coloured Plates. 10s.0rf.net. Medicine Ulcer of the Stomach and Duodenum. By Samuel Fenwick, M.D., F.R.C.F., and W. Soltau Fenwick, M.D., B.S. 5d Illustrations, 10s. Or/. By the same Authors. Cancer and other Tumours of the Stomach. 70 Illustrations. 10s. 6rZ. On Carbohydrate Metabolism, with an Appendix on the Assimilation of Carbohydrate into Protekl and Fat followed by^ the Fundamental Principles and the Treatment of Diabetes dialectical y discussed. By Frederick W. Pavy, M.D., LL.D., F.R.S., F.R.O.F., Con- sulting Physician to Guy's Hospital. With 8 Plates. Demy 8vo. 6s.net. The Schott Methods of the Treatment of Chronic Diseases of the Heart, with an account of the Nauheim Baths and of the Therapeutic Exercises. By W. Bezly Thome, M.D., M.R.C.P. Fifth Edition. Illustrated. 5s. net. The Clinical Examination of Urine, with an Atlas of Urinary Deposits. By Bindley Scott, M.A., M.D. 41 original Plates (mostly in colours). 15s. net. Urine Examination made easy : a Method of Examining Urine, with the common Tests fully described. By Thomas Caeruthers, M.B., Ch.B. Is. 6d. net. Rational Organotherapy, with Reference to Urose= miology. Translated from the Russian Text by Professor Dr. A. von Poehl, Professor Prince J. vox Taucitanoff, Dr. Alf von Poehl, and Dr. P. Wachs. Vol. I. 8vo. 7s. 6d. net. On Gallstones, or Cholelithiasis. By E. M. Brockbank, M.D.Vict., M.R.C.P.Lond., Honorary Physician to the Ancoats Hospital, Manchester. 7s. Obstinate Hiccough: the Physiology, Pathology, and Treatment, based on a collection of over 150 cases from British and Foreign Works. By L. F. B. Knuthsen, M. D.Edin. 6s. On Syphonage and Hydraulic Pressure in the Large Intestine, with their Bearing upon the Treatment of Constipation, Appendicitis, etc. By Ralph Wixnington Lefi wich, M.D. 3s.net. Uric Acid as a Factor in the Causation of Disease. By Alexander Haig, M.D., F.R.C.P , Physician to the Metropolitan Hospital and the Royal Hospital for Children and Women. Seventh Edition. 75 Illustrations. 11<. net. Uric Acid in the Clinic. A Clinical Appendix to " Uric Acid as a Factor in the Causation of Disease." 5s.net. Uric Acid, an Epitome of the Subject. Second Edition. 2*. Oil. net. Diet and Food considered in relation to Strength and Power of Endurance, Training, and Athletics. Sixth Edition. 2». net. J. & A. CHURCHILL Surgery ThepPJsctisce of Surgery. By W. GL Spencer, M.S., G E l;« UPR°? % LTUr?e0n to St" Bartholomew's Hospital, ; assisted Dy u . w. Akdhwes, M.D., Lecturer on Pathnlno-c- St- Bartholomew's Hospital. Numerous ; By the same Author. On Diseases of the Rectum and Anus, including the Fifth Edition of the Jacksonian Prize Essay on Cancer. Third Edition. With 13 Plates and Si Illustrations. 10s. 6rf. net. \ Also Cancer of the Rectum, especially considered with ) regard to its Surgical Treatment. Jacksonian Prize Essay. FifthEdition. \ With 13 Plates and several Engravings. 5s. net. < Diseases of the Rectum, Anus, and Sigmoid Colon. By F. Swikfohd EnwiHDs, F.R.C.S., Senior Surgeon to St. Mark's ( Hospital for Fistula and other Diseases of the Bectum. Third Edition. ( 102 Illustrations. 10s. Grl. net. ) A Manual of Minor Surgery and Bandaging. Fourteenth Edition of Heath's. By Bilton Pollaed, F.B.C.S., Surgeon ) to University College Hospital. 250 Engravings. 7*. 