UBRA.lV BACTERIOLOGY AND THE PUBLIC HEALTH BACTERIOLOGY AND THE PUBLIC HEALTH BY GEORGE NEWMAN, M.D., F.E.S.K, D.P.H. \N FORMERLY DEMONSTRATOR OF BACTERIOLOGY IN KING'S COLLEGE, LONDON, ETC. MEDICAL OFFICER OF. HEALTH OF THE METROPOLITAN BOROUGH OF FINS1XURY JOINT-AUTHOR OF " BACTERIOLOGY OF MILK." ILLUSTRATED THIRD EDITION PHILADELPHIA P. BLAKISTON'S SON AND CO. 1012 WALNUT STREET 1904 N5 BIOLOGY LIBRARY G Printed in Great Britain. ~~___^ ^Ct:r ^^^^* PREFACE THOUGH nominally a third edition of Bacteria in Relation to the Economy of Nature, Industrial Processes, and ttye Public Health, this is, speaking generally, a new book. Several new chapters have been added, and the whole has been enlarged and revised. The book is an attempt to set forth a simple general statement of our present knowledge of bacteria, especially as they are related to the public health. Theoretical and practical text-books of bacteriology abound, but as a rule they deal largely, and rightly so, with laboratory methods and technique. The general student of hygiene and the medical officer of health require, however, an elementary book in which, whilst ample laboratory facts are recorded, the subject is viewed broadly and particularly as it concerns the practical everyday problems of health and preventive medicine. This book is aimed to meet that requirement. I am indebted to many friends and colleagues for suggestions and criticisms, and for a number of illustrations. In addition to a number of cliches used in former editions, some of which were kindly lent by the Scientific Press, Limited, from the Atlas of Bacteriology, by Slater & Spitta, I have to express my obligations to the Controller of His Majesty's Stationery Office, the Secretary of the Eoyal Commission on Sewage Disposal, and the Chairman of the Main Drainage Committee of the London County Council, for permis- sion to use several blocks illustrating sewage bacteria derived from cultures obtained by my friend, Dr Houston, in the course of his sewage investigations. I am in a similar way much indebted to Mr vii o f \ viii PREFACE Fouler ton of the Middlesex Hospital, and Dr Harold Spitta of St George's Hospital, for the use of some excellent photographs. My colleague, Mr Harold Swithinbank, has kindly allowed me to use three coloured plates of "acid-fast" cultures from our book on the Bacteriology of Milk, and he has also supplied me with several original plates. To each of these gentlemen I am glad to have the oppor- tunity of expressing my sincere thanks. G. N. LONDON, August 1904. INTRODUCTION THE science, of biology has for its object the study of organic beings, and for its end the knowledge of the laws of their growth, organisation, and function. From the earliest times of man that life has been studied and the observations recorded. Thus there has come slowly to be a considerable accumulation of knowledge concerning the various forms (morphology) and functions (physiology) of organised life. In the midst of this gradual accumulation of facts we begin to see incoherence becoming coherent, chaos becoming cosmos, and apparent chance and accident becoming law. Bacteriology is a part, a chapter, of general biology, and is concerned with the facts, as at present known, of some of the lowest forms of micro-organic life. Owing to a variety of circumstances, the chief of which is the relation of these micro-organisms to disease, the study of bacteria has assumed a place among the branches of biology of somewhat exceptional importance. The application of biology to daily life and its problems has in recent years been nowhere more marked than in the realm of bacteriology, where the great names of Pasteur, Koch, and Lister, represent the modern epochs of advance. Turn where we will, we shall find the work of the unseen hosts of bacteria daily claiming more and more attention from practical people, and thus biology, even when concerned with the work of microscopic cells, is coming to occupy a new place in the minds of men. Its evolution begins to form part of the general social evolution. Certainly the recent development of bacteriology forms a remark- able feature in the scientific advance of our time. Not only in the diagnosis and treatment of disease, nor even in the various applications of preventive medicine, but in every increasing degree and sphere micro-organisms are recognised as agents of good or ill no longer to be ignored. They occur in our drinking water, in our milk supply, in the air we breathe. They ripen cream, and flavour butter. They purify sewage, and remove waste organic products from the land. They are the active agents in a dozen industrial fermentations. They « (12 x INTRODUCTION assist in the fixation of free nitrogen, and they build up assimilable compounds. Their activity assumes innumerable phases and occupies many spheres, probably more frequently proving itself beneficial than injurious, for bacteria are both economic and industrious in the best sense of the terms. Yet bacteriology has its limitations. It is well to recognise this, for the new science has in some measure suffered in the past from over-zealous and sanguine friends. It cannot achieve everything demanded of it, nor can it furnish a causal agent for every disease to which human flesh is liable. It is a science which even yet is fuller of hope than of proved and established knowledge, for we are at present but upon the threshold of the matter. As in the neighbouring realm of chemistry, it is to be feared that bacteriology has not been without its alchemy. The interpretations and conclusions which have been drawn from time to time respecting bacteriological findings have led to alarmist or optimist views which have not, by later investigations, been fully confirmed. For the science has had devotees who have fondly believed, like the Alchemists, that the twin secret of "transmuting the baser metals into gold," and of indefinitely prolonging human life, was at last to be known. Neither the worst fears of the alarmist nor the sanguine hopes of the optimist have been verified. Science does not progress at such speed or with such kindly accommodation. It holds many things in its hand, but not finally life or death. It has not yet brought to light either " the philosopher's stone " or " the vital essence." What has already been said affords ample reason for a wider dissemination of the elementary facts of bacteriological science. But there are other reasons of a more practical nature. Municipalities and other bodies are expending public moneys in water analysis, in the examination of milk and the control of its supply, in the inspection of cows and dairies, in the bacterial treatment of sewage, in pro- tecting the oyster trade, in the ventilation of workshops and factories, in disinfection, in the prevention of epidemic diseases, and in other branches of public health administration. Furthermore, our increasing colonial possessions with their tropical diseases, and the growth of preventive medicine generally, make an increasing claim upon public opinion and those engaged in raising the physical condition of the people. The successful accomplishment and solution of these questions depends in measure upon a correct appreciation of the elements of bacteriology. The present is a transition period in this department of knowledge. A very large body of facts has been collected, and there has been a natural tendency to draw somewhat sweeping deductions which subsequent knowledge has not supported. What is now required is that our experience in the laboratory and outside should be patiently INTRODUCTION xi and repeatedly checked and tested. If the science of bacteriology is to be built solidly, the two necessities of accumulating accurate facts and making generalisations and deductions must proceed side by side, the former being well established before the latter are accepted. It is the danger of a new science that too much is expected of it. Bacteriology, except in a few well-defined spheres, cannot yet stand alone as reliable basis for legislation. The bacteriologist must be content at present to serve as indicator rather than as olictator. The detection, for instance, of certain bacteria in milk or in oysters is an indication, and not an absolute proposition, of unsatisfactory dairying or oyster culture. Common sense and a broad view of all the ascertain- able facts must guide those whose business it is to apply the findings of bacteriology to preventive measures. In the pages that follow, a large number of statements occur as to the external circumstances and conditions affecting the life of bacteria, and to understand these rightly and hold them in right proportion to each other, it is necessary to bear in mind that many, if not most of them, are of relative importance. They are of value, not as isolated units, but as parts of a whole. It is their co-ordination, relativity, and correlation which must be sought after. Again, the presence of a diphtheria bacillus in the throat of a healthy man appears at first sight to be a fact of absolute and critical importance until the life-history of the bacillus is inquired into and determined, and the relation of the healthy tissues to the performance of its function understood. The bacteriologist and worker in preventive medicine can never afford to neglect the inter-relationship which exists between the seed and the soil. It is not wholly the one or the other with which he has to deal as a practical man. It is the combination and the inter-action between the two. If that principle, and the relativity of our know- ledge of bacteria and the rdle which they play are borne in mind, there is little to fear from a transition period. Whilst there can then be no doubt as to the advantage of a wide dissemination of the ascertained facts concerning bacteria, especially in relation to water, air, milk, and other foods, it must not be forgotten that only patient and skilled observation, and experimental research in well-equipped laboratories, can advance this branch of science or indeed train bacteriologists. The lives of Darwin and Pasteur adequately illustrate this truth. As the world learns its intimate relation to science, and the inter-dependence between its life and scientific truth, States and public authorities may be expected more heartily to support science. CONTENTS CHAPTEK I THE BIOLOGY OF BACTERIA PAGE Early work — Place of Bacteria in Nature— Biology of Bacteria ; Morphology, Composition, Reproduction, Influence of External Conditions — Light —Modes of Bacterial Action— Seed and Soil — Specificity of Bacteria — Association, Antagonism, Attenuation — Bacterial Diseases of Plants 1-32 CHAPTER II BACTERIA IN WATER Quantity of Bacteria in Water— Quality of Water Bacteria : (a) Ordinary Water Bacteria ; (6) Sewage Bacteria ; B. coli communis ; (c) Patho- genic Bacteria in Water — Interpretation of the Findings of Bacteri- ology—Natural Purification of Water— Artificial Purification of Water —Sand Filtration— Domestic Purification of Water . . . 33-72 CHAPTER III BACTERIA IN THE AIR Methods of Examination of Air — Conditions of Bacterial Contamination of Air : (1) Dust and Air Pollution ; (2) Moisture or Dampness of Surfaces : Bacteria in Sewer Air ; (3) the Influence of Gravity ; (4) Air Currents. The Relation of Bacteria to CO2 in the Atmosphere : in Workshops, in Bakehouses, in Railway Tubes, in the House of Commons . .73-91 xiii xiv CONTENTS CHAPTEE IV BACTERIA AND FERMENTATION PAGE Early Work— Kinds of Fermentation r (1) Alcoholic Fermentation, Asco- spores, Pure Cultures, Films ; (2) Acetous Fermentation ; (3) Lactic Acid Fermentation ; (4) Butyric Fermentation ; (5) Ammoniacal Fer- mentation— Diseases of Wine and Beer : Turbidity, Ropiness, Bitter- ness, etc. — Industrial Applications of Bacterial Ferments . . 92-115 CHAPTEK V BACTERIA IN THE SOIL Methods of Examination — Methods of Anaerobic Culture— Place and Function of Micro-organisms in Soil — Denitrification, Nitrification, Nitrogen- fixation, Bacterial Symbiosis — Saprophytic and Pathogenic Organisms in Soil— Tetanus— Quarter-Evil— Malignant (Edema— The Relation of Soil to Bacterial Diseases, such as Typhoid Fever . . 116-150 CHAPTEE VI THE BACTERIOLOGY OF SEWAGE AND THE BACTERIAL TREATMENT OF SEWAGE Composition of Sewage— Quantity and Quality of Bacteria in Sewage— Treat- ment of Sewage : (1) Disposal without Purification ; (2) Chemical Treat- ment ; (3) Bacterial Treatment — Evolution of Bacterial Methods — Septic Tank Method— Contact-Bed Method— Manchester Experiments— Effect of Bacterial Treatment on Pathogenic Organisms . . 151-177 CHAPTEE VII BACTERIA IN MILK AND MILK PRODUCTS General Principles— Sources of Pollution— Number of Bacteria in Milk — Influence of Time and Temperature— Species of Bacteria found in Milk — Fermentations of Milk — Pathogenic Organisms in Milk— Milk-borne Disease: Tuberculosis, Typhoid Fever, Scarlet Fever, Sore-Throat Illnesses, Cholera, Epidemic Diarrhoea— Preventive Measures— Pro- tection of Milk Supply — Control of Milk Supply : Refrigeration, Strain- ing, Sterilisation, Pasteurisation — Specialised Milk — Bacteria in Milk Products— Cream-Ripening — Butter-Making— Cheese-Making— Abnor- mal Cheese-Ripening— Poisonous Cheese . . . . 178-252 CONTENTS xv CHAPTEK VIII BACTERIA IN OTHER FOODS PAGE 1. Shell-fish, Oysters, Cockles, Clams, and their Relation to Disease; Symptoms of Oyster-borne Disease ; Channels of Infection ; Preven- tive Methods — 2. Meat Poisoning ; Tuberculous Meat — 3. Ice-cream and Ice— 4. Bacterial Infection of Bread— 5. Miscellaneous Foods, Watercress, etc. . . . . » . . 253-279 CHAPTEE IX BACTERIA AND DISEASE Growth of Knowledge of Bacteria as Disease Producers— Channels of Infec- tion— How Bacteria cause Disease — Diphtheria: Conditions of Infec- tion— Scarlet Fever, Typhoid Fever, Epidemic Diarrhoea : Conditions of Infection— Suppuration and Abscess Formation— Anthrax— Pneu- monia—Influenza— Actinomycosis— Glanders . . . 280-324 CHAPTEE X TUBERCULOSIS AS A TYPE OF BACTERIAL DISEASE Pathology and Bacteriology of Tuberculosis — The Bacillus of Koch — Animal Tuberculosis, Bovine, Avian, etc. — Bovine and Human Tubercle Bacilli compared — Inter-communicability — Diagnosis of Bovine Tubercle — The Prevention of Tuberculosis— Pseudo-Tuberculosis— Acid-fast Bacteria Allied to the Tubercle Bacillus : in Man, in Animals, in Butter and Milk, in Grass— Differential Diagnosis— Streptothrix Group . 325-369 CHAPTEE XI THE ETIOLOGY OF TROPICAL DISEASES Malaria : Forms of Malarial Fever, the Mosquito Theory, Prevention of Malaria — Cholera : Methods of Diagnosis — Plague : Symptoms, Rats and Plague, Bacteriology, Administrative Considerations— Leprosy — Yellow Fever— Malta Fever— Sleeping Sickness— Beri-beri . 370-404 xvi CONTENTS CHAPTEK XII THE QUESTION OF IMMUNITY AND ANTITOXINS PAGE Bacterial Products — Toxins — Question of Immunity — Kinds of Immunity — Theories of Immunity— Applications of Immunity — Vaccination for Small-pox: Effect of Vaccination — Pasteur's Treatment for Rabies- Inoculations for Cholera, Typhoid, and Plague— Antitoxin Treatment of Diphtheria and its Effects . . . . . 405-431 CHAPTEE XIII DISINFECTION General Principles — Means of Disinfection : by Heat ; by Chemicals — Practical Disinfection: Rooms, Walls, Bedding, Clothing, Excreta, Books, Linen, Stables, etc.— Disinfection of Hands— Disinfection after Special Diseases : Phthisis, Small-pox, Scarlet Fever, Diphtheria, Typhoid, Plague . . . . . . . 432-451 APPENDIX ON TECHNIQUE ...... 453-488 INDEX 489-497 LIST OF FIGURES FKJ. PAGE 1. Various Forms of Bacteria .; . -.'-.. , . 7 2. Diagram of Sarcina - . . : . . . .8 3. Diagrams of Normal and Polymorphic Forms of Tubercle Bacilli . . . . . . . 10 4. Various Forms of Spore Formation and Flagella . . 13 5. Inoculating Needles . . . . . .17 6. Media for Surface and Depth Culture . . . ,17 7. Method of Producing Hydrogen by Kipp's Apparatus for Cultivation of Anaerobes . . . .23 8. Koch's Steam Steriliser . . . . . 24 9. Diagrams of B. typhoms and B. coli . . . .47 10. Pasteur-Chamberland Filter . . . . .71 11. Miquel's Flask . .. . . . .74 12. Sedgwick's Sugar-tube . . . . .75 13. Diagram of Ascospore Formation . . . .98 14. Gypsum Block . . . . . . .98 15. Diagram of S. ceremsice . . . . .102 16. Diagram of S. ellipsoidens . . . . .102 17. Diagram of S. pastorianus . . . . .102 18. Frankel* s Tube , . . . . ,118 19. Rootlet of Pea with Nodules . . . . 133 20. Diagram of Bacillus of Symptomatic Anthrax . . .143 21. A Plan of Septic Tank and Filter-beds . . .167 22. Contact-beds , , , .... 169 23. "Ulax" Filter . . . . , . . 229 24. Diagram of Bacillus diphtherice . . . . . . 289 25. Diagram of Types of Streptococci . . . .312 xvij xviii LIST OF FIGURES FIG. PAGE 26. Diagram of Micrococcns tetragonm- . . . .313 27. Diagram of Gonococcus ..... 314 28. Diagram of Bacillus of Anthrax and Blood Corpuscles. . 316 29. Quartan Malaria Parasite . . . . .373 30. Tertian Malaria Parasite . . . . .374 31. Malignant Malaria Parasite . . . . . 374 32. Anopheles maculipennis . . . . . . 377 33. Diagram of Culex and Anopheles . . . . 378 34. Human and Mosquito Cycles of the Malaria Parasite . ' . 380 35. Diagram of the Comma Bacillus of Cholera . . . 385 36. Suspended Spinal Cord . . . . .421 37. Flask used for Preparation of the Toxin of Diphtheria . 426 38. PetriDish . . . . . ., . 453 39. A Diagram of Colonies of Bacteria on a Gelatine Plate . 454 40. The Hanging Drop . . . . . .455 41. Drying Stage for Fixing Films . . . .456 42. Types of Liquefaction of Gelatine . . . . 457 43. Levelling Apparatus for Koch's Plate . . . ^ ; 464 44. Moist Chamber for Koch's Plate . . . .464 45. Wolfhugel's Counter . . . . . 465 46. Filter-brushing Method . . . . . 466 47. Buchner Tube . .-'.'. . . .466 48. Another Form of Buchner Tube 478 LIST OF PLATES [Note. — Photographs marked with an asterisk (*) have been kindly lent by the Scientific Press Company; those marked f are taken by permission from the Report of Royal Commission on Sewage Disposal; and those marked J from Reports to the London County Council. ] PLATE 1. A Form of Room Temperature Incubator . . To face page 18 2. Hot-air Steriliser and Blood-heat Incubator :. „ 24 3. B. colt communis *; Proteus vulgaris J . . „ 46 4. B. coli communis ; Gas Production in Gelatine f „ 50 5. Small Centrifuge ; Sedgwick's Sugar-tube „ 74 6. Air-Plate Culture from Labourer's Cottage . „ 76 7. Air- Plate Cultures from Bakehouses „ 86 8. Saccharomyces cerevisice ; Ascospores ; Pathogenic Yeast ..*... , „ 98 9. Buchner's Tube ;• Kipp's Apparatus for Anaerobic Culture . . . . „ 116 10. A Vacuum Method of Anaerobic Culture . . „ 118 11. Nitrous Organism; Nitric Organism; Nitrogen- fixing Organisms . . . . „ 128 12. Nitrogen-fixing Bacteria in Nodule on Rootlet of Pea . •'•'••• • • « 134 13. B. tetani \*; B. mycoides*; Streptothrix actinomyces ; B. mallei. . . . . . „ 140 14. Sewage Proteus, Organism and Plate Culture J . „ 154 15. Sewage Streptococci t and Streptococcus pyogenes * . „ 156 16. B. mesentericus, Organism and Plate Culture J . „ 158 17. B. anthracis, from Septic Tank Liquor and in Gelatine Culture (impression) f . . „ 176 18. B. tuberculosis (old culture); Tubercle Bacilli in Cow's Udder . 204 xx LIST OF PLATES PLATE 19. B. diphtheric?; B. von II of maim . . . To face page 288 20. B.typhosus; B. typhosus (flagella)*; Widal-Griiber Reaction * ; B. typhosus in Human Mesenteric Gland. . . 302 21. B. enteritidis sporogenes\; "Enteritidis change" in Milk Cultures! „ 307 22. B. anthracis (Stab Culture) f; B. anthracis from Blood*; Frankel' s Pneumococcus . , „ 318 23. B. tuberculosis, from Sputum,* Tissues, and Culture „ 328 24. Comparative Cultures of Tubercle Bacillus (Bird, Mammal, Butter) „ 350 25. Cultures of Butter Bacillus of Rabinowitsch and Mailer's Milk Bacillus (Chromo) . . „ 360 26. Cultures of B. friburgemis, Nos. I. and II. (Chromo) „ 362 27. Cultures of Butter Bacilli of Binot and Grassberger (Chromo) ...... 364 28. Comparative Cultures of Acid- fast Bacteria (Grass and Manure) ...... 366 29. Streptothrix luteola (Foulerton) ; Streptothrix hominis (Foulerton) .... ,,368 30. B. leprce ; B. pestis*; Staphylococcus pyogenes aureus t, 398 31. Apparatus for Filter-brushing Method in ^Water Examination 466 OF THE UNIVERSITY BACTERIOLOGY AND PUBLIC / HEALTH CHAPTEE I THE BIOLOGY OF BACTERIA* Early work — Place of Bacteria in Nature — Biology of Bacteria; Morphology, Composition, Reproduction, Influence of External Conditions — Light — Modes of Bacterial Action — Seed and Soil — Specificity of Bacteria — Association, Antagonism, Attenuation — Bacterial Diseases of Plants. THE first scientist who demonstrated the existence of micro-organisms was Antony von Leeuwenhoek. He was born at Delft, in Holland, in 1632, and enthusiastically pursued microscopy with primitive instruments. He corroborated Harvey's discovery of the circulation of the blood, in the web of a frog's foot ; he defined the red blood corpuscles of vertebrates, the fibres of the lens of the human eye, the scales of the skin, and the structure of hair. He was neither educated nor trained in science, but in the leisure time of his occupation as a linen-draper he learned the art of grinding lenses, in which he became so proficient that he was able to construct a microscope of greater power than had been previously manufactured. The compound microscope dates from 1590, and when Leeuwenhoek * We propose throughout to use the term bacterium (pi. bacteria) in its generic meaning, unless especially stated to the contrary. It will also be synonymous with the terms microbe, germ, and micro-organism. The term bacillus will, of course, be restricted to a rod-shaped bacterium. A 2 THE BIOLOGY OF BACTERIA was about forty years old, Holland had already given to the world both microscope and telescope. Eobert Hooke did for England what Hans Janssen had done for Holland, and established the same conclusion that Leeuwenhoek arrived at independently, viz., that a simple globule of glass mounted between two metal plates which were pierced with a minute aperture to allow rays of light to pass was a contrivance which would magnify more highly than the recognised microscopes of that day. It was with some such instru- ment as this that the first micro-organisms were observed in a drop of water. It was not until more than a hundred years later that these " animalcula," as they were termed, were thought to be anything more than accidental to any fluid or substance containing them. Plenciz, of Vienna, was one of the first to conceive the idea that decomposition could only take place in the presence of some of these "animalcula." This was in the middle of the eighteenth century. Just about a century later, by a series of important discoveries, it was established beyond dispute that these micro-organisms had an intimate causal relation to fermentation, putrefaction, and disease. Spallanzani, Pasteur, and Tyndall are the three workers who more than others contributed to this discovery. Spallanzani was an Italian who studied at Bologna, and was in 1*754 appointed to the Chair of Logic at Eeggio. But his inclinations led him into the realm of natural history. Amongst other things, his attention was directed to the doctrine of spontaneous generation, which had been propounded by Needham a few years previously. In 1768 Spallanzani became Professor of Natural History at Pavia, and whilst there he demon- strated that if infusions of vegetable matter were placed in flasks and hermetically sealed, and then brought to the boiling point, no living organisms could thereafter be detected, nor did the vegetable matter decompose. When, however, the flasks were but slightly cracked, the air gained admittance, then invariably both organisms and decomposition appeared. Schwann, the founder of the cell- theory, and Schultze, both showed that if the air gaining access to the flask were either calcined or drawn through strong acid the result was the same as if no air entered at all, namely, there were no organisms and there was no decomposition. The result of these investi- gations was that scientific men began to believe that no form of life arose de novo (aliogenesis), but had its source in previous life (biogenesis). It remained for Pasteur and Tyndall to demonstrate this beyond dispute, and to put to rout the fresh arguments for spontaneous generation which Pouchet had advanced as late as 1859. Pasteur collected the floating dust of the air, and found by means of the microscope many organised particles, which he sowed on suitable infusions, and thus obtained rich crops of "animalcula." He also demonstrated that these organisms existed in varying SPONTANEOUS GENERATION 3 degrees in different atmospheres, few in the pure air of the Mer de Glace, more in the air of the plains, most in the air of towns. He further proved that it was not necessary to insist upon hermetic sealing or cotton filters to keep these living organisms in the air from gaining access to a flask of infusion. If the neck of the flask were drawn out into a long tube and turned downwards, and then a little upwards, even though the end be left open, no con- tamination gained access. Hence, if the infusion were boiled, no putrefaction would occur. The organisms which fell into the open end of the tube were arrested in the condensation water in the angle of the tube ; but even if that were not so, the force of gravity acting upon them prevented them from passing up the long arm of the tube into the neck of the flask. A few years after Pasteur's first work on this subject, Tyndall conceived a precise method of determining the absence or presence of dust particles in the air by passing a beam of sunlight through a glass box before and after its walls had been coated with glycerine. Into the floor of the box were fixed the mouths of flasks containing an infusion. These were boiled, after which they were allowed to cool, and might then be kept for weeks or months without putrefying or reveal- ing the presence of germ life. Here all the conditions of the in- fusions were natural, except that in the air above them there was no dust. The sum-total of result arising from these investigations was to the effect that no spontaneous generation was possible, that the atmosphere contained unseen germs of life, that the smallest of organisms responded to the law of gravitation and adhered to moist surfaces, and that micro-organisms were in some way or other the cause of putrefaction. The final refutation of the hypothesis of spontaneous generation was followed by an awakened interest in the unseen world of micro- organic life. Investigations into fermentation and putrefaction followed each other rapidly, and in 1863 Davaine claimed that Pollender's bacillus of anthrax, which was found in the blood and tissues of animals which had died of anthrax, was the cause of that disease. From that time to this, in every department of biology, bacteria have been increasingly found to play an important part. They cause changes in milk, and flavour butter; they decompose animal matter, yet build up the broken-down elements into com- pounds suitable for use in nature's economy; they assist in the fixation of free nitrogen ; they purify sewage ; in certain well- established cases they are the cause of specific disease, and in many other cases they are the probable cause. No doubt the disposal of spontaneous generation did much to arouse interest in this branch of science. Yet it must not be forgotten that the advance of the 4 THE BIOLOGY OF BACTERIA microscope and bacteriological method and technique have played a large share in this development. The sterilisation of culture fluids by heat, the use of aniline dyes as staining agents, the introduction of solid culture media (such as gelatine and agar), and Koch's " plate " method, have all contributed not a little to the enormous advance of bacteriology. The Place of Bacteria in Nature As we have seen, for a considerable period of time after their first detection these unicellular organisms were considered to be members of the animal kingdom. As late as 1838, when Ehrenberg and Dujardin drew up their classification, bacteria were placed among the Infusorians. This was in part due to the powers of motion which these observers detected in bacteria. It is now, of course, recognised that animals have no monopoly of motion. But what, after all, are the differences between animals and vegetables so low down in the scale of life ? Chiefly two : there is a difference in life-history (in structure and development), and there is a difference in pabulum. A plant secures its nourishment from much simpler elements than is the case with animals ; for example, it obtains its carbon from the carbonic acid gas in air and water. This it is able to do, as regards the carbon, by means of the green colouring matter known as chloro- phyll, by the aid of which, with sunlight, carbonic acid is decomposed in the chlorophyll corpuscles, the oxygen passing back into the atmosphere, the carbon being stored in the plant in the form of starch or other organic compound. The supply of carbon in the chlorophyll-free plants, amongst which are the bacteria, is obtained by breaking up different forms of carbohydrates. Beside albumen and peptone, they use sugar and similar carbohydrates and glycerine as a source of carbon. Many of them also have the capacity of using organic matters of complex constitution by converting such into water, carbonic acid gas, and ammonia. Their hydrogen comes from water, their nitrogen from the soil, chiefly in the form of nitrates. From the soil, too, they obtain other necessary salts. Now all these substances are in elementary conditions, and as such plants can absorb them. Animals, on the other hand, are only able to utilise compound food products which have been, so to speak, prepared for them, for example albuminoids and proteids. They cannot directly feed upon the elementary substances forming the diet of vegetables. This distinction, however, did not at once clear up the difficult matter of the classification of bacteria. It is true, they possess powers of motion, are free from chlorophyll, and even feed occasionally upon products of decomposition — three physiological characters which UNIVERSITY OF CLASSIFICATION OF would ally them to the animal kingdom. Yet by their structure and capsule of cellulose and by their life-history and mode of growth they unmistakably proclaim themselves to be of the vegetable kingdom. In 1853 Cohn arrived at a conclusion to this effect, and since that date bacteria have become more and more limited in clas- sification and restricted in definition. Even yet, however, we are far from a scientific classification of bacteria. Nor is this matter for surprise. The development in this branch of biology has been so rapid that it has been impossible to assimilate the facts collected. The facts themselves by their remarkable variety have not aided classification. Names which a few years ago were applied to individual species, like Bacillus subtilis, or Bacterium termo, or Bacillus coli, are now representative, not of individuals, but of families and species. Again, isolated character- istics of certain microbes such as motility, power of liquefying gelatine, size, colour, and so forth, which at first sight might appear as likely to form a basis for classification, are found to vary not only between similar germs, but in the same germ. Different physical conditions have so powerful an influence upon these microscopic cells that their individual characters are constantly undergoing change. For example, bacteria in old cultures assume a different size, and often a different shape, from younger members of precisely the same species; Bacillus pyocyaneus produces a green to olive colour on gelatine, but a brown colour on potato ; the bacillus of Tetanus is virulently pathogenic, and yet may not act thus unless in com- pany with certain other micro-organisms. Hence it will at once appear to the student of bacteriology that, though there is great need for classification amongst the six or seven hundred named "species" of microbes, our present knowledge of their life-history is not yet advanced enough to form more than a provisional arrangement. We know that bacteria are allied to Hyphomycetes on the one hand and Saccharomycetes on the other, and that they have no differentiation into root, stem, or leaf ; we know that they are fungi (having no chlorophyll), in which no sexual reproduction occurs, and that their mode of multiplication is by division. From such facts as these we may build up a classification as follows : — [VEGETABLE KINGDOM. THE BIOLOGY OF BACTERIA VEGETABLE KINGDOM. Thallophyta. [ = The lowest forms of vegetable life. No differentiation into root, stem, or leaf.] I Muscineae. I I Pteridophyta. Phanerogam ia. Algae. [= Chlorophyll present.] Fungi. [ = No Chlorophyll.] Hymenomycetes. Hyphomycetes. Blastoraycetes. Schizomycetes (Mushrooms, etc.) (Moulds.) (Yeasts, etc.) [ = multiplication by cell division or by spores] or Bacteria. Ml) Coccacese ;; — r( cells. (2) Bacteriacese — and threads. (3) Leptotrichese. (4) Cladotrichese. * Migula has suggested that the Schizomycetes should be subdivided into Coccacece, Bacteriacecv, Spirillac (spirilla, spirochreta), Chlamydobacteriacece (Streptothrix, Crenothrix, Cladothrix), and Ikggiatoa. Morphology: Structure and Form Having now located micro-organisms in the economy of nature, we may proceed to describe their subdivisions and form. For practical convenience rather than theoretical accuracy, we may accept the simple division of the family of bacteria into three chief forms, viz. : — ( (1) Round cell form — coccus. Lower Bacteria-! (2) Eod form — 'bacillus. ( (3) Thread form — spirillum. Higher Bacteria — Leptothrix, Streptothrix, Cladothrix, etc. A classification dependent as this is upon the form alone is not by any means ideal, for it ignores all the complicated functions of bacteria, but it is, as we have said, practically convenient. 1. The Coccus. — This is the group of round cells. They vary in size as regards species, and as regards the conditions, artificial or natural, under which they have been grown. Some are less than 2- WOTF °f an incn m diameter ; others are half as large again, if the word large may be used to describe such minute objects. No regular standard can be laid down as reliable with regard to their size. Hence the subdivisions of the cocci are dependent not upon the individual elements so much as upon the relation of those elements to each other. A simple round cell of approximately the size already named is termed a microcowus (jumcpos, small). Certain species of FORMS OF BACTERIA inicrococci always or almost always occur in pairs, and such a com- bination is termed a diplococcus. Some diplococci are united by a thin capsule, which may be made apparent by special methods of staining; in others no limiting or uniting membrane can be seen with the ordinary high powers of the microscope. Again, one fre- quently finds a species which is exactly described by saying that two inicrococci are in contact with each other, and move and act as one individual, but otherwise show no alteration ; whilst others are seen which show a flattening of the side of each micrococcus which Is in relation to its partner. Perhaps the diplococci in an even greater degree than the micro- cocci respond to external conditions both as regards size and shape. It must further be borne in mind that a dividing micrococcus assumes the exact appear- ance of a diplococcus during the transition stage of the fission. Hence, with the exception of several well - marked species of diplococci, this form is somewhat arbitrary. The third kind of micrococcus is that formed by a number of elements in a twisted chain, named streptococcus (orrpeTTTo?, twisted). This form is produced by cells dividing in one axis, and remaining in contact with each other. It occurs in a number of different species, or what are supposed by many authorities to be different species, owing to their different effects. Morphologically all the streptococci are similar, though a somewhat abortive attempt has been made to divide them into two groups, according as to whether they were long chains or short. As a matter of fact, the length of streptococci depends in some cases upon biological properties, in others upon external treatment or the medium of cultivation which has been used. Sometimes they occur as straight chains of only half a dozen elements ; at other times they may contain thirty or forty elements, and twist in various ways, even forming rosaries. The elements, too, differ not only in size, but in shape, appearing occasionally as oval FIG. 1.— DIAGRAMS OF VARIOUS FORMS OF BACTERIA. 1. Micrococcus. 2. Diplococcus. 3. Streptococcus. 4. Staphylococcus. 7. Sarcina. 5. Leuconostoc, show- 8 Bacillus. ing Arthrospores. 9. Spirillum. 6. Merismopedia. 8 THE BIOLOGY OF BACTERIA cells united to each other at their sides. The fourth form is consti- tuted by the micrococci being arranged in masses like grapes, the staphylococcus ((rTav\i?, a bunch of grapes). The elements are often smaller than in the streptococcus, and the name itself describes the arrangement. There is no matrix and no capsule. This is the commonest organism found in abscesses, etc. The sarciiia is best classified amongst the cocci, for it is composed of them, in packets of four or multiples of four, produced by divi- sion vertically in two planes. If the division occurs in one plane, we have as a result small squares of round cells known as merismopedia. In both these conditions it frequently happens that the contiguous sides of the elements of packets become faceted or straightened against each other. It may happen, too, particularly in the sarcincc, that FIG. 2,-Diagram of sarcina. segmentation is not complete, and that the elements are larger than in any other class of cocci. They stain very readily. Nearly all the cocci are non-motile, though Brownian movement (see p. 11) may readily be observed. 2. The Bacillus. — This group consists of rods, having parallel sides and being longer than they are broad. They differ in every other respect according to species, but these two characteristics remain to distinguish them. Many of them are motile, others not. The ends or poles of a bacillus may be pointed, round, or almost exactly square and blocked. They all, or nearly all, possess a capsule. Individuals of the same species may differ greatly, according to whether they have been naturally or artificially grown, and pleomorphic forms are abundant. 3. The Spirillum. — This wavy-thread group is divisible into a number of different forms, to which authorities have given special names. It is sufficient, however, to state that the two common forms are the non-septate spiral thread (e.g. the Spirillum Obermeier of relapsing fever), which takes no other form but a lengthened spirillum ; and the spirillum which breaks up into elements or units, each of which appears comma-shaped (e.g. the cholera bacillus). The degree of curvature in the spirilla, of course, varies. They are the least important of the lower bacteria. The Higher Bacteria group includes more highly organised members of the Schizomycetes. They possess filaments, which may be branched, and almost always have septa and a sheath. Perhaps the most marked difference from the lower bacteria is in their POLYMORPHISM 9 reproduction. In the higher bacteria we may have what is in fact a flower — terminal fructification by conidia. In this group of vegetables we have the Beggiatoa, Leptothrix, Cladothrix, and, at the top, the Streptothrix. It has been demonstrated that Strcptothrix actinomycotica and Streptothrix madurce are the organismal cause, respectively, of Actinomycosis and Madura-foot, two diseases which had hitherto been obscure. Polymorphism (or Pleomorphism). — This term is used to designate an inconstancy of form or a tendency towards biological variation. Vibrios may become spirilla, the ray fungus passes through a coccoid and bacillary stage, and the diphtheria bacillus may either be long, short, straight, or clubbed. This diversity of form appears to belong to many species, and is transmitted from generation to generation ; or the various forms may occur in succession, and represent different stages in the life-history. In B. diphtherice, B. pestis, and B. tuber- culosis and other forms, polymorphism undoubtedly occurs. It is particularly marked in very old cultures of the last named. The ordinary well-known bacillus may grow out into threads with bulbous endings, granular filaments, "drumsticks," and diplococcal forms. It is now known that amongst the causes of polymorphism are certain adverse conditions of medium or other physical influences (moisture, temperature, age, etc.), and thus some bacteria, especially bacilli or vibrios, become altered in shape, losing their ordinary form. On transferring such aberrant and abnormal forms to fresh medium or favourable conditions, they are generally able to assume their original morphology. Indeed the aberrant form is in all probability only a stage in their life-history. Involution forms usually imply degeneration. Biology of Bacteria Composition. — From what we have seen of the pabulum of micro-organisms, we should conclude that in some form or other they contain the elements nitrogen, carbon, and hydrogen. All three substances are combined in the mycoprotein or protoplasm of which the body of the microbe consists. This is generally homogeneous, proteid material, and there is no sign of a nucleus. It possesses a marked affinity for aniline dyes, and by this means organisms are stained for the microscope. Besides the variable quantity of nitrogen present, mycoprotein may also contain various mineral salts. The uniformity of the cell-protoplasm may be materially affected by disintegration and segmentation due to degenerative changes. Vacuoles, which it is necessary to differentiate from spores, also may appear from a like cause. Vacuolation may also occur as a result of a process of osmosis in salt solutions, the protoplasm of the bacillus becoming contracted and disintegrated (plasmolysis). 10 THE BIOLOGY OF BACTERIA Two other signs of degeneration are the appearance of granules in the body of the cell-protoplasm known as metachromatic granules, owing to their different staining propensities, and the polar bodies which are seen in some species of bacteria. Surrounding the mass of mycoprotein, we find in most organisms a capsule or membrane composed, in part at least, of cellulose. This sheath plays a protective part in several ways. During the adult stage of life it protects the mycoprotein, and holds it together. At the time of reproduction or degeneration it not infrequently swells up, and forms a viscous hilum or matrix, inside which are formed the new sheaths of the younger generation. It may be rigid, and so maintain the normal shape of the species, or, on the other hand, flexible, and so adapted to rapid movement of the individual. Here, then, we have the major parts in the constitution of a bacillus — its body, mycoprotein ; its capsule, cellulose. But, further f FIG. 3.— Diagrams of Normal and Polymorphic Forms of Tubercle Bacilli. than this, there are a number of additional distinctive characteristics as regards the contents inside the capsule which call for mention. Sulphur occurs in the Beggiatoa which thrive in sulphur springs. Starch is commoner still. Iron as oxide or other combination is found in several species. Many contain pigments, though these are generally the "innocent" bacteria, in contradistinction to the disease-producing. A pigment has been found which is designated lacterio-purpurin. According to Zopf, the colouring agents of bacteria are the same as, or closely allied to, the colouring matters occurring widely in nature. Migula holds that most of the bacterial pigments are non-nitrogenous bodies. There are a very large number of chromogenic bacteria, some of which produce exceedingly brilliant colours. Among some of the commoner forms possessing this character are Bacillus et micrococcus molaccus, B. et M. aurantiacus (orange) ; B. et M. luteus ; M. roseus (pink) ; many of the Sarcince ; B. aureus ; B. fluorescens liqucfaciens et BACTERIAL POWERS OF MOTION 11 non-liquefaciens (green); B. pyocyaneus (green); B. prodigiosus (blood-red). Motility. — When a drop of water containing bacteria is placed upon a slide, a clean cover-glass* superimposed, and the specimen examined under an oil immersion lens, various rapid movements will generally be observed in the micro-organisms. These are of four chief kinds : (1) A dancing, stationary motion known as Brownian movement. This is molecular, and depends in some degree upon heat and the medium of the moving particles. It is non-progressive, and is well seen in gamboge particles. (2) An undulatory, serpentine movement, with apparently little advance being made. (3) A rotatory movement, which in some water bacilli is very marked, and consists of spinning round, sometimes with considerable velocity, and maintained for some seconds or even minutes. (4) A progressive, darting movement, by which the bacillus passes over some con- siderable distance. The conditions affecting the motility of bacteria are but partly understood. Heating the slide or medium accelerates all movement. A fresh supply of oxygen, or indeed the addition of some nutrient substance, like broth, will have the same effect. There are also the somewhat mysterious powers by which cells possess inherent attraction or repulsion for other cells, known as positive and negative ckemiotaxis. These powers have been observed in bacteria by Pfeiffer and Ali-Cohen. The essential condition in the motile bacilli is the presence of flagella* These cilia, or hairy processes, project from the sides or from the ends of the rod, and are freely motile and elastic. Some- times only one or two terminal flagella are present ; in other cases, like the bacillus of typhoid fever, five to twenty may occur all round the body of the bacillus, varying in length and size, sometimes being of greater length even than the bacillus itself. It is not yet established as to whether these cilia are prolongations of capsule only, or whether they contain something of the body protoplasm. Migula holds the former view, and states that the position of flagella is constant enough for diagnostic purposes. They are but rarely recognisable except by means of special staining methods. Micfococcus agilis (Ali-Cohen) is one of the rare cases of a coccus which has flagella and powers of active motion. Modes of Reproduction. — Budding, division, and spore formation are the three chief ways in which Schizomycetes and Saccharomycetes (yeasts) reproduce their kind. Budding occurs in many kinds of yeast-cells, and generally takes place when the nutriment and * A.flagellum is a hair-like process arising from the poles or sides of the bacillus. It must not be confused with a, filament, which is a thread-like growth of the bacillus itself. 12 THE BIOLOGY OF BACTERIA environment are favourable. The capsule of a large, or "mother" cell, shows a slight protrusion outwards, which is gradually -enlarged into a " daughter " yeast, and later on becomes constricted at the neck. Eventually it separates as an individual. The protoplasm of the spores of yeasts differs, as Hansen has pointed out, according to the conditions of culture. Division, or fission, is the commonest method of reproduction. It occurs transversely. A small indentation occurs in the capsule, which appears to make its way slowly through the whole body of the bacillus or micrococcus until the two parts are separate, and each contained in its own capsule. It has been pointed out already that in the incomplete division of micrococci we observe a stage precisely similar to a diplococcus. So also in the division of bacilli an appear- ance occurs described as a diplobacillus. Simple fission requires but a short period of time to be complete. Hence multiplication is very rapid, for within half an hour a new adult individual can be produced. It has been estimated that at this rate one bacillus will in twenty-four hours produce millions of similar individuals; or, expressed otherwise, Cohn calculated that in three days, under favourable circumstances, the rate of increase would be such as to form a mass of living organisms weighing many tons, and numbering billions of individuals. Favourable conditions do not occur, fortunately, to allow of such increase, which, it is evident, can only be roughly estimated. But the above facts illustrate the enormous fertility of micro-organic life. When we remember that in some species it requires 10,000 or 15,000 fully-grown bacilli placed end to end to stretch the length of an inch, we see also how exceed- ingly minute are the individuals composing these unseen hosts. Spore formation may result in the production of germinating cells inside the capsule of the bacillus, endospores, or as modified individuals, arthrospores. The body of a bacillus, in which sporulation is about to occur, loses its homogeneous character and becomes granular, owing to the appearance of globules in the protoplasm. In the course of three or four hours the globule enlarges to fill the diameter of the rod, and assumes a more concentrated condition than the parent cell. At its maturity, and before its rupture of the bacillary capsule, a spore is observed to be bright and shining, oval and regular in shape, with concentrated contents, and frequently causing a local expansion of the bacillus. In a number of rods lying endwise, these local swellings produce a beaded or varicose appearance, even simulating a streptococcus. In the meantime the rod itself has become slightly broader and pale. Eventually it breaks down by segmentation or by swelling up into a gelatinous mass. The spore now escapes and commences its individual existence. Under favourable circumstances it will germinate. The tough capsule gives way at one point, MODES OF REPRODUCTION 13 O X generally at one of the poles, and the spore sprouts like a seed. In the space of about one hour's time the oval refractile cell has become a new bacillus. One spore produces by germination one bacillus. Spores never multiply by fission, nor reproduce themselves. Hueppe has stated that there are certain organisms (like LeuconostoCy and some streptococci) which reproduce by the method of arthrospores. Defined shortly, this is simply an enlargement of one or more cell elements in the chain which thus takes on the function of maternity. On either side of the large coccus may be seen the smaller ones, which it is supposed have contributed of their proto- plasm to form a mother cell. An arthrospore is said to be larger, more re- fractile, and more resistant than an ordinary endospore. Many bacteriologists of re- pute have declined hitherto definitely to accept arthro- spore formation as a proved fact. Spore formation in bac- teria is not to be considered as a method of multipli- cation. The general rule is undoubtedly that one bacillus produces one spore, and One Spore germinates Flo> ^-DIAGRAMS OF VARIOUS FORMS OF SPORE FORMATION illtO 0116 bacillus. It is a AKD FLAGELLA. reproduction, not a mul- tiplication. Indeed, the whole process is of the nature of a resting stage, and is due (a) to the arrival of the adult bacillus at its biological zenith, or (6) to the con- ditions in which it finds itself being unfavourable to further vegeta- tive growth, and so it endeavours to perpetuate its species. Most authorities are probably of the latter opinion, though there is not a little evidence for the former. Exactly what conditions are favour- able to sporulation is not known. Nutriment has probably an intimate effect upon it. The temperature must not be below 16° C., nor much above 40° C. Oxygen, as we have seen, is favourable, if not necessary, to many species, which will in cultivation in broth rise to the surface and lodge in the pellicle to form their seeds. Moisture, too, is considered a necessity. Koch found that spore formation in B. anthracis occurred in six A. Stages in formation of spore and its after development. B. Spirillum with terminal flagella. 14 THE BIOLOGY OF BACTERIA hours. The spores may be situated in the middle of the bacillus (as in B. anthracis, B. acidi lutyrici, etc.), towards one end (Bacillus of Malignant (Edema), or actually terminal (B. tetani). Those spores produced inside the capsule of the bacillus are termed endospores. Hueppe has described the spores of certain streptococci as arthrospores. The spores of yeast are termed ascospores. The spores of all bacillary species possess, however, certain characters in common. They are as follow. The spore is generally oval, though more spherical in the Hyphomycetes : it is bright and glistening in aspect ; it is often greater in diameter than the bacillus giving rise to it ; its capsule is thicker and stronger than the capsule of the parent bacillus; and it is generally held that the contained protoplasm is more concentrated, so to speak, than that of the bacillus. These two last characters are of chief importance to us, for it is owing to them that spores possess such marked power of resistance. Cohn has suggested that the capsule of a spore is in reality a double envelope, an inner one of fatty and an outer one of gelatinous nature, and it is owing to this that its resistance to heat and dessication is due. The protoplasm of the spore contains, of course, the essential constituents of the mother cell. It is the method by which "the continuity of germ plasm " is secured in these lowly forms of life. Under favourable circumstances this spore-protoplasm will germinate into a new bacillus. It should be understood that whilst holding the view that spores are a resting stage during adverse conditions,* we fully recognise that certain favouring external conditions are essential * Yeast can be effectually starved by cultivating on a small block of plaster- of-Paris kept moist under a bell jar ; under these circumstances the yeast is supplied with nothing but water. In a few days the protoplasm of yeast cells thus circumstanced becomes filled with vacuoles and fat cells. The protoplasm has been undergoing destructive metabolism, and, there being nothing to supply new material, has diminished in quantity and at the same time been partly converted into fat. Both in plants and animals fatty degeneration is a more or less constant phenomenon of starvation, and to this bacteria are no exception. After a time the protoplasm collects towards the centre of the cell, and divides simultaneously into four masses arranged like a pyramid of four billiard balls, three at the base and one above. These are the ascospores, and sooner or later they are liberated by the rupture of the mother-cell wall. Certain of the Streptothrix family also " sporulate " when they find themselves, like yeast upon gypsum, surrounded by an unfavourable environment. Again, in old cultures, it will be found that when the food supply has been exhausted the bacteria have either sporulated or have died. For these reasons sporulation may be looked upon not as a method of multiplication but one of reproduction, of carrying on the species under adverse conditions. With regard to the rapid formation of spores under apparently favourable circumstances (B. filamentosus^ B. anthracis, etc. ), it must be borne in mind that the medium may not be by any means so favourable as appears to be the case (Flugge). It is clear that the food supply immediately around many of the bacteria in a culture must soon be exhausted. Besides, there is the toxic influence early at work, often as an inimical agency acting unfavourably towards the bacillus producing it. So that the appearance of spores in such a culture may still be due to conditions which are actually unfavourable. INFLUENCE OF EXTERNAL CONDITIONS 15 to spore formation. Of these, there are at least three of which bacteriologists have knowledge, namely, moisture, oxygen, and a certain temperature. Fluid media forms an excellent nidus for sporulation so long as some oxygen can gain access to the sporulating germs. But many organisms will not sporulate if lying deep in such a medium. In moulds and yeasts oxygen is essential, and for some spore-bearing bacilli a supply of oxygen is a sine qud non (the exceptions are strict anaerobes like B. tetani, B. butyricus, etc.) of sporulation. Prazmowski has pointed out that it is characteristic of these forms that they are non-motile during sporulation. B. tetani, B. lutyricus, and other strict anaerobes continue to remain motile during sporing. Temperature exerts a marked influence on the process.* In the case of B. subtilis, an organism frequently present in milk, spore formation did not occur below 6° C. ; at 18° C. it required two days ; at 22° C. one day ; and at 30° C. only twelve hours, f • When free in the field of the microscope, spores must be dis- tinguished from fat cells, micrococci, starch cells, some kinds of ova, yeast cells, and other like objects. Spores are detected frequently by their resistance to ordinary stains and the necessity of colouring them by special staining methods. When, however, a spore has taken on the desired colour, it retains it with tenacity. In addition to their shape, size, thickened capsule, and staining characteristics, spores also resist desiccation and heat in a much higher degree than bacilli not bearing spores. It has been suggested that bacteria should be classified according to their method of spore formation. The Influence of External Conditions on the Growth of Bacteria In the earliest days of the study of micro-organisms it was observed that they mostly congregate where there is suitable food for their nourishment. The reason why fluids such as milk, and dead animal matter such as a carcase, and living tissues such as a man's body, contain many microbes, is because each of these three media is favourable to their growth. Milk affords almost an ideal food and environment for microbes. Its temperature and con- stitution frequently meet their requirements. Dead animal matter, too, yields a rich diet for certain species (saprophytes). In the living tissues bacteria obtain not only nutriment, but a favourable * Koch has shown in the case of B. anthracis that at least 16° C. is necessary for spore formation, and at this temperature limited formation of spores did not occur until after seven days. At 21° C. spores had formed after seventy-two hours, at 25° C. after thirty- five to forty hours, and between 30° C. and 40° C. in about twenty-four hours ; • the best and strongest cultivations were obtained from 20° to 25° C. f Fliigge. — Micro-organisms. Translation by W. Watson Cheyne, 1890, p. 539. 16 THE BIOLOGY OF BACTERIA temperature and moisture. Outside the human body it has been the endeavour of bacteriologists to provide media as similar to the above as possible, and containing many of the same elements of food, in order that the life-history may be carried on outside the body and under observation. By means of cover-glass preparations for the microscope we are able to study the form, size, motility, flagella, spore formation, and peculiarities of staining, all of which characters aid us in determining to what species the organism under examination belongs. By means of artificial nutrient media we may further learn the characters of the organism in " pure culture," * its favour- able temperature, its power or otherwise of liquefaction, of curdling of milk, or of gas or acid production ; its behaviour towards oxygen ; its power of producing indol, pigment, and other bodies ; as well as its thermal death-point and resistance to light and disinfectants. It is well known that under artificial cultivation an organism may be greatly modified in its morphology and physiology, and yet its conformity to type remains much more marked than any divergence which may occur. Nutritive Medium.f The basis of many of these artificial media is broth. This is made from good lean beef, free from fat and gristle, which is finely minced up and extracted in sterilised water (one pound of lean beef to every 1000 c.c. of water). It is then filtered and sterilised. To provide peptone beef-broth, ten grammes of peptone and five grammes of common salt are added to every litre of acid beef-broth. It is rendered slightly alkaline by the addition of sodium car- bonate or sodium hydrate, and is filtered and sterilised. In glycerine-broth 6 to 8 per cent, of glycerine has been added after filtration, in glucose-broth 1 or 2 per cent, of grape-sugar. This latter is used for anaerobic organisms. The use of broth as a culture medium is of great value. It is undoubtedly the best fluid medium, and in it may not only be kept pure cultures of bacteria which it is desired to retain for a length of time, but in it also emulsions and mixtures may be placed preparatory to further examination. Gelatine consists of broth solidified by the addition of 100 grams of best French gelatine to the litre. Its advantage is twofold : it is trans- parent, and it allows manifestation of the power of liquefaction. When we speak of a liquefying organism we mean a germ having the power of producing a pepton- ising ferment which can at the temperature of the room break down solid gelatine into a liquid. Grape-sugar gelatine is made like grape-sugar broth. Agar was introduced as a medium which would not like gelatine melt at 25° C. , but remain solid at blood-heat (37'5° C. ; 98 '5° F.). It is a seaweed generally obtained in dried strips from the Japanese market. Ten to fifteen grammes are added to every litre of peptone-broth. Glycerine and grape-sugar may be added as elsewhere. Blood agar is ordinary agar with fresh sterile blood smeared over its surface. Blood serum is drawn from a jar of coagulated horse-blood, in which the serum has risen to the top. This is collected in sterilised tubes and coagulated in a special apparatus (the serum inspissator). Potato is prepared by scraping ordinary potatoes, washing in corrosive sublimate, and sterilising. It may then be cut into various shapes con- venient for cultivation. Upon any of these forms of solid media the characteristic * A "pure culture" is a growth, in an artificial medium outside the body, of one species of micro-organism only. f The facts here given are obviously only general indications. The accurate preparation of medium is of vital importance in Bacteriology, and for its accomplish- ment text-books should be consulted (Eyre's Bacteriological Technique, 1 25-1 74). TEMPERATURE 17 growth of the organism can be observed. Of the nutrient elements required, nitrogen is obtained from albumens and proteids, carbon from milk-sugar, cane-sugar, «or the splitting up of proteids; salts (particularly phosphates and salts of potassium) are readily obtain- able from those incorporated in the f~ media ; and the water which is required ^^ V_ is obtainable from the moisture of the media. FIG. 5. — INOCULATING NEEDLES. Platinum wire fused into glass handles.. There are two common forms of test-tube culture, viz., on the surface and in the depth of the medium. In the former the medium is sloped, and the inocu- lating needle is drawn along its surface ; in the latter the needle is thrust vertically downwards into the depth of the solid medium. Plate cultures and anaerobic cultures will be described at a later stage. Temperature. — -When the medium, has been inoculated the culture is placed at a temperature which will be favourable. For every species of bacteria there is a favour- able temperature, termed the optimum temperature. This is usually the tempera- ture of the natural habitat of the organism. Two standards of temperature are in use in bacteriological laboratories. The one, room temperature, varies from 18°-22° C. ; the other is Hood-heat, and varies from 35°-38° C. (Plates 1 and 2). It is true some species will grow below 18° C., and others above 38° C. The pathogenic (disease-producing) bacteria thrive best as a rule at 37° C., and the non- pathogenic at the ordinary temperature of the room. The different degrees of tempera- ture are obtained by means of incubators. For the low temperatures gelatine is chosen as a medium, for the higher temperatures agar. Most bacteria grow well at room temperature (about 60° F.), but they will grow more luxuriantly and speedily at blood-heat. Whilst these are the ordinary limits of temperature affecting bacteria, they do not by any means include the extremes of heat and cold which micro-organisms can withstand. The average thermal death-point is about 55° C, but certain species, termed thermophilic, E FIG. 6.— Media for 'Surface and Depth Culture. 18 THE BIOLOGY OF BACTERIA isolated from the intestine, horse manure, etc., grow at 60°-70° C. On the other hand, investigations have shown that bacteria can withstand exceedingly low temperatures. Koch showed that the cholera vibrio was not killed by a temperature of —32° C. In 1900, Swithinbank exposed cultures of the tubercle bacillus to the temperature of liquid air (—193° C.) for continuous periods varying from six hours to forty-two days, without their vitality being affected ; and in the same year MacFadyen and Eowland found that Proteus vulgaris, B. coli, and several other species were not killed after an exposure of ten hours to a temperature of liquid hydrogen ( — 252° C). It will thus be seen that bacteria can withstand great alternations of temperature. From a public health point of view, it is important to remember that organisms can exist in freezing mixtures and ice, retaining their vitality and virulence. For example, B. coli and the typhoid bacillus can exist from the low temperatures above mentioned to 80° C., although the usual thermal death-point for these species is between 50°-60° C.* Moisture has been shown to have a favourable effect upon the growth of microbes. Drying will of itself kill many species (e.g. the spirillum of cholera), and other things being equal, the more moist a medium is, the better will be the growth upon it. Thus it is that the growth in broth is always more luxuriant than that on solid media. Yet the growth of Bacillus subtilis and some other species are an exception to this rule, for they prefer a dry medium. Desiccation as a rule diminishes virulence and lessens growth. But some species can withstand long-continued drying without injury. Light acts as an inhibitory, or even germicidal, agent. This fact was first established by Downes and Blunt in a memoir to the Eoyal Society in 1877. They found by exposing cultures to different degrees of sunlight that the growth of the culture was partially or entirely prevented, being most damaged by the direct rays of the sun, although diffuse daylight acted prejudicially. Further, these same investigators proved that the rays of the spectrum which acted most inimically upon bacteria were the blue and violet rays, next to the blue being the red and orange-red rays. The action of light, they explain, is due to the gradual oxidation which is induced by the sun's rays in the presence of oxygen. Duclaux, who worked at this question at a later date, concluded that the degree of resistance to the bactericidal influence of light, which some bacteria possess, might be due to difference in species, difference in culture media, and difference in the degrees of intensity of light. Tyndall tested the growth of organisms in flasks exposed to air and light on the Alps, * For the latest researches on this point, see Proc. Roy. Soc., 1900 and 1901 ; and the Thirty-fourth Annual Report of the State Board of Health, Massachusetts, 1903, pp. 269-281. Dewar commenced experiments of this character in 1892. A FORM OF PASTEITR'S LARGE INCUBATOR FOR CULTIVATION AT ROOM TEMPERATURE. [To face page 18. EFFECT OF LIGHT 19 and found that sunlight inhibited the growth temporarily. A large number of experimenters on the Continent and in England have worked at this fascinating subject since 1877, and though many of their results appear contradictory, we may be satisfied in adopting the following conclusions respecting the matter : — (1) Sunlight has a deleterious effect upon bacteria, and to a less extent on their spores. (2) This inimical effect can be produced by light irrespectively of rise in temperature. (3) The ultra-violet rays are the most bactericidal, and the infra-red the least so, which indicates that the phenomenon is due to chemical action. (4) The presence of oxygen and moisture greatly increase this action, the process being largely an oxidation. (5) Sunlight also acts prejudicially upon the culture medium, and thereby exerts an injurious action on the culture. (6) The time occupied in the bactericidal action depends upon the intensity of the light and tne inherent vitality of the organism. (7) With regard to the action of light upon pathogenic organisms, some results have recently been obtained with Bacillus typJiosus. Janowski maintains that direct sunlight exerts a distinctly depressing effect on typhoid bacilli. At present more cannot be said than that sunlight and fresh air are two of the most powerful agents we possess with which to combat pathogenic germs. A very simple method of demonstrating the influence of light is to grow a pure culture in a favourable medium, either in a test- tube or upon a glass plate, and then cover the whole with black paper or cloth. A little window may then be cut in the protec- tive covering, and the whole exposed to the light. Where it reaches in direct rays, it will be found that little or no growth has occurred; where, on the other hand, the culture has been in the dark, abundant growth occurs. In diffuse light the growth is merely somewhat inhibited. A number of experiments in this direction were made at Lawrence, Massachusetts,* with cultures of typhoid and B. coli. In two experiments, each with typhoid bacillus and B. coli, water dilutions were made from fresh cultures of the germs, 1 c.c. of this water being placed in Petri dishes in the sun for definite periods. After exposure, the water in the plates was mixed with agar, and all plates were incubated twenty-four hours at 38°, after which the number of colonies was counted. In one experiment the water dilution of typhoid was mixed with melted agar, and plates made as * Thirty -fourth Ann. Rep. State Ed. of Ilealth of Massachusetts, 1903, p. 275. 20 THE BIOLOGY OF BACTERIA usual. After the agar had set, these plates were then exposed to the sunlight. In one experiment with B. coli, the water culture was not exposed to the sunlight in plates, but the exposure was made in a clear, white glass bottle of the Blake pattern, holding 100 c.c., samples being taken from this at the proper intervals, and plated as usual. In all cases control cultures were made under exactly the same conditions as were the cultures exposed, these, however, being pro- tected from the sunlight by a heavy, opaque cloth, or some similar material. The temperature of these cultures was, of course, consider- ably lower than was the temperature in the sun. The numbers of bacteria in the controls showed the usual variation to be expected under the circumstances, usually a slight reduction in numbers being noted during two or three hours' standing, although in one instance the numbers increased quite materially. The data of these control cultures are not shown in the accompanying tables. The brightness of the sun also varied considerably, and attempts were made to measure the amount of light by photographic means, but these measurements were unsatisfactory, and the data are not included here. With typhoid, from 95 to 99 per cent, of all the germs were destroyed by ten to fifteen minutes' exposure to direct sunlight. A few germs may resist the sunlight for a somewhat longer time ; usually, however, all the germs were destroyed by three or more hours' exposure to bright sunlight. The results of the experiments with typhoid are shown in the following tables : — TABLE showing Elimination of Typhoid Germs in Water on Exposure to Sunlight. Experiment 24. Experiment 25. Bacteria. Average. Bacteria. Average. Start . 698 734 716 592 532 562 15 minutes 66 4 35 13 5 9 30 minutes 17 1 9 4 4 4 45 minutes 0 1 1 3 0 2 1 hour . 6 2 4 21 5 13 H hours 2 0 1 5 3 4 2 hours . 0 0 0 4 3 4 4 hours . 3 1 2 0 0 0 6 hours . 0 0 0 0 0 0 EFFECT OF LIGHT 21 TABLE showing Elimination of Typhoid Germs in A gar Platen on Exposure to Sunlight. Exposure. Temperature. Experiment 26. Bacteria. Average. Start . ^ 608 642 625 10 minutes 91° F. 2 3 3 20 minutes 83 7 2 5 30 minutes 81 0 0 0 40 minutes 83 0 0 0 50 minutes 83 00 0 1 hour . 78 1 0 1 1 hour, 10 minutes 74 00 0 1 hour, 20 minutes 71 00 0 1 hour, 30 minutes 69 0 0 0 1 hour, 40 minutes 68 o o 0 1 hour, 50 minutes 71 0 0 0 2 hours 68 0 0 0 With B. coli the results have been somewhat more variable, prob- ably due to more changeable conditions. In one experiment, some- thing over 80 per cent, of the germs were destroyed by fifteen minutes' exposure, all being destroyed after four hours. The results of this experiment were undoubtedly influenced greatly by clouds in the sky, so that at times the sunlight was not very bright, after about two and one-half hours the sun being entirely overcast. In one experiment about 96 per cent, of the germs were eliminated at the end of fifteen minutes, and after thirty minutes all of the germs were destroyed. In these two experiments the water cultures were exposed in plates, the results being shown in the following table : — TABLE showing Elimination of B. coli in Water Cultures on Exposure to Sunlight. Experiment 78. Experiment 79. Exposure. Tem- perature. Bacteria per c.c. Average. Tem- perature. Bacteria per c.c. Average. Start . 78 70,000 72,800 71,400 445,400 824,300 634,850 15 minutes 78 6,564 16,166 12,365 106 19,100 31,800 25,400 30 minutes 80 4,473 2,663 3,568 107 0 0 0 45 minutes 80 2,130 0 1,065 110 0 0 0 1 hour 80 590 0 295 100 0 0 0 1 .V hours 82 2,130 93 1,111 108 0 0 0 2 hours 62 10 70 40 105 0 0 0 3 hours 100 0 0 0 4 hours 61 0 0 0 78 0 0 0 22 THE BIOLOGY OF BACTERIA In one experiment the water was exposed in bottles. In this case about 98 per cent, of the germs were destroyed after fifteen minutes, the cultures varying somewhat. The germs persisted in the water in considerable numbers for two hours and in small numbers up to four hours, after five hours the sample being completely sterilised. The results of this experiment are shown as follows : — TABLE shotting Change in Numbers of B. coli in Water in Bulk on Exposure to Sunlight. Experiment 81 Exposure. Temperature. Bacteria per c.c. Average. Start . 94 2,360,000 1,620,000 1,990,000 15 minutes 94 30,000 43,200 36,600 30 minutes 92 85,300 22,000 53,650 45 minutes 92 44,000 55,000 49,500 1 hour . 94 53,700 45,300 49,500 1^ hours 96 35,800 34,100 34,950 2 hours . 109 57,400 76,400 66,900 3 hours . 95 450 1,172 786 4 hours . 102 3 5 4 5 hours . 76 0 0 0 It has been found that the electric light has but little action upon bacteria, though that which it has is similar to sunlight. Eecent experiments with the Rontgen rays have not given bactericidal results. In 1890 Koch stated that tubercle bacilli were killed after an exposure to direct sunlight of from a few minutes to several hours. The influence of diffuse light would obviously be much less. Professor Marshall Ward has experimented with the resistant spores of Bacillus anthracis by growing these on agar plates and exposing to sunlight. From two to six hours' exposure had a germicidal effect.* It should be remembered that several species of sea-water bacteria themselves possess the property of phosphorescence. Pfliiger was the first to point out that it was such organisms which provided the phosphorescence upon decomposing wood or decaying fish. To what this light is due, whether capsule, or protoplasm, or chemical product, is not yet known. The only facts at present established are to the effect that certain kinds of media and pabulum favour or deter phosphorescence. * See Trans. .Tenner Inst. (second series), 1899, p. 81. MEANS OF STERILISATION 23 Aerobiosis. — Pasteur was the first to lay emphasis upon the effect which free air had upon micro-organisms. He classified them according to whether they grew in air, aerobic, or whether they flourished most without it, anaerobic. Some have the faculty of growing with or without the presence of oxygen, and are designated SLS facultative aerobes or anaerobes. As regards the cultivation of anaerobic germs, it is only necessary to say that hydrogen, nitrogen, or carbonic acid gas may be used in place of oxygen, or they may FIG. 7.— Method of producing Hydrogen by Kipp's Apparatus for Cultivation of Anaerobes (see p. 117. be grown in a medium containing some substance which will absorb the oxygen (see p. 117). Means of Sterilisation. — As this term occurs frequently even in books of an elementary nature, and as it is expressive of an idea which must always be present to the mind of the bacteriologist, it may be desirable to make allusion to it here. Chemical substances, perfect filtration, and heat are the three means at our command in order to secure germ-free conditions of apparatus or medium. The first two, though theoretically admissible, are practically seldom used, the former of the two because the addition of chemical substances annuls or modifies the operation, the latter of the two on account of the great practical difficulties in securing efficiency. Hence in the investigations involved in bacteriological research heat is the common sterilising agent. A sustained temperature of 70° C. (158° F.) will kill all bacilli; even 58° C. will kill most kinds. Boiling at 100° C. (212° F.) for five minutes will kill anthrax spores, and for thirty to sixty minutes will kill all bacilli and their spores. This difference in the thermal 24 THE BIOLOGY OF BACTERIA death-point between bacilli and their spores enables the operator to obtain what are called "pure cultures" of a desired bacillus from its spores which may be present. For example, if a culture contains spores of anthrax and is contaminated with micrococci, heating to 70° C. (158° F.) will kill all the micrococci, but will not affect the spores of an- thrax, which can then grow into a pure culture of anthrax bacilli. Frac- tional or discontinuous sterilisation depends on the principle of heating to the sterilising point for bacilli (say 70° C.) on one day, which will kill the bacilli, but leave the spores uninjured. But by the following day the spores will have germinated into bacilli, and a second heating to 70° C. will kill them before they in their turn have had time to sporulate. Thus the whole will be sterilised, though at a temperature below boiling. Successful sterilisation, therefore, depends upon killing both bacteria and their spores, and nothing short of that can be considered as sterilisa- tion. The following methods are those generally used in the laboratory. For dry heat (which is never so in- jurious to organisms as moist heat*) : (a) the Bunsen burner, in the flame of which platinum needles, etc., are steril- ised; (b) hot-air chamber, in which flasks and test-tubes are heated to a temperature of 150°-170° C. for an hour or more. For moist heat : (c) boiling, for knives and * It will be observed that there is a marked difference between the effects of dry heat and moist heat. Moist heat is able to kill organisms much more readily than dry, owing to its penetrating effect on the capsule of the bacillus. Dry heat at 140° C. (284° F.), maintained for three hours, is necessary to kill the resistant spores of Bacillus anthracis and B. subtilis, but moist heat at forty degrees less will have the same effect. It is from data such as these that in laboratories and in disinfecting apparatus moist heat is invariably preferred to dry heat For with the latter such high temperatures would be required that the articles being disinfected would be damaged. Koch states the following figures for general guidance : Dry heat at a temperature of 120° C. (248° F.) will destroy spores of mould fungi, micrococci, and bacilli in the absence of their spores ; for the spores of bacilli 140° C. (284° F.), maintained for three hours, is necessary; moist heat at 100° C. (212° F.) for fifteen minutes will kill bacilli and their spores. FIG. 8.— Koch's Steam Steriliser. PLATE 2. MODES OF BACTERIAL ACTION 25 instruments; (d) Koch's steam steriliser, by means of which a crate is slung in a metal cylinder, at the bottom of which water is boiled; (e) the autoclave, which is the most rapid and effective of all the methods. This is in reality a Koch steriliser, but with apparatus for obtaining high pressure. The last two (d, e} are used for sterilising the nutrient media upon which bacteria are culti- vated outside the body. Blood serum would, however, coagulate at a temperature over 60° C. (124° F.), and hence a special steriliser has been designed to carry out fractional sterilisation daily for a week at about 55° C.-580 C. Modes of Bacterial Action In considering the specific action of micro-organisms, it is desir- able, in the first place, to remember the two great functional divisions of saprophyte and parasite. A saprophyte is an organism that obtains its nutrition from dead organic matter. Its services, of whatever nature, lie outside the tissues of living animals. Its life is spent apart from a " host." A parasite, on the other hand, lives always at the expense of some other organism which is its host, in which it lives or upon which it lives. There is a third or inter- mediate group, known as " facultative," owing to their ability to act as parasites or saprophytes, as the exigencies of their life may demand. The saprophytic organisms are, generally speaking, those which contribute most to the benefit of man, and the parasitic the reverse, though this statement is only approximately true. In their relation to the processes of fermentation, decomposition, nitrification, etc., we shall see how great and invaluable is the work which saprophytic microbes perform. Their result depends, in nearly all cases, upon the organic chemical constitution of the substances upon which they are exerting their action, as well as upon the varieties of bacteria them- selves. Nor must it be understood that the action of saprophytes is wholly that of breaking down and decomposition. As a matter of fact, some of their work is, as we shall see, of a constructive nature ; but, of whichever kind it is, the result depends upon the organism and its environment. This, too, may be said of the pathogenic species, all of which are in a greater or less degree parasitic. It is well known how various are the constitutions of man, how the bodies of some persons are more resistant than those of others, and how the invading microbe will meet with a different reception according to the constitution and idiosyncrasy of the body which it attacks. Indeed, even after invasion the infectivity of the special disease, whatever it happens to be, will be materially modified by the tissues. When we come to turn to the micro-organisms which are pathogenic parasites or THE If klf WET*** 26 THE BIOLOGY OF BACTERIA we shall further have to keep clear in our minds that their action is complex, and not simple. In the first place, we have an infection of the body clue to the bacteria themselves. It may be a general and widespread infection, as in anthrax, where the bacilli pass, in the blood or lymph current, to each and every part of the body ; or it may be a comparatively local one, as in diphtheria, where the invader remains localised at the site of entrance. But, be that as it may, the micro-organisms themselves, by their own bodily presence, set up changes and perform functions which may have far-reaching effects, It is obvious that the wider the distribution the wider is the area of tissue change, and vice versd. Yet there is something of far greater importance than the mere presence of bacteria in human or animal tissues, for the secondary action of disease-producing germs — and possibly it is present in other bacteria — is due to their poisonous products, or toxines, as they have been termed. These may be of the nature of ferments, and they become diffused throughout the body, whether the bacteria themselves occur locally or generally. They may bring about very slight and even imperceptible changes during the course of the disease, or they may kill the patient in a few hours. Latterly bacteriologists have come to understand that it is not so much the presence of organisms which is injurious to man and other animals as it is their products, which cause mischief ; and the amount of toxic product bears no known proportion to the degree of invasion by the bacteria. The various and widely differing modes of action in bacteria are therefore dependent upon these three elements (1) the tissues or medium, (2) the bacteria or agents, and (3) the products of the bacteria or toxins ; and in all organismal processes these three elements act and react upon each other. Seed and Soil. — It is of essential importance to the right under- standing of the role which bacteria play in the production of disease to give full place to the part taken by the soil on which they are implanted. Few ideas in bacteriology are more erroneous, or likely to lead to graver misconception, than to suppose that bacteria produce the same effect under all conditions, and that the human tissues play a small part. One might equally well expect seed to behave in the same way in all kinds of soil. We know that as a fact, seeds only flourish under certain conditions, and that the soil is only second in importance to the seed-life itself. It is somewhat the same in the production of disease. The early school of pre- ventive medicine declared for the health of the individual and laid the emphasis upon predisposition ; the modern school have declared for the infecting agent, and have laid emphasis upon the bacillus. The truth is to be found in a right perception of the action and interaction of the tissues and the bacillus. B. diphtheric? in one person's throat (A) sets up diphtheria, in another person's throat SEED AND SOIL 27 (B) lies quiescent, producing no apparent disease. The cause of this extraordinary fact may be a question of different virulence in the two bacilli, but is much more likely to be due to the greater vigour and power of resistance of the mucous membrane of B's throat. Sewer air, as we shall see subsequently, does not contain many bacteria, and probably does not frequently convey germs of disease. But this does not prove that the inhalation of sewer air will not weaken the throat, and so form a favourable nidus for organisms resting there, or organisms shortly to be inhaled from dust or mucous particles from the throat of a diseased person. Which is the more important preventive method, to maintain the resistance of the individual or to waylay the infecting organism, is a nice point we need not attempt to decide. Obviously, both objects should be kept in view. Phthisis is another example. Thousands of persons inhale the tubercle bacillus who are not attacked by the clinical disease of consumption. This fortunate result is due to the resistant tissues of the healthy lung, and the lesson to be derived is to maintain such resistance at its maximum. This evidently is, in part, the scientific explanation of Koch's dictum, " It is the overcrowded dwellings of the poor that we have to regard as the real breeding places of tuberculosis ; it is out of them that the disease always crops up anew, and it is to the abolition of these conditions that we must first and foremost direct our attention if we wish to attack the evil at its root and to wage war against it with effective weapons."* Part of the explanation of these words is doubtless that it is in such places that the tubercle bacillus breeds and passes from one person to another. But every sanitarian knows that the effect of such environment is to lower the natural resistance, to weaken the lung, impoverish the blood, and undermine the constitution, and thus a suitable nidus is supplied to the invading bacillus. "A perfectly healthy lung is seldom if ever primarily infected with the tubercle bacillus " (Wood- head). But the evidence of bacteriology as to the part played by the soil is even stronger than at first sight appears. For we now know, by experiment, that micro-organisms which in some animals produce acute disease rapidly ending in death, result only in mild disease in other animals, and in yet a third group produce no apparent disease whatever. This is not due to variation in virulence but to variation in soil. The advance of bacteriology has been so rapid and marked by such striking discoveries that there has been a tendency to over-rate altogether the potentiality of the bacillus apart from its medium. The latest findings in the study of comparative culture work, of immunity and of the production of antitoxins have, however, demon- * Trans. Brit. Cong, of Tuberculosis, 1901, vol. i., p. 31. 28 THE BIOLOGY OF BACTERIA strated beyond all doubt the enormous part played by the medium or soil in which the micro-organism is growing.* Specificity of Bacteria A species may be denned as a group of individuals which, however many characters they share with other individuals, agree in present- ing one or more characters of a peculiar and hereditary kind with some certain degree of distinctness.-)- There is no doubt that separate species of bacteria occur and tend to remain as separate species. Bub it must be remembered that species are merely arbitrary divisions which present no deeper significance from a philosophical point of view than is presented by well-marked varieties, out of which they are in all cases believed to have arisen, and from which it is often a matter of individual opinion whether they shall be separated by receiving a specific label. What degree or character of variation shall be considered as sufficient for the demarcation of a species of bacteria ? B. coli and B. typliosus have certain distinctive features, which are accepted as factors of provisional differentiation. But they have many points in common, the peculiarity and heredity of which are not as yet determined. And they have many allies, para-typhoid and para-colon organisms, in the same way as the tubercle bacillus possesses many allies, both bovine and human, among acid-fast species having similar characters but differing in degree of virulence. The fact is, that our present knowledge of these matters is very small, and it is impossible to dogmatise. The future may reveal some unlooked-for relationships, and organisms now classified as morphologically separate may ultimately prove to be nearly related. Further, it may be found that their respective action in the human body is not greatly dissimilar (the production, of diarrhoea, for example, by the colon group). Medium and tissue have their effect in the production of variations of greater or lesser mark in bacteria. B. typhosus may, in the course of sub- culture, become morphologically indistinguishable from B. coli, and its pathogenicity may also be reduced. The tubercle bacillus in old culture and in saprophytic existence becomes almost indistinguish- able from the streptothrix family. Streptococcus conglomeratus on certain media simulates in a marked degree the Klebs-Loffler diph- theria bacillus, and by passage through a mouse loses its streptococcal * The writer has been impressed in particular as to the truth of this view by observation of a number of epidemics, by the study of a long series of cultures of the same bacillus on different media, and by antitoxin production. But the same conclusion has been reached from other premises. See a suggestive paper by Sir W. J. Collins, M.D., in the Jour, of the Sanitary Institute 1902 (Oct.), xxiii., pt. iii., p. 335. f Darwin and After Darwin, G. J. Romanes, F.R.S., vol. ii., p. 231. ASSOCIATION OF ORGANISMS 29 form (Gordon). The Klebs-Lofiler bacillus in its turn may be greatly modified in morphology and pathogenicity by environment. Nor is the change necessarily in descending order. Non-pathogenic organisms may possibly become pathogenic. We do not know. The subject is one full of difficulty in a transition period of knowledge in any branch of science. But there is no reason to suppose that bacteria are exceptional in nature and outside the influence of natural selection ; and it is not improbable that the views of the early bacteriologists will have to be very much revised, and that eventually it will be found that many "species" of micro-organisms are in reali ty varieties of a single species showing involution and pleomorphic forms. At the same time it should be recognised that amongst the lowliest forms of life specific distinctions are, as a rule, less definite, and less permanent, than amongst forms of life much higher in the organic scale. The Association of Organisms At a later stage we shall have an opportunity of discussing Symbiosis and allied conditions. Here it is only necessary to draw attention to a fact that is rapidly becoming of the first importance in bacteriology. When species were first isolated in pure culture it was found that they behaved very differently under varying circumstances. This modification in function has been attributed to differences of environment and physical conditions. Whilst it is true that such external conditions must have a marked effect upon such sensitive units of protoplasm as bacteria, it has recently been proved that one great reason why modification occurs in pure artificial cultures is that the species has been isolated from amongst its colleagues and doomed to a separate existence. One of the most abstruse problems in the immediate future of the science of bacteri- ology is to learn what intrinsic characters there are in species or individuals which act as a basis for the association of organisms for a specific purpose. Some bacteria appear to be unable to perform their ordinary role without the aid of others.* An example of such association is well illustrated in the case of Tetanus, for it has been shown that if the bacilli and spores of tetanus alone obtain entrance to a wound the disease does not follow the same course as when with the specific organism the lactic acid bacillus or the common organisms of suppuration or putrefaction also gain entrance. There is here evidently something gained by association. Again the viru- * The three different degrees of association have been expressed by the following terms : Symbiosis, the co-operation for a mutual advantage, not obtained other- wise ; metabiosis, where one organism prepares the way for another ; antibiosis (antagonism of bacteria), where one of the two associated organisms is directly or indirectly injuring the other. 30 THE BIOLOGY OF BACTERIA lence of other bacteria is also increased by means of association. The Bacillus coli is an example, for, in conjunction with other organisms, this bacillus, although normally present in health in the alimentary canal, is able to set up acute intestinal irritation, and various changes in the body of an inflammatory nature. It is not yet possible to say in what way or to what degree the association of bacteria influences their role. That is a problem for the future. But whilst we have examples of this association in Streptococcus and the bacillus of diphtheria, B. coli and yeasts, Tetanus and putrefactive bacteria, Diplococcus pneumonice and Proteus vulgaris, and Streptococcus erysipelatis and Proteus vulgaris, we cannot doubt that there is an explanation to be found of many, hitherto unknown, results of bacterial action. This is the place in which mention should also be made of higher organisms associated for a specific purpose with bacteria. There is some evidence to support the belief that some of the Leptotrichese (Crenothrix, Beggiatoa, Leptothrix, etc.) and the Cladotricheae (Cladothrix) perform a preliminary disintegration of organic matter before the decomposing bacteria commence their labours. This occurs apparently in the self-purification of rivers, as well as in polluted soils. Antagonism of Bacteria (Antibiosis). — Study of the life- history of many of the water bacteria will reveal the fact that they can live and multiply under conditions which would at once prove fatal to other species. Some of these water organisms can indeed increase and multiply in distilled water, whereas it is known that other species cannot even live in distilled water owing to the lack of pabulum. Thus we see that what is favourable for one species may be the reverse for another. Further, we shall have opportunity of observing, when consider- ing the bacteriology of water and sewage, that there is in these media in nature a keen struggle for the survival of the fittest bacteria for each special medium. In a carcase it is the same. If saprophytic bacteria are present with pathogenic, there is a struggle for the survival of the latter. Now whilst this is in part due to a competition owing to a limited food supply and an unlimited popula- tion, as occurs in other spheres, it is also due in part to the inimical influence of the chemical products of the one species upon the life of the bacteria of the other species. Moreover, in one culture medium, as Cast has pointed out, two species will often not grow. When Pasteur found that exposure to air attenuated his cultures, he pointed out that it was not the air perse that hindered growth, but it was the introduction of other species which competed with the original. The growth of the spirillum of cholera is opposed by Bacillus pyogcnes foetidus. B. anthracis is, in the body of animals, opposed by either B. pyocyaneus or Streptococcus erysipelatis, and yet or ££j ANTAGONISM AND ATTENUATION 31 it is aided in its growth by B. prodigiosus. B. aceti is under certain circumstances antagonistic to B. coli. In several of the reports of the late Sir Eichard Thome issued from the Medical Department of the Local Government Board, we have the record of a series of experiments performed by Dr Klein upon the subject of the antagonisms of microbes. From this work it is clearly demonstrated that whatever opposition one species affords to another it is able to exercise by means of its poisonous properties. These are of two kinds. There is, as is now widely known, the poisonous product named the toxin, into which we shall have to inquire in more detail at a later stage. There is also in many species, as several workers have pointed out, a poisonous constituent, or constituents, included in the body protoplasm of the bacillus, and which he therefore terms the intracellular poison. Now, whilst the former is different in every species, the latter may be a property common to several species. Hence those having a similar intracellu- lar poison are antagonistic to each other, each member of such a group being unable to live in an environment of its own intracellular poison. Further, it has been suggested that there are organisms possessing only one poisonous property, namely, their toxin — for example, the bacilli of Tetanus and Diphtheria — whilst there are other species, as above, possessing a double poisonous property, an intracellular poison and a toxin. In this latter class would be included the bacilli of Anthrax and Tubercle. There can be no doubt that these complex biological properties of association and antagonism, as well as the parasitic growth of bacteria upon higher vegetables, are as yet little understood, and we may be glad that any light is being shed upon them. In the biological study of soil bacteria in particular may we expect in the future to find examples of association, even as already there are signs that this is so in certain pathogenic conditions. In the alimentary canal, on the other hand, and in conditions where organic matter is greatly predominating, we may expect to see further light on the subject of antagonism. Attenuation of Virulence or Function. — It was pointed out by some of the pioneer bacteriologists that the function of bacteria suffered under certain circumstances a marked diminution in power. Later workers found that such a change might be artificially pro- duced. Pasteur introduced the first method, which was the simple one of allowing cultures to grow old before sub-culturing. Obviously a pure culture cannot last for ever. To maintain the species in characteristic condition it is necessary frequently to sub-culture upon fresh media. If this simple operation be postponed as long as possible consistent with vitality and then performed, it will be found that the sub-culture is attenuated, i.e., weakened. Another mode is 32 THE BIOLOGY OF BACTERIA to raise the pure culture to a temperature approaching its thermal death-point. A third way of securing the same end is to place it under disadvantageous external circumstances, for example in a too alkaline or too acid medium. A fourth method is to pass it through the tissues of an insusceptible animal. Thus we see that, whilst the favourable conditions which we have considered afford full scope for the growth and performance of functions of bacteria, we are able .by a partial withdrawal of these, short of that ending fatally, to modify the character and strength of bacteria. In future chapters we shall have opportunity of observing what can be done in this direction. Bacterial Diseases of Plants Eeference has been made to the associated work of higher vegetable life and bacteria. The converse is also true. Just as we have bacterial diseases affecting man and animals, so also plant life has its bacterial diseases. "Wakker, Prillieux, Erwin Smith, and others have investigated the pathogenic conditions of plants due to bacteria, and though this branch of the science is in its very early stages, many facts have been learned. Hyacinth disease is due to a flagellated bacillus. The wilt of cucumbers and pumpkins is a common disease in some districts of the world, and may cause widespread injury. It is caused by a micro-organism which fills the water-ducts. Wilting vines are full of the same sticky germs. Desiccation and sunlight have a strong prejudicial effect upon these organisms. Melon Uight must not be confused with the bacterial wilt of cucumbers and melons. The blight disease is caused by Plasmopara cubensis, a sporulating fungus. Bacterial brown-rot of potatoes and tomatoes is another plant disease probably due to a bacillus. The bacillus passes down the interior of the stem into the tubers, and brown-rots them from within. There is another form of brown-rot which affects cabbages. It blackens the veins of the leaves, and a woody ring which is formed in the stem causes the leaves to fall off. This also is due to a micro-organism, which gains entrance through the water-pores of the leaf, and subsequently passes into the vessels of the plants. It multiplies by simple fission, and possesses a flagellum. Certain diseases of Sweet Corn have been investigated by Stewart, and traced to a causal bacillus possessing marked characters. Professor Potter believes that white-rot of the turnip is produced by Pseudomonas destructans, a liquefying, motile, aerobic bacillus. CHAPTEE II BACTERIA IN WATER Quantity of Bacteria in Water— Quality of Water Bacteria : (a) Ordinary Water Bacteria ; (6) Sewage Bacteria ; B. coll communis ; (c) Pathogenic Bacteria in Water — Interpretation of the Findings of Bacteriology — Natural Purification of Water— Artificial Purification of Water— Sand Filtration— Domestic Puri- fication of Water. The collection of samples, though it appears simple enough, is sometimes a difficult and responsible undertaking. Complicated apparatus is rarely necessary, and fallacies will generally be avoided by observing two directions. In the first place, the sample should be chosen as representative as possible of the real water or conditions we wish to examine. Some authorities advise that it is necessary to allow the tap to run for some minutes previously to collecting the sample; but if we desire to examine chemically for lead or biologically for micro-organisms in the pipes, then such a proceeding would be injudicious.* If it is well water that is to be examined, the well should be pumped for some minutes before taking the sample. If it is river water which is to be examined, it is important to collect the sample without incorporating any deposit. In short, we must use common sense in the selection and obtaining of a sample, following this one guide, namely, to collect as nearly as possible a sample of the exact water, the quality of which it is desired to learn. In the second place, we must observe strict * Water from a house cistern is rarely a fair sample of a town supply. It should be taken from the main. If taken from a stream or still water, the collect- ing bottle should be held about a foot below the surface before the stopper is removed. 33 34 BACTERIA IN WATER bacteriological cleanliness in all our manipulations. This means that we must use only sterilised vessels or flasks for collecting the sample, and in the manipulation required we must be extremely careful to avoid any pollution of air or any addition to the organisms of the water from unsterilised apparatus. A flask polluted in only the most infinitesimal degree will entirely vitiate all results. Vessels may be sterilised by heating at 150° C. for two or three hours. If this is impracticable the vessel may be washed with pure sulphuric acid, and then thoroughly rinsed out in the water which is to be examined. Accompanying the sample should be a more or less full statement of its source. There can be no doubt that, in addition to a chemical and bacteriological report of a water, there should also be made a careful examination of its source. This may appear to take the bacteriologist far afield, but until he has seen for himself what " the gathering ground " is like, and from what sources come the feeding streams, he cannot judge the water as fairly as he should be able to do. The configuration of the gathering ground, its subsoil, its geology, its rainfall, its relation to the slopes which it drains, the nature of its surface, the course of its feeders, and the absence or presence of cultivated areas, of roads, of houses, of farms, of human traffic, of cattle and sheep — all these points should be noted, and their influence, direct or indirect upon the water, carefully borne in mind. When the sample has been duly collected, sealed, and a label affixed bearing the date, time, and conditions of collection and full address, it should be transmitted with the least possible delay to the laboratory. Frequently it is desirable to pack the bottles in a small ice-case for transit. Miquel, Pakes, and others have constructed special forms of packing-cases, and these have their advantages. But the ordinary bottle of water may be quite satisfactorily con- veyed, as a rule, packed in sawdust and ice. On receipt of such a sample of water the examination must be immediately proceeded with, in order to avoid, as far as possible, the fallacies arising from the rapid multiplication of germs. Multiplication of Bacteria in Water. — In almost pure water, at the ordinary temperature of a room, Frankland found that organisms multiplied as follows : No. of Germs. Hours. per c.c. 0 . . •« 1,073 6 ... 6,028 24 . . * 7,262 48 . » . 48,100 Another series of observations revealed the same sort of rapid MULTIPLICATION OF WATER BACTERIA 35 increase of bacteria. On the date of collection the micro-organisms per c.c. in a deep-well water (in April) were seven. After one day's standing at room temperature the number had reached twenty-one per c.c. After three days under the same conditions it was 495,000 per c.c. At blood-heat the increase would, of course, be much greater, as a higher temperature is more favourable to multiplication. But this would depend in part also upon the degree of impurity in the water, a pure water decreasing in number of germs on account of the exhaustion of the pabulum, whereas, for the first few days at all events, an organically polluted water would show an enormous increase in bacteria. It is desirable to remember that organisms, in an ordinary water, do not continue to increase indefinitely. Cramer, of Zurich, examined the water of the Lake after it had been standing in a vessel for different periods, with the following results: — Hours and Days of No. of Micro-organisms Examination. per c.c. 0 hours 143 24 „ 3 days 8 ,, 17 „ 70 „ 12,457 328,543 233,452 17,436 2,500 In a general way it may be said that foul waters, rich in putrescible animal matter, show a rapid increase of bacteria ; surface waters, such as river water, show a slow and persistent multiplication of organisms ; and deep -well waters and spring water show comparatively little increase in contained bacteria. Indeed it may be said that the condition of a water is partly indicated by the rapidity or slowness with which its bacteria increase. A low temperature (5° C.) undoubtedly diminishes the multiplication, and there are other conditions such as exposure to air, movement, and antagonism of organisms which exert an indirect effect. As will be inferred from what has been said, the most important condition affecting the number of bacteria in a water is the organic matter contained in it.* -The Bacteriology of Water In many natural waters there will be found varied contents even in regard to flora alone: algce, diatoms, spirogyrce, desmids, and all * For suggestions and hints on points of technique in the systematic examination of a water, see Delepine's Bacteriological Survey of Surface Water Supplies : Jour, of State Medicine, 1898, vol. vi., pp. 145, 193, 241, 289 ; and Bacteriological Examina- tion of Water, by W. H. Horrocks. (See also present volume, pp. 463-473.) 36 BACTERIA IN WATER sorts of vegetable detritus. Many of these organisms are held responsible for certain disagreeable tastes and odours. The colour of a water may also be due to similar causes. Dr Garrett, of Cheltenham, has recorded the occurrence of redness of water owing to a growth of Crenothrix polyspora, and many other similar cases make it evident that not unfrequently great changes may be produced in water by contained microscopic vegetation. With the exception of water from springs and deep wells, all unfiltered natural waters contain numbers of bacteria.* The actual number roughly depends, as we have seen, upon the amount of organic pabulum present, and upon certain physical conditions of the water. In some species multiplication does not appear to depend on the presence of much organic matter, and, indeed, sonle bacteria can live and multiply in almost pure water ; e.g., Microeoccus aquatilis and Bacillus erythrosporus. Again, others depend not upon the quantity of organic matter, but upon its quality. And frequently in a water of a high degree of organic pollution it will be found that bacteria have been restrained in their development by the competi- tion of other species monopolising the pabulum. It will be necessary to deal with the subject under the two subdivisions of (1) quantity and (2) quality of bacteria found in water. Quantity of Bacteria in Water Percy Frankland has quoted in his book •(• a number of records of the quantity of organisms found in various waters. These tables five the returns for the rivers Seine (Miquel), Ehone, Saone (Koux), pree (Frank), Isar (Prausnitz), Limmat (Schlatter), Ehine (Mcers), etc. Here it is unnecessary to do more than give typical illustra- tions, and for comparative purposes English rivers may be taken. Prof. Frankland himself collected water from the river Thames at various times and seasons, and some of his results were as follow : — * Bacteria, of course, exist in the water of the sea. Near land, as might be expected, the number is greatest, and diminishes rapidly further out to sea. Currents sometimes bring them to the surface from a depth of 596 fathoms (Fischer). At a depth of 100-200 fathoms bacteria have been found in large numbers. The comparative paucity at the surface is due to the germicidal effect of sunlight. Ocean bacteria vary widely -in size and shape. Apparently, typical cocci and bacilli are never met with on the high seas. Spirilla and zooglooa masses are common. Most sea bacteria are motile and furnished with flagella ; some are anaerobes. f Micro-organisms in Water (1894), pp. 89-116. NUMBER OF BACTERIA IN WATER 37 River Thames Water Collected at Hampton. Number of Micro-organisms obtained from 1 c.c. of Water. Mouth. January February March April . May . June . July . August September October November December 45,000 15,800 11,415 12,250 4,800 8,300 3,000 6,100 8,400 8,600 56,000 63,000 1887. 30,800 6,700 30,900 52,100 2,100 2,200 2,500 7,200 16,700 6,700 81,000 19,000 92,000 40,000 66,000 13,000 1,900 3,500 1,070 3,000 1,740 1,130 11,700 10,600 Another example from the river Lea was as follows : — River Lea Water Collected at Chingford. Number of Micro-organisms obtained from 1 c.c. of Water. Month. January February March April . May . June . July . August September October November December 1886. 39,300 20,600 9;025 7,300 2,950 4,700 5,400 4,300 3,700 6,400 12,700 121,000 1887. 37,700 7,900 24,000 1,330 2,200 12,200 12,300 5,300 9,200 7,600 27,000 11,000 31,000 26,000 63,000 84,000 1,124 7,000 •2,190 2,000 1,670 2,310 57,500 4,400 " During the summer months these waters are purest as regards micro-organisms, this being due to the fact that during dry weather these rivers are mainly composed of spring water, whilst at other seasons they receive the washings of much cultivated land " (Frank- land). Prausnitz has shown that water differs, as would be expected, according to the locality in the stream at which examination is made. His investigations were made from the river Isar before and after it receives the drainage of Munich :— Above Munich Near entrance of principal sewer 13 kilometres from Munich . 22 33 No. of Colonies per c.c. 531 227,369 9,111 4,796 2,378 38 BACTERIA IN WATER Frankland has shown that the river Dee affords another example, even more perfect, of pollution and restoration repeated several times until the river becomes almost bacterially pure. Professor Boyce and his colleagues have recently made an examination of the river Severn before and after its waters pass the town of Shrewsbury.* Their findings may be represented briefly as follows : — Average Total Average Position of Examination. No. of Bacteria No. of B. coli per c.c. per c.c. At Asylum, 2 miles above Shrewsbury Waterworks, opposite Shrewsbury 7,000 13,000 13 46 Ferry i., 0'6 of a mile lower down 20,000 177 English Bridge, 1 '6 of a mile lower down 23,000 321 Ferry iii., 2 '5 miles lower down 19,000 600 Uffington, 4*7 miles lower down 17,000 142 Alcham, 9 miles lower down . 13,000 48 Cressage, 16 miles lower down 5,000 36 This table and that of Prausnitz — and many other workers have produced similar records — illustrate the effect of (a) local pollution, and (&) river purification, upon the bacterial content of water, to which subsequent reference wili be made. The record respecting the Severn includes also the indication of sewage pollution by the presence of B. coli. An elaborate examination has also been made of the water of the river Thames and the Thames estuary, by Houston, and the report dealing with it is full of information on the subject, to which reference should be made.f Lastly, the accompanying table (pp. 39 and 40), for 1902 and 1903, deals with the London water supply as examined by Crookes and Dewar. J It is concerned, it should be added, only with numerical results. This record, compiled from the monthly reports respecting the three waters supplied to the metropolis, illustrates many interesting points upon which we have not space to dwell fully. A few notes, however, upon an actual example are more useful than much theoretical information, and therefore a brief study of these figures may be made. In the main the table illustrates two points more clearly than the preceding tables. The first is the effect of filtration, and the second is the effect of season, upon the number of bacteria in water. In respect to the former, comment is needless. It is only necessary to * Royal Commission on Sewage Disposal, Second Report, 1902, p. 99. t Ibid., Fourth Report, 1904', vol. iii., pp. 1-75. J Metropolitan Water Supply, 1902 and 1903. BACTERIA IN LONDON WATER 39 1 g ^J "3 Jfi «+H OH CU * J3 |v& jj § | g" glls % H H H P a 3 BACTERIA IN LONDON WATER 41 examine the returns to recognise the marked reduction in the number of bacteria, in some cases amounting to 98 per cent., brought about by filtration. In respect to the latter, the effect of season, some note is required. It will be seen that during 1902 the figures are fairly uniform throughout the year, showing, on the whole, a rise in winter and spring, and a fall during summer. But in 1903 the returns show wide variation which calls for explanation, which is as follows : — The water supply in December 1902, on the whole, maintained an equal microbic purity to that of November. This exceptional condition of the supply, the com- paratively small number of bacteria, for the winter months is no doubt due to the absence of floods in the Thames Valley, and to the unusually mild character of the season. "If the large deficit in the rainfall is made up," wrote Crookes and Dewar, * * no doubt there will be in the near future a period when the filtration of the London waters will require more than usual care. As the general filter-beds are, however, in good working order, we believe these difficulties, should they arise, will be overcome satisfactorily." The standard of general organic purity during 1902, as defined by chemical methods, was maintained. As regards the month of December, the Thames-derived Companies showed decided differences among themselves, which, as the supply comes from the same source, were essentially connected with differences in storage capacity and variations in the structure of the filter-beds, although the latter is of less importance in removing soluble organic matter. Crookes and Dewar add: "The longer our experience of the bacteriological method, as applied to the analysis of the filtered supply, and the wider its application, the more we are convinced of its primary importance as a safeguard to the public. It enables us to define in a much more delicate way than is possible by chemical analysis what is an efficiently filtered water, and thereby enables the chemist to warn the engineer the moment any one of his filters show signs of defective working. Whether the supply as regards the organic matter in solution varies more or less according to the season of the year, is of relatively small moment as compared with the knowledge that the microbic impurity is reduced to a minimum. " That was the position at the end of 1902. But at the turn of the year, owing to the great increase in the rainfall, the microbes in the unfiltered Thames water rose from about 6000 to 13,000, that is, the bacteriological impurity about doubled, whereas the unfiltered New River water underwent little or no alteration. The result of this increase was that the filters of the Thames-derived Companies, which were not working at their best, furnished a larger number of samples from the filter wells, showing an increase in the number of bacteria which was the inevitable result of an increased rainfall. Things remained thus until April, when in comparison with the month of March the bacteriological quality showed considerable improvement, a result which might have been anticipated from the advent of summer, and the improved natural conditions associated with vegetable growth ; a state of things which generally improves the quality of the water obtained from such collecting areas as the valleys of the Thames and Lea. But in June, when the number of bacteria ought to have been low, as ordinarily there would be a small rainfall, an exceptional condition of things arose. The total excess of rainfall amounted to 49 '9 per cent, on the thirty years' average, so that during the month of June actually 22 -5 per cent, or an amount approaching one-half of the previous excess, of rain fell in the valley of the Thames. Such an amount of rain is altogether exceptional in twenty- two years' experience. The result was that the proportion of vegetable matter in solution, and therefore the colour of the water, were both quite exceptional for the summer months. Nevertheless, the general filtration was adequately and effectively performed, as is shown by the bacteriological results. Similar conditions prevailed in August and October. The exceptional rainfall, which amounted to sixty per 43 BACTERIA IN WATER cent, in excess of the average, kept the colour and amount of soluble vegetable matter in solution abnormally high. In December, owing to the continued rains, the New River and Thames unfiltered waters contained a maximum number of bacteria. In dry weather the number per c.c. had been as low as 149 and 2013 respectively, but owing to seasonal changes they had risen to 861 and 27,216 bacteria per c.c. respectively. From these various records we find that in the result the number of bacteria in river water depends upon a variety of circumstances, amongst which the most important direct conditions are four, namely, (1) local pollution, (2) natural purification (to which subsequent reference will be made), (3) season and rainfall, and (4) sedimentation and filtration. Behind these direct conditions we have also seen that time, temperature, light, exposure to air, and the presence of organic matter play an essential part. Bacteriological Examination of Water. — [See Appendix, p. 463.] Quantitative Standard. — In arriving at a conclusion respecting the number of organisms in a water and their bearing upon its suitability for use, it should be remembered that a chemical report and a bacteriological report are desirable before forming an opinion. The former is able to tell us the quantity of salts and condition of the organic matter present : the latter the number and quality of micro- organisms. Neither can take the place of the other, and, generally speaking, both are more or less useless until we can learn, by inspec- tion and investigation of the source of the water, the origin of the organic matter or contamination. Hence a water report should con- tain not only a record of physical and microscopical characters, of chemical constituents, and of the presence or absence of micro- organisms, injurious and otherwise, but it should also contain infor- mation obtained by personal investigation of the source. Only thus can a reasonable opinion be expected. Moreover, it is generally only possible to form an accurate judgment of a water by watching its history ; that is to say, not from one examination only, but from a series of observations. The writer has examined a certain water supply for thirty-six consecutive months. In 1901 the average number of bacteria per c.c. was 93, in 1902, 136, and in 1903, 57. This shows a stable bacterial content which in itself is favourable. A water yielding a steady standard of bacterial content is a much more satisfactory water, from every point of view, than one which is unstable, one month possessing 50 bacteria per c.c. and another month 5000. It is obvious that rainfall and drought, soil and trade effluents, time and temperature, will have their influence in materially affecting the bacterial condition of a water. Miquel and others have suggested standards which allow " very pure water" to contain up to 100 micro-organisms per c.c. Pure water must not contain more than 1000, and water containing up to NUMERICAL STANDARDS 43 100,000 bacteria per c.c. is contaminated with surface water or sewage. Mace gives the following table : — Bacteria per c.c. Very pure water . . 0 to 50 Good water . - . 50 „ 500 Passable (mediocre) water 500 „ 3,000 Bad water. . »' 3,000 ,, 10,000 Very bad water . k 10,000 „ 100,000 and over. Koch first laid emphasis on the quantity of bacteria present as an index of pollution, and whilst different authorities have all agreed that there is a necessary quantitative limit, it has been impossible to arrive at a settled standard of permissible impurity. Besson adopts the standard suggested by Miquel, and on the whole French bacteriologists follow suit. They also agree with him, generally speaking, in not placing much emphasis upon the numerical estima- tion of bacteria in water. In Germany and England it is the custom to adopt a stricter limit. Koch in 1893 suggested 100 bacteria per c.c. as the maximum number of bacteria which should be present in a properly filtered water. Miquel holds that not more than ten different species of bacteria should be present in a drinking water, and such is a useful standard. The presence of many rapidly liquefying bacteria or organisms associated with sewage or surface pollution would, even though present in fewer numbers than a standard, condemn a water. From a consideration of all the facts it will be seen that it is impossible to judge alone by the numbers. As the science of bacteriology advances less emphasis is laid upon quantitative estimation, for the reason that it is impossible to gauge the quality of a water only by such estimation. The character of the organisms present and the relative abundance of each species is of more importance than quantitative estimations. Such estimations of water bacteria, based upon the counting of colonies in plate cultures, are of little value, and are in no sense an adequate bacteriological examination of a water. It is such "examinations" which have brought bacteriology into disrepute, for it is certain that estimations of this kind are frequently not even approximately correct, nor do they furnish any final indication as to safety or otherwise of a water supply. At the same time it should not be forgotten that, other things being equal and constant, a low number of organisms tends to indicate that a water has not been contaminated with organic matter or the addition of foreign bacteria, and has not been in a condition to favour multiplication of bacteria, and vice versd. Broadly speaking, it must be true that a water containing a large degree of organic matter, the pabulum of bacteria, will contain a higher number of bacteria than a water containing a 44 BACTERIA IN WATER low degree, and this, of course, is the reason for quantitative estimations. Quality of Water Bacteria The species of bacteria found in water vary widely. Many of them are common in pure water, and may be strictly termed " water bacteria " ; others are as clearly " sewage bacteria," with an allied group belonging to the soil and washed into rivers, or wells, by rain, and which may be described as "surface bacteria"; and a third group are the pathogenic bacteria, which have under exceptional conditions been isolated from water. Prof. Marshall Ward, in his fifth report to the Water Kesearch Committee of the Koyal Society, drew up a classification of water bacteria,* which was adopted two years later by Boyce and HilLf In 1899 Johnson and Fuller made other groups,! and many other workers have sug- gested classifications. The two most recent have been constructed by Horrocks of Net ley § and Jordan of Chicago. (| Both authorities recognise that provisional classification is all that is at present possible. Their groups are as follows : — CLASSIFICATION OF HORROCKS. GROUP i. Fluorescent bacilli. ii. B. aquatilis sulcatus. iii. B. subtilis and " Potato bacilli." iv. B. liquefaciens. v. Chromogenic (red) bacilli. vi. Chromogenic (yellow) bacilli. vii. Chromogenic (blue) bacilli, viii. Chromogenic (milk-white) bacilli. ix. Chromogenic (brown) bacilli. x. Micrococci. xi. Sarcinae. xii. Spirilla. xiii. Denitrifying and nitrifying bac- teria. xiv. B. coll communis. xv. B. enteritidis sporogenes. xvi. Staphylococci. xvii. Streptococci, xviii. The Proteus group. xix. Sewage bacteria. xx. B. typhosus. CLASSIFICATION OF JORDAN. GROUP i. B. coli communis. ii. B. lactis aerogenes. iii. Proteus, iv. B. enteritidis. v. B. fl,uorescens liquefaciens. vi. B. fluorescens non-liquefacients. vii. B. subtilis. viii. Non-gas forming, non-fluorescent, non-sporulating, liquefy gela- tine and acidify milk, ix. Similar to Group viii., but milk rendered alkaline. x. Similar to Group viii. , but gelatine not liquefied. xi. Similar to Group ix. , but gelatine not liquefied, xii. Similar to Group xi., but the reaction of milk not altered, xiii. Chromogenic bacilli, not included in above groups. xiv. Chromogenic Staphylococci. xv. Non-chromogenic Staphylococci. xvi. Sarcinse. xvii. Streptococci. * Proc. Roy. Soc., 1897, Ixi., p. 415. t Jour, of Path, and Bact., 1899, vi., p. 32. J Jour, of Exp. Med., 1899, iv., p. 609. § An Introduction to the Bacteriological Examination of Water, 1901, p. 42 et sey. II Jour, of Hygiene, 1903, vol. iii., tfo. 1, p. 5. QUALITY OF WATER BACTERIA 45 Both the above quoted authorities furnish a large body of facts illustrative of the characteristics of the various groups suggested, to which the reader is referred for further particulars. Broadly it may be said that the organisms classified in twenty groups by Horrocks are divisible into a few general divisions. Groups i.-xii. are the ordinary water bacteria ; Group xiii. is the denitrifying and nitrify- ing organisms found in soil, water, etc.; Groups xiv.-xix. are the sewage bacteria ; and Group xx. represents the pathogenic group of organisms occurring occasionally in water. Brief reference will now be made to these four groups, with the exception of the second, which will be dealt with subsequently. (a) Ordinary Water Bacteria. — These are organisms usually found in pure or approximately pure waters. They are common in well waters and unpolluted river water. They include the common fluorescent bacilli, liquefying and non-liquefying, and which create an iridescent green colour in the nutrient media. In this class also are B. aquatilis sulcatus, the "potato bacilli" (B. mesentericus, vulgatus, fuscus, et ruler), the " hay bacilli " (B. subtilis, B. mycoides, B. megatherium), the liquefying bacilli common in unfiltered waters, the chroinogenic organisms (B. prodigiosus, B. lactis erythrogenes, B. rubescens, B. arborescens, B. aquatilis, B. aurantiacus, B. violaceus, etc.), and the micrococci, sarcinae, and ordinary water spirilla.* The presence of these species of bacteria in water, unless in very exceptional numbers, indicates little of importance. They vary according to season, geological formation over or through which the water passes, surface washings, aeration of the water, forms of vegetation existing in the water, and many other similar natural conditions. The fluorescent and non-gas-producing and non-liquefy- ing bacilli are generally less abundant in recently polluted waters than in purer waters, and non-chromogenic staphylococci more abundant. (b) Sewage Bacteria. — This group includes B. coli communis and its allies, the Proteus family, B. enteritidis sporogenes of Klein, and certain streptococci and staphylococci. They will be treated of subsequently in a chapter devoted to the bacteriology of sewage (see pp. 152-157). Exception will, however, be made in the case of B. coli communis, as this organism is perhaps the most important in relation to water. It will, therefore, be considered here. In the first place the chief biological and cultural facts may be stated, and in the second place a general note may be added. * The biological characters of these various groups of water bacteria will be found in Frankland's Micro-organisms in Water, pp. 399-508 ; Lehmann and Neumann's Bacteriology, vol. ii., pp. 133-381; Crookshank's Bacteriology and Infective Diseases ; Horrocks' Bacteriological Examination of Water, pp. 42-80 ; and in the systematic works of Sternberg, Fliigge, Besson, Mace, etc. 46 BACTERIA IN WATER BACILLUS COLI COMMUNIS (Escherich) Source and habitat — An organism of wide distribution, normally present in the excreta of man and animals. Abundant in crude sewage (100,000 per c.c. in London Sewage, Houston). In polluted water, milk, soil, etc. Morphology — A short rod with round ends; size and shape may vary in same colony; polymorphism, depending upon age of culture, products of culture, com- position of medium, etc., 2 to 3 /z long, 0'5 to 0'6 IM broad ; sometimes oval, hardly longer than broad. Usually single, but occasionally in pairs, bundles, or even chains and threads (Plate 3). Staining reaction— Ordinary aniline dyes. Decolorised by Gram. (Schmidt states that B. coli from fatty stools of infants holds the Gram.) Capsule — Present. Flagella—3 or 4 in number, fragile, short, and not wavy. Sometimes only a terminal one ; sometimes several long ones ; but polar staining and vacuolation frequently present in old cultures, or cultures grown under unfavourable con- ditions. Motility — Present, especially" in young cultures, but not, as a rule, so active as B. typhosus ; oscillatory rather than progressive. Sometimes apparently absent. Spore formation — None. Biology : cultural characters (including biochemical features} — Grows best at 37° C. , but will also grow at room temperature. Gordon showed that many varieties of B. coli exist with many minor modifications (Jour, of Path, and Bact., 1897). In gelatine plate cultures the colonies appear generally within 24 hours at 20° C. The deep colonies appear as small white dots, the surface colonies as delicate, slightly granular films of an irregularly circular shape. They are bluish-white by reflected, and amber colour by transmitted light. The diameter of the colony is 1 to 2 mm. The colonies are transparent, and sometimes iridescent, especially towards the periphery, but at the centre and over the entire surface in old cultures an opacity due to a greater thickness of the bacterial growth is observed (Plate 4). It has been observed that species derived from water grow in transparent colonies, whereas those from the alimentary canal or excreta may show opacity of the colony, which characteristic disappears if the culture be passed through milk. About the second or third day the surface colonies attain a diameter of 5 to 6 mm. , and become marked by concentric, or radiating, or irregular markings. The surrounding gelatine very frequently acquires a dull, cloudy, faded appearance, and the edges of the colony become more crenated and thinner. The whiteness of ±he colony turns to yellow. There is no liquefaction of the gelatine. In gelatine stab-cultures the organism grows rapidly. On the surface, in twenty- four hours, the growth is often 2 to 3 mm. in diameter, and closely resembles a surface colony in a plate culture, though more luxuriant. A thick white growth extends along the whole length of the track of the needle, and not infrequently gas bubbles or fissures appear. The gelatine is not liquefied, even in old cultures. In gelatine streak cultures growth is also abundant. In twenty-four hours the elongated milky surface colony may be 5 mm. in diameter. It consists of a delicate faintly-granular film with transparent and irregular margins. Down the centre longitudinally the growth is thicker and therefore more opaque. Irregular thick- enings, foldings, and corrugations may occur in old cultures. Sometimes the film shows iridescence, and the medium, though not liquefied, becomes clouded. The growth, as on the plate cultures, is bluish-white by reflected, and yellowish-amber in colour by transmitted light. In 25 per cent, gelatine at 37° C.— In 48 hours the melted gelatine remains clear, but a thick pellicle forms on the surface (Klein). Gelatine shake cultures become turbid, and within twenty-four hours at 20° C. are riddled with bubbles of gas, which are generally more numerous and larger towards the foot of the tube. They increase in size by the second day, sometimes even forming fissures. The gas is mainly carbonic acid. The presence of a small per cent, of fermentable sugar in the medium increases the gas production (Plate 4.). On potato-gelatine the colonies of B. coli are similar in appearance to those PLATE 3. Bacillus coli communis. From agar culture, 48 hours at 37° C. x 1000. Proteus vulgaris. Impression preparation from " swarming islands" on gelatine, 20 hours at 20° C. X 3000. [To face page BACILLUS COLI 47 occurring on ordinary gelatine, except that they grow more slowly, are more circumscribed, and of a characteristic brown colour (Houston). On carbol-gelatine ('05 per cent, of phenol), the growth does not differ from ordinary gelatine cultures except that it is delayed. Broth— In less than twelve hours at 37° C. the medium becomes uniformly turbid. It may be very pronounced. Frequently there is also at a later stage a marked amorphous flocculent sediment consisting of bacteria. Only a faint film forms on the surface, which rarely becomes a pellicle. There is a foetid odour, and sometimes gas formation. In glucose, lactose, saccharose broth (2 per cent), and glucose-formate broth (Pakes), and bile-salt broth (M'Conkey), the growth is abundant, and gas is produced. In phenolated broth ('05 per cent, of phenol), and in broth containing formalin (1 to 7000), there is also growth. On agar at 37° C. the organism grows rapidly, producing thin, moist, translucent creamy greyish-white colonies of irregular shape and size. The colonies grow more rapidly on the surface than in the depth of the medium. The same appearances occur on agar at 20° C. , except that the growth is delayed. Gas bubbles frequently occur in the condensation fluid. Litmus lactose agar (2 per cent.) — The medium is turned red in twenty-four hours, B. Typlwsus. . B. coli. FIG. 9.— Diagrams of B. typhosus and B. coli. and the surface growth becomes tinged slightly with the reddened litmus. Numer- ous gas bubbles are produced in the medium. On potato at 37° C. there is produced in twenty-four hours a thick, moist, yellowish-grey growth, becoming brown in old cultures. The colour varies widely in degree, sometimes being richer than at other times. The potato becomes changed in colour near the growth. If the potato is not fresh, or its reaction has been made alkaline, the growth of B. coli may be almost colourless. There are, of course, a very large number of bacteria which produce a growth on potato not readily distinguishable from B. coli. Litmus milk — Usually an acid curdling of the milk occurs in twenty-four hours at 37° C., though sometimes slightly delayed. The bluish-purple colour changes to pink, then the whole of the milk is turned into a solid compact coagulum, the milk itself becoming white. Later the redness extends from the surface downwards until the whole contents of the tube are bright red in colour. On blood serum at 37° C. an abundant white glistening layer is rapidly developed, somewhat similar to the growth on agar. There is no liquefaction. water). The reaction -eight hours at 37° C., but in any case is generally kept at 37° C. for five days. The "red reaction " may be obtained by adding to such a culture 1 c.c. of a 0'02 per cent solution of potassium nitrite, and 0'5 c.c. of strong sulphuric acid. If the colour (due to 48 BACTERIA IN WATER nitroso-indol) does not appear at once, the culture may be incubated for a brief period. Reduction of nitrate. — B. coli is a vigorous denitrifying organism. In twenty- four hours at 37° C. the reduction of nitrates to nitrites is well marked. (Bouillon 5 per cent., KNO3 0*1 per cent., water 94*9 per cent.). Aerobic or facultative anaerobic. Vitality and powers of resistance, not considerable, but more than the typhoid bacillus. The following table of comparative features of B. coli and B. typhoons is a provisional scheme of some of the differences between a typical B. coli and a typical typhoid bacillus. As is pointed out elsewhere, the Coli group is large and its characteristics vary according to origin, race, cultivation, and many other conditions. In some ways the table is misleading, as it is exceptional to find a bacillus which gives all these features, but the table is inserted for reference, because in a general way it states the broad differences between the types : — Comparative Features B. typhosus. Morphology— Bacillus of unequal lengths; some filaments. Flagella — Long, wavy, spiral, numerous (9 to 18) ; movement very active. On gelatine and agar — Angular, irregular, slightly raised colonies ; slow growth ; medium remains clear. In gelatine — In ordinary gelatine and in lactose gelatine no gas is produced (at 20° CA No liquefaction. Milk — Not curdled by the bacillus (at 37° C.). No acid production. Indol — In bouillon and Witte's peptone water, no production of indol. Bouillon containing 0'3 per cent. Phenol, or Formalin (1 : 7000) — No growth. Lactose — bouillon at 37° C. — No gas pro- duction. Neutral-red glucose-agar — No change. Glucose or lactose media, shake cultures — No gas production. Potato— An "invisible growth" if the potato is acid in reaction. 25 per cent, gelatine at 37° 0.— Strongly and uniformly turbid (Klein). No pellicle. Eisner's iodised potato-gelatine — Slow growth ; small transparent colonies. Proskauer and Capaldis Medium, No. 1 — No growth ; no change in reaction. Widal's reaction— Bacilli became motion- less and agglutinated when suspended in blood serum from a typhoid patient. (See Appendix.) M'Conkey's lactose agar — Surface colonies transparent ; medium clear. Vitality in icater or sewage— B. typhosus soon ceases to multiply and more or less readily dies. Pfeiffer's inoculation test with anti-typhoid serum — Negative result. of B. coli and B. typhosus B. coli. Bacillus shorter and thicker; filaments rare. Shorter, stiffer, few (average 3), move- ment less active, and sometimes almost absent. Colonies with even margin, homogenous, much larger and quicker growth, medium becomes turbid or coloured. Under the same circumstances abundant gas is produced. No liquefaction. Milk is curdled, within 24 to 48 hours at 37° C. Abundant acid production. Indol is present as a rule. Grows well and uniformly throughout medium. Gas production occurs. Marked green fluorescence. Marked gas production. Thick, yellowish-white growth, later be- coming brown in colour. Gelatine remains clear within 48 hours, but a thick pellicle forms on the surface. Rapid growth ; large brown colonies. Growth ; acid reaction. B. coli remains motile and not aggluti- nated. Surface colonies white with yellow centre ; haze on medium. B. coli retains vitality and power of self-multiplication . Positive result, variable symptoms ac- cording to virulence of bacillus. ARTIFICIAL PURIFICATION OF WATER 65 bicarbonate of lime, is converted into insoluble normal carbonate of lime by the addition of a suitable quantity of limewater. Carbonates of lime and magnesia are soluble in water containing free carbonic acid, but when fresh lime is added to such water it combines with the free C02 to form the insoluble carbonate, which falls as a sediment : — CaCO3 + CO2 + CaH2O2 (limewater) = 2 CaCO3 + H2O. As the carbonate falls to the bottom of the tank it carries down with it the organic particles. Hence sedimentation is brought about by means of chemical precipitation. It is obviously a mechanical process as regards its action upon bacteria. Nevertheless its action is well-nigh perfect, and 400 bacteria per c.c. may be reduced to 4 or 5 per c.c. We shall refer to this same action when we come to speak of bacterial purification of sewage. Alum has been frequently used to purify water which contain much suspended matter. Five or six grains of alum are added to each gallon of water, plus some calcium carbonate by preference. Precipitation occurs, and with it sedimentation of the bacteria, as before. But, as Babes has pointed out, alum itself acts inimically on germs ; in such treatment, there- fore, we get sedimentation and germicidal action combined. As a matter of actual practice, however, sedimentation alone is rarely sufficient to purify water. It is true that the collection of water in large reservoirs permits subsidence of suspended matters, affords time for the action of light, and the suicidal competition among the common water bacteria. But in small collections of water it is otherwise. Here filtration is the most important and most reliable method. Sand filtration, as a means of purifying water, has been practised since the early part of last century. But it was not till 1885 that Percy Frankland first demonstrated the great difference in bacterial content between a water unfiltered and a water which had passed through a sand filter (only about 3 per cent, of the bacteria originally present being left in the water). Previous to this time the criterion of efficiency in water purification had been a chemical one only, and the presence or absence of bacteria in any appreciable quantity was described not in mathematical terms, but in indefinite descriptive words, such as " turbid," " cloudy," etc. It is needless to say that this difference in estimation was largely due to the introduction by Koch of the gelatine-plate method of examination. As a result of investi- gation Percy Frankland formulated the following conclusions as regards the chief factors influencing the number of microbes passing through the filter. The efficiency of filtration, he held, depended upon (a) the storage capacity for unfiltered water, by which it was possible to obtain the preliminary advantage of subsidence ; (b) the thickness of fine sand through which the filtration is carried on; E 66 BACTERIA IN WATER (e) rate of filtration ; (d) the renewal of the filter-beds. After a certain time the filter-bed becomes worn out and inefficient, and at such times renewal is necessary. Not only may the age of the filter act prejudicially, but the extra pressure required will tend to force through it bacteria which ought to have remained in the filter. In 1890 a special study of filtration was made by the Massa- chusetts State Board of Health, and in annual reports published from 1890, a number of experiments are recorded which have proved of classic importance, and which should be consulted by the student or practical worker desiring to acquire a thorough grasp of the principles of biological filtration. There it is shown that water can be filtered through sand filters at the rate of 3,000,000 gallons per acre daily and 9 9 '9 5 per cent, of the bacteria removed. In actual practice it was found that the finer sands were more effective than the coarser, and under moderate pressure 1 foot of sand was as effec- tive as 5 feet. Over 80 per cent, of the bacteria removed were found in the upper inch of sand and 55 per cent, in the upper quarter inch. If the surface of the filter was scraped, it was shown that an increased number of bacteria passed through the filter, which was therefore much less effectual. Subsequently, Koch emphasised the importance of this vital layer. But it was the Massachusetts Board that first proved by experiment that the oxidation which occurs in a filter-bed was due to the nitrifying organisms in the surface or scum layer. When nitrification is established in a filter, the rate of filtration within certain limits was found to exert comparatively little effect upon the removal of the organic matter. In 1893 Koch brought out his monograph upon Water Filtration and Cholera, and his work had a deservedly great influence upon the whole question. He showed how the careful filtration of water supplied to Altona from the Elbe saved the town from the epidemic of cholera which came upon Hamburg as a result of drinking unfiltered water, although Altona is situated several miles below Hamburg, and its drinking water is taken from the river after it has received the sewage of the latter. Now, from his experience of water filtration, Koch arrived at several important conclusions. In