6rf. net. Injuries and Diseases of the Jaws. By Christopher Heath, F.R.C.S. Fourth Edition. Edited by Heney Pekct Dean, , M.S., F.R.C.S., Assistant Surgeon to the London Hospital. 187 Wood ) Engravings. Ms. ( By the same Author. Clinical Lectures on Surgical Subjects delivered at University College Hospital. First Series, 6s. ; Second Series, 6«. An Essay on the General Principles of the Treat- ment of Spinal Curvatures. By B. Heathek Bigg, F.B.C.S.Edin. ( Illustrated by Photographs and Sketches. 5s. net. ( < J. & A CHURCHILL " O Surgery 0 Anaesthetics ) The Operations of Surgery. By W. H. A. Jacobson, M Ch.Oxon., F.R.C.S., Consulting Surgeon Guy's Hospital, and R. P. Rowlands, M.S.Lond., F.R.C.S.. Assistant Surgeon and Surgeon to the Orthopaedic Department, Guy's Hospital. Filth Edition. 2 vols. 777 Illustrations. 42s. net. Abdominal Pain: its Causes and Clinical Signifi= cance. By Alfred Ernest Maylard, M.B.Lond. and B.S., Senior Surgeon to' the Victoria Infirmary, Glasgow. Second Edition. 7s. Gd. net. > By the same Author. l ) Abdominal Tuberculosis. 57 Illustrations. 12s. 6cl. net. Clinical Essays and Lectures. By Howard Marsh, F.R.C.S., Professor of Surgery in the University of Cambridge, late Surgeon to St. Bartholomew's Hospital. 26 Illustrations. 7s. 6rf. Hare = lip and Cleft Palate. By B. W. Murray, E.R.C.S., Surgeon, David Lewis Northern Hospital, late Surgeon, Liver- pool Infirmary for Children. 25 Hlustrations. 3s. By the same Author. Hernia, its Cause and Treatment. 27 Illustrations. is. 6rf. net. Modern Bullet = Wounds and Modern Treatment, with Special Regard to Long Bones and Joints, Field Alliances and First Aid. Part of the Alexander Essay for 1903. By Major F. Smith, 1 D.S.O., R.A.M.C. 3s.net. Surgical Emergencies : together with the Emer= gencies attendant on Parturition and the Treatment of Poisoning. By Paul Swain, F.R.C.S., Surgeon to the South Devon and East Cornwall Hospital. Fifth Edition. 14,9 Engravings. 6s. Chloroform : a Manual for Students and Practi- tioners. By Edward Lawrie, M.B.Edin., Lieut.-Col. I.M.S., Residency Surgeon, Hyderabad. Illustrated. 5s. net. O J. & A. CHURCHILL o Neurology 0 Urinary Disorders A Text=Book of Nervous Diseases. By W. Aldren Paralysis and other Nervous Diseases in Childhood HoaSft£ %'« ?y JtHES,T^TI-,0R. M.D., F.R.C.P., Physician, National 12° Grf net Paralysed and EPdeptic, Queen Square. 74 Illustrations. ) V " ' s A Manual of Diseases of the Nervous System. ) By Silt William B. Gowers, M.D., F.R.S. Vol. I.— Nerves and Spinal Cord. Third Edition, by the Author and James Taylok, M.D., F.R.C.P. 192 Engravings. 15». By the same Author. Subjective Sensations of Sight and Sound, Abio= trophy, anil other Lectures on Diseases of the Nervous System. 18 Illus- \ trations. On. net. ) Also Epilepsy and Other Chronic Convulsive Diseases: their Causes, Symptoms, and Treatment. Second Edition. 10s. 6rf. ( Also The Borderland of Epilepsy, Faints, Vagal Attacks, Vertigo, Migraine, Sleep Symptoms, and their Treatment. 4s. M. net. Text=Book of Nervous Diseases and Psychiatry. By Chaulks L. Dana, A.M., M.D., LL.D., Professor of Nervous J Diseases in Cornell University Medical College. Seventh Edition. { With 3 Plates and 201 Text-figures. 25*. net. ) Selected Papers on Stone, Prostate, and other Urinary Disorders. By Reginald Hauuison, F.R.C.S., Surgeon to St. Peter's Hospital. 15 Illustrations. 5». By E. HffBBT Fenwick, F.R.C.S., Surgeon to the London Hospital. A Handbook of Clinical Cystoscopy. 31 Plates aiid Hi Figures in the Text. (Reprinting). Atlas of Electric Cystoscopy. 34 Coloured Plates. Zlg. net. The Value of Radiography in the Diagnosis and Treatment of Urinary Stone. With 80 Plates. 10«. Grf. net. Obscure Diseases of the Urethra. 63 Illustrations. 6s. 6d. Operative and Inoperative Tumours of the Urinary Bladder ; a Clinical and Operative Study based on 500 cases. 39 Illus- ) trations. 5?. net. Tumours of the Urinary Bladder. Fas. I. 5s. net. ) Ulceration of the Bladder, Simple, Tuberculous, and ) Malignant: a Clinical Study. Illustrated. 5.-. O J. & A. CHURCHILL O Midwifery a Gynaecology Medical Jurisprudence The Practice of Midwifery. By Alfred L. Galabin, M A. M D., F.R.C.P., Consulting Obstetric Physician to Guy's Hospital ; and G F. B&ackeb, M.D., F.R.C.S., Obstetric Physician to University College Hospital. Seventh Edition. 503 Engravings. 18s.net. Manual of Midwifery. By T. W. Eden, M.D., C M Edin , F.R.C.P.Lond., Obstetric Physician and Lecturer on Practical Midwifery, Charing Cross Hospital. Second Edition. 42 Plates and 231 Illustrations in the" Text. 12s. 6d. net. j A Short Practice of Midwifery, embodying the Treatment adopted in the Rotunda Hospital, Dublin. By Henrt Jellett, M.D., B.A.O.Dub., King's Professor oE Midwifery, Trinity College, / Dublin. Fifth Edition. 4 Coluuied Plates and 200 Illustrations. 10*. 6d. net. By the same Author. A Short Practice of Midwifery for Nurses, with a Glossary of the Medical Terms used in the Book and the Regulations of the CM. B. Third Edition. 4 Coloured Plates and 1G1 Illustrations. ? 6*. Sd. net. A Manual for Midwives. By C. J. 1ST. Longridge, M.D., M.R.O.P., F.R.C.S., Examiner Central Midwives' Board. 3 Plates and 47 Illustrations. 3*. (S. A Lecture on Dysmenorrhcea. By R. A. Gibbons, M.D., F.R.C.S.E., Physician to the Grosvenor Hospital for Women. 2«. ) net. S Lectures on Medical Jurisprudence and Toxicology. By Fred. J. Smith, M.D., F.R.C.P., F.R.C.S.Eng., Physician to, and / Lecturer on, Forensic Medicine and Toxicology at the London Hospital. Second Edition. 8s. Grf. net. Medical Jurisprudence: its Principles and Practice. By Alfred S. Taylor, M.D., F.R.O.P., F.R.S. Fifth Edition, by Fred. J. Smith, M.D., F.R.C.l*., Lecturer on Forensic Medicine at the London ( Hospital. 2 vols. 39 Engravings. 30s. net. ( J. &c A. CHURCHILL n q Ophthalmology Medical Ophthalmoscopy: a Manual and Atlas. fLp7„>w,1AM R- Goweks M.D., F.R.C.P., F.R.S. Fourth Edition, fhl i t th? assist,ance °f Makcus Gunn, M.B., F.R.C.S., Surgeon to the Royal London Ophthalmic Hospital. Autotype Plates and Woodcuts. Manual of Ophthalmic Surgery and Medicine. By W. H. H. Jkssop, M.A., F.R.C.S., Senior Ophthalmic Surgeon to St Bartholomew's Hospital. Second Edition. 8 Plates and 155 other Illustrations. 9s. 6rf. net. Refraction of the Eye: a Manual for Students. By Gustavus Haktkidge, F.R.C.S., Surgeon to the Royal Westminster Ophthalmic Hospital. Fourteenth Edition. 109 Illustrations, also Test- types, etc. 5s. net. By the same Author. The Ophthalmoscope : a Manual for Students. Fifth Edition. 68 Illustrations and 4 Plates. 4s.net. Diseases of the Eye : a Manual for Students and Practitioners. By J. Hehbeut Parsons, D.Sc, M.B., B.S., F.R.O.S., / Assistant Ophthalmic Surgeon, University College Hospital ; Assistant / Surgeon, Royal London (Moorhelds) Ophthalmic Hospital. 308 Illustra- ) tions and 15 Plates. 10s. 6rf. net. ■ . ) By the same Author. ( Elementary Ophthalmic Optics, including Ophthal= moscopy and Retinoscopy. 00 Illustrations. 6s. 6rf. ( Sight = Testing Made Easy, including Chapter on Retinoscopy. -By W. \V. Hardwioke; M.I)., M.R.C.P., late Hon. ( Physician, Jlolesey and Hamptou Court Cottage Hospital. Second ( Edition. 12 Engravings. 2~nr^ , m ■ , Medicine, University £ feS ga%F»* On the Causes and Continuance of Plague in Honp; Konff. By W. J. Simpson. M.D., P.B.O.P. Numerous ('harts and Din-rams 10s.net. Report on Plague in the Gold Coast in 1008. illustrated. 2s, net. On the Outbreak of Yellow Fever in British Honduras in 1005. By Sir Rotjeht Bovck, M.B., F.R.S. 3s. Gd. net. A Handbook on Leprosy. By S. P. Impey, M.D., late Supt., Rohben Island Leper Asylum, Cape Colony. 38 Plates. 12s. A Manual of Diseases of the Skin, with an Analysis of 20,000 Consecutive Cases and a Formulary. By Duncan E Bulkley M.D., New York. Fourth Edition. 6«. Gd. ■' ' Skin Diseases of Children. By Geo. H. Fox, M.D., Clinical Professor of Diseases of the Skin, College of Physicians and Surgeons, New York. 12*. Gd. On Maternal Syphilis, including the Presence and Recognition of Syphilitic Pelvic Disease in AVomen. By John A. Sntw- Mackenzie, M.D. Coloured Plates. 10s. Gd. The Diagnosis and Treatment of Syphilis. By Tom Robinson, M.D.St. And., Physician to the Western Skin Hospital. Second Edition. 3s. Gd. By the same Author. The Diagnosis and Treatment of Eczema. Second Edition. 3s. 6d. Ringworm, and some other Scalp Affections : their Cause and Cure. By Haydn Brown, L.R.C.P.Ed. 5s. J. & A. CHURCHILL - u Chemistry * Physics The Elements of Chemistry. By M. M. Pattison Mdir, M.A. Illustrated. IOi. U. net. The Analyst's Laboratory Companion : a Collection ofSS Data for Chemists .and Students. By Alfred E. Johnson, B Sc F I.C. Third Edition. 6s. 6(7. net. Commercial Organic Analysis: a Treatise on the [Prospectus on application.] Volumetric Analysis ; or, the Quantitative Estima- tion of Chemical Substances by Measure. By Francis Sot .on, F.C.S., FIC Ninth Edition. 121 Engravings. 20s.net. A Manual of Chemistry, Theoretical and Practical. By Sir William A. Tii.de*. D.Sc, F.R.S., Professorof Chemistry m the Royal College of Science, London. 2 Plates and 143 Woodcuts. 10s. Valentin's Practical Chemistry. By Dr. W. R. Hodgkin^ok. F.R.S.E., Professor of Chemistry and Physics in the oVdhance College, Woolwich. Tenth Edition. 14 Plates and 83 Figures in the Text. 10s. net. A Handbook of Physics and Chemistry. By Herbert E. Corbin, B.Sc.Lond., and Archibald M. Stewart, B.Sc.Lond. Third Edition. 165 Illustrations. 6s. 6rZ. net. A Treatise on Physics. Bv Andrew Gray, LL.D., F R.S., Professor of Natural Philosophy in the University of Glasgow. Vol.1. Dynamics and Properties of Matter. 350 Illustrations. Ids. Qualitative Analysis and Practical Chemistry. By Frank Clowes, D.Sc.Lond., Emeritus Professor of Chemistry m the University College, Nottingham. Eighth Edition. 102 Engravings, is. 6rf. net. Quantitative Analysis. By Frank Clowes, D.Sc.Lond., and J. Bernard Coleman, Assoc. R. C, Sc. Dublin : Professor of Chemistry, South-West London Polytechnic. Eighth Edition. Idd Engravings. 10s. del. net. By the same Authors. Elementary Practical Chemistry. Parti. Fifth Edition. General Chemistry. 75 Engravings. 2s.