the first place, he maintained that the portion of the filter-led which really removed micro-organisms effectively was the slimy membranous organic layer upon the surface of the sand. This layer is produced by a deposit from the still unpurified water lying immediately above it. The most vital part of the filter- bed is this organic layer, which, after formation, should not be dis- turbed until it requires removal owing to its impermeability. A filter-bsd, as is well-known, consists of, say, 3 feet of sand and 1 foot of coarse gravel. The water to be filtered is collected into large reservoirs, where subsidence by gravitation occurs. From thence it FILTRATION OF WATER 67 is led by suitable channels to the surface of the filter-bed. Having passed through the 3 or 4 feet of the bed, it is collected in a storage reservoir and awaits distribution. Such being the principles of construction, it will be apparent that the action of the whole process is both mechanical and chemical. Mechanically by subsidence, much suspended matter is left behind in the reservoir. Again, mechanically, much of that which remained suspended in the water when it reached the filter-bed is waylaid in the substance of the sand and gravel of the filter-bed. The next change is a chemical one. Oxidation of the organic matter occurs to some extent as the water passes through the sand. Until recently this chemical action and the double mechanical action (sedimentation and straining) was believed to be the complete process, and its efficiency was tested by chemical oxidation and alteration, and absence of the suspended matter. Now, however, it is recognised that the second portion of the chemical action is vastly the more important, indeed, the only vital part of the process. This is the chemical effect of the layer of scum and mud on the surface of the sand at the top of the filter- bed. The mechanical part of this layer is, of course, the holding back of the particulate matter which has not subsided in the reservoir ; the vital action consists in what is termed nitrification of unoxidised substance, which is accomplished in this layer of organic matter. We shall deal at some length with the principles of nitrification when we come to speak of soil. But we may say here that by nitrification is understood a process of oxidation of elementary compounds of nitrogen, by which these latter are built up into stable bodies which can do little or no harm in drinking-water. The action of a filter-bed may, therefore, be summarised as follows : — There is (1) subsidence of the grosser particles of impurity in the settling tank ; (2) mechanical obstruction to impurities in the interstices of the scum, sand, and gravel in the filter; (3) oxidation of organic matter by the oxygen held in the pores of the sand and gravel; (4) nitrification in the vital scum layer, which is accomplished by micro-organisms themselves. This latter is now considered to be incomparably the most important part of the filter. That being so, its removal, except when absolutely necessary, is to be avoided as detrimental to the efficiency of the filter. New filters have obviously but little of this action. Kiimmel found that when a filter had new sand placed upon it the number of bacteria in the filtered water was as follows:- Perc>c- Before cleaning . . 42 One day after cleaning . Two days after cleaning . Three days after cleaning Four days after cleaning Five days after cleaning Six days after cleaning . 1880 752 208 156 102 84 68 BACTERIA IN WATER Hence it is necessary to allow a new filter-bed to act for a short period (say four days) before the filtered water is used for domestic purposes, in order to allow a fresh film, the organic layer, to be formed. This must also be borne in mind after a filter-bed has been cleaned.* To maintain this nitrifying action of a filter in efficiency, Koch suggested, in the second place, that the rate of filtration must not exceed four inches per hour. At the Altona water-works this rate of filtration was maintained, and the number of organisms always remained below 100 per c.c., which, as we have seen, is the standard. Thirdly, it is important that periodic bacteriological examinations should be made. Koch's emphasis upon this point is well known, and the cumulative experience of bacteriologists only further supports such a course being taken. Clark and Gage of the Lawrence Experimental Station, claim that the test for the presence of B. coli is a more delicate indication of filter efficiency when filtering polluted water than tests for the total number of bacteria present. Fourthly, Koch maintained that the thickness of the sand of the filter-bed should never be less than one foot. Fifthly, if it be true that efficient sand filtration is a safeguard against putrefactive and disease-producing germs, then there can be but one criterion of efficiency, viz., their absence in the filtered water, which can only be ascertained by regular examination. But it is not alone for pathogenic germs that filtration is proposed. Hence Koch laid down that filtered water containing more than 100 micro- organisms of any kind per c.c. is below the standard of purity, and should not, if possible, be distributed for drinking purposes. In this country chemical analysis, with a more or less cursory microscopic examination, has been almost invariably accepted as reliable indication of the condition of the water. But such an examination is not really any more a fair test of the working of the filter than it is of the actual condition of the water. It is true, the quantity of organic matter can be estimated and the condition in which it exists in combination obtained; but it cannot tell us what a bacteriological examination can tell us, viz., the quantity and quality of living micro-organisms present in the water. Upon this fact, after all, an accurate conclusion depends. There is abundant evidence to show that no valuable opinion can be passed upon a water except by both a chemical and a bacteriological examination, and further by a personal investigation, outside the laboratory, of the origin of the water and its liabilities to pollution. So convinced was Koch of the efficiency of sand filtration as protection against disease-producing germs, that he advocated an adaptation of this plan in cases where it was found that a well yielded infected water. Such pollution in a well may be due to * See also Thirty-fourth Ann. Rvp. State Ed. of Health, Massachusetts, 1903, p. 228. FILTRATION OF WATER 69 various causes ; surface-polluted water oozing into the well is probably the commonest, but decaying animal or vegetable matter might also raise the number of micro-organisms present almost indefinitely. Koch's proposal for such a polluted well was to fill it up to its highest water level with gravel, and above that, up to the surface of the ground, with fine sand. Before the well is filled up in this manner it must, of course, be fitted with a pipe passing to the bottom and connected with a pump. This simple procedure of filling up a well with gravel and sand interposes an effectual filter-bed between the subsoil-water and any foul surface-water percolating downwards. Such an arrangement yields as good, if not better, results than an ordinary filter-bed, on account of there being practically no disturbance of the bed nor injury done to it by frost. The evidence that filter-beds remove pathogenic bacteria has not only been demonstrated by experiment but by actual experience. At Lawrence, Hamburg, Mount Vernon, and other towns, a marked decline in water-borne typhoid fever has occurred as a result of filtration. The effect of filtration upon the number of bacteria was demon- strated in the results which Sir Edward Frankland arrived at in his investigation of London waters so long ago as 1887.* Mean of Monthly Examinations for the Year. Micro-organisms per c.c. Average % of Name of Company. Source of Supply. Micro- organisms removed by At After After Source. Storage. Filtration. Filtration. The Chelsea Co. /Thamesan \Hampton / 16,138 1,067 34 98-96 West Middlesex Co. . 16,138 1,788 58 99-40 Southwark & Vauxhall Co. 11 16,138 - 80 97-72 f 623 ] Grand Junction Co. . M 16,138 2,500 100 \ 98-46 \ 96 J Lambeth Co. »> 16,138 7,820 75' 99-50 In 1899 the Massachusetts Board of Health found that by continuous filtration through 45 inches of sand (size 0'23 mm.) 99*49 per cent, of the bacteria were removed ; and by intermittent filtration 99*08 per cent, of the organisms were removed. In 1902 the intermittent filter removed 98'7 per cent, of the total bacteria, 99'9 per cent. B. coli, and 100 per cent. B. typhosus. The continuous * Report on the Metropolitan Water Supply, 1887. 70 BACTERIA IN WATER filter removed 98'7 per cent, of the total bacteria, 99*8 per cent, of B. coli, and 99'9 of the typhoid bacillus.* The teaching of these figures could, with great ease, be emphasised again and again if such was necessary ; but sufficient has been said to show that sand filtration, when carefully carried out, offers a more or less absolute barrier to the passage of bacteria, whether non- pathogenic or pathogenic. Domestic Purification of Water Something may, however, be added, from a bacteriological point of view, relative to what is called domestic purification. There is but- one perfectly reliable method of sterilising water for household use, viz., boiling. As we have seen, moist heat at the boiling-point main- tained for a few minutes will kill all bacteria and their spores. The only disadvantages to this process are the labour entailed and the "flat" taste of the water. Nevertheless, in epidemics due to bad water, it is desirable to revert to this simple and effectual purification. There are a large number of domestic filters on the market with, in many cases, but little difference between them. The materials out of which they are made are chiefly the following: carbon and charcoal, iron (spongy iron or magnetic oxide), asbestos, porcelain and other clays, natural porous stone, and compressed siliceous and diatomaceous earths. From an extended research in 1894 by Prof. Sims Woodhead and Dr Cartwright Wood, who repeated and extended experiments by Freudenreich, Schofer, and others, our knowledge of the quality of these substances as protectives against bacteria has been largely increased.-)- They concluded that a^ filter failed to act in one of two ways. It was either pervious to micro- organisms, or its power of filtering became modified owing to (a) structural alteration of its composition, or (b) to the growing through of the micro-organisms, which had been demonstrated by previous workers. The conditions which chiefly influence the growth of bacteria through a filter appear to be the temperature, the inter- mittent use of the filter, and the species of bacteria. The higher the temperature and the longer the organisms are retained in the filter the more likely is it that they will grow through, and in the next usage of the filter appear in the filtrate. As to the species, those multiplying rapidly and possessing the power of free motility will naturally appear earlier in a filtrate than others. Woodhead and Wood concluded that out of 18 different kinds of domestic filter, each of which had its supporter, the Pasteur-Chamberland candle filters * Thirty-fourth Ann. Rep. State Bd. of Health, Massachusetts, 1903, pp. 224 and 269. t Brit. Med. Jour., 1894, i., pp. 1053, 1118, 1182, 1375, 1486. DOMESTIC FILTRATION OF WATER (composed of porcelain formed by a mixture of kaolin and other clays) were the only filters out of the substances named above which were reliable and protective against bacteria. They tested over three dozen of the Pasteur filters, and " in every case these gave a sterile filtrate." Pare cholera bacillus in suspension (5000 bacilli to every c.c.) and typhoid bacillus in suspension (8000 per c.c.) were passed through these filters, and not a single bacillus was detectable in the filtrate. The Berkefeld filter (siliceous earth) came second on the list as an effective filter, and had but the one fault of not being a "continuous" steriliser. A certain Parisian filter ("Porcelaine d'Amiante "), made of unglazed porcelain, ren- dered water absolutely free from bacteria. Its action was, however, very slow. Setting aside these three efficient filters, we are face to face with the fact that most filters do not produce germ-free filtrates, even though they are nomin- ally guaranteed to do so. It is professed for animal charcoal, which is widely used, that it absorbs oxygen, and so fully oxidises whatever passes through it. This may be so at first, but after a little use it does more harm than good. It appears to add nitrogen and phosphates to water, which are both nutritive substances on which bacteria grow, and it readily absorbs im- purities from the air. As a matter of experiment and practice, it has been found by Frankland, Woodhead, and others, that charcoal actually adds to the number of germs after it has been in use for some time. Subsequent experiments were made in this country by Lunt and Horrocks. Lunt working in 1897 investigated the power of the Berkefeld filter to intercept pathogenic bacteria, especially the typhoid bacillus. He concluded as a result of his inquiry that on the first day an efficiently sterilised Berkefeld filter gave an absolutely sterile filtrate, but that on the second or third day of using some water bacteria passed through. For thirty- nine days the B. coli did not pass through, though the organism could be detected on the outside of the filtering candle. Lunt found that the action of the filter depended very much upon its method of use: forcing or intermittently pumping water through the filter resulted in a filtrate containing bacteria, whilst if the same filter was steadily used a germ-free filtrate was obtained. In short, the result of Limb's work was to show the necessity for a frequent sterilisa- tion of the filter, for though it allows ordinary water bacteria to FIG. 10.— PASTEUR- CHAM BERLAND FILTER. Attached to Water Supply. 72 BACTERIA IN WATER pass on the second or third day, B. coli and the typhoid bacillus do not appear with them in the filtrate until a subsequent date. Probably, if reliance is to be placed upon such a filter from a bacteriological point of view, daily sterilisation is advisable. Experiments of a similar nature have been done by Horrocks,* who arrives at the following conclusions. First, the B. typhosus is not able to grow through the walls of a Pasteur-Chamberland candle, and if proper care be taken to prevent the direct passage of organisms through flaws in the material and imperfections in the fittings, the Pasteur-Chamberland filter ought to give complete protection from water-borne disease. Secondly, typhoid bacilli can grow through the walls of Berkefeld candles, the time required for the passage being largely dependent on the nutriment supplied to the organisms by the filtering fluid. Possibly the weakness of the candle from a bacteri- ological point of view is due to the large size of the lacunar spaces, which cannot be avoided if a fair delivery is to be obtained, but which " appears to militate against the immobilising and devitalising influences which operate so strongly in filters made with very narrow lacunar spaces." Thirdly, Horrocks concluded that when a highly polluted liquid containing typhoid bacilli is filtered through a Berkefeld candle the bacilli may appear in the filtrate in four days. Consequently, it is necessary to sterilise these candles every third day. The method of sterilisation of filters is not washing or brushing or any other kind of cleansing or soaking in water, but by exposing them to steam or boiling them. * Bacteriological Examination of Water, 1901, pp. 273-280. CHAPTEE III BACTERIA IN THE AIR Methods of Examination of Air — Conditions of Bacterial Contamination of Air : (1) Dust and Air Pollution ; (2) Moisture or Dampness of Surfaces : Bacteria in Sewer Air ; (3) the Influence of Gravity ; (4) Air Currents. The Relation of Bacteria to CO2 in the Atmosphere : in Workshops, in Bakehouses, in Rail- way Tubes, in the House of Commons. THE basis of the usual methods in practice for bacterially examining air is to pass the air over or through some nutrient medium. By this means the contained organisms are waylaid, and finding them- selves under favourable conditions of pabulum, temperature, and moisture, commence active growth, and thus reveal themselves in characteristic colonies. These are examined by the microscope and sub-culture. Eeturns of the number of bacteria in the sample taken may be made for the sake of information, but little or no conclusion of value can be drawn from such data. The standard recognised in Europe is the cubic metre or litre, and one may report, for example, of the air of a room containing 500 or 'more germs per cubic metre. Methods of Examination of Air 1. The Plate Method. — Koch adopted the simplest of all the culture methods, viz^ exposing a plate of gelatine or agar for a longer or shorter time to the air of which examination is desired. By gravity the suspended bacteria fall on the plate and start growth. As a matter of quantitative exactitude, this method is not to be recommended, but it frequently proves an excellent method for qualitative estimation. It will be found in practice that nutrient agar is better for the purpose than nutrient gelatine. Greater latitude is obtained both in point of temperature and length of incubation, and the result is uncomplicated by the, at times, very rapid liquefaction of the gelatine by liquefying organisms. Care should be taken in preparing the plates to allow them to cool on a level surface, and at least 15 c.c. 73 74 BACTERIA IN THE AIR of the medium should be employed for each Petri dish in order to ensure an even surface and sufficient depth of medium all over the plate. After exposure the plates are, under ordinary circumstances, best left at room temperature during the development of the colonies, but if it is desired to examine the bacteria alone it will be found well to favour the growth of these at the expense of the moulds, by first incubating the dish at a temperature of 37° C. for, say, eighteen hours. In any case the plate should be shielded from light, or otherwise many of the chromogeriic organisms will not assume their typical coloration. Should it be desired to photo- graph the plates in order to obtain a permanent record, the growth should be arrested, and the organisms killed about the third or fourth day of incubation. The best method of doing this is to reverse the dish, and to pour upon a piece of blotting paper placed on the inner surface of the lid, which will now be undermost, a sufficient quantity of Formalin to saturate it. The results of this method of examination may be expressed per square foot per minute, the area of the Petri dish / 22 \ being calculated f = (radius)2 x — J. 2. The Flask Method of Mquel. — Pasteur was the first to analyse air by the culture method, and he adopted a plan which, in principle, is ivashing the air in some fluid culture medium which will retain all the particulate matter, which may then be cultured directly or sub-cultured into any favourable medium. Miquel has con- trived a simple piece of apparatus for the carrying out of this principle. It consists of a flask with a central tube through its own neck for the entrance of the air. On one side of the flask is a tube to be con- nected with the aspirator, on the other side of the flask a tube through which to pour off the contained fluid at the end of the process. In the flask are placed 30 c.c. of sterilised water (or, indeed, if it be pre- ferred, sterilised broth). The entrance tube is now unplugged, and the aspirator draws through a fair sample of the air in the room (say ten litres). This air perforce passes through the water, and by the exit tube to the aspirator, and is thereby washed, leaving behind in the water its bacteria. The aspiration is then stopped, and the entrance tube closed. The water (plus bacteria) is now poured out into test-tubes of media or plated out on Petri's dishes. Provided that the appar- atus has been absolutely sterilised, and that only sterilised water is used, any colonies developing upon the Petri dish are composed of micro-organisms from the air examined. 3. The Method of Hesse. — This method is somewhat akin to Pouchet's aeroscope, but is in addition a culture method. Hesse's tube is 50-70 cm. long and 3-5 cm. bore throughout. At one end is an indiarubber stopper bored for a glass tube to the aspirator. The other end is open. Before using, the tube is sterilised, and 40 or 50 c.c. of sterilised gelatine are placed in it. The tube is now rapidly rotated in a groove on a block of ice or under a cold-water tap, and by this simple means the gelatine becomes fixed and forms a layer inside the tube throughout. We have therefore, so to speak, a tube of glass with a tube of gelatine inside it. The apparatus is now ready for use. It is fixed on the1 tripod, and 10-20 litres of air are drawn through, and the tube is properly plugged and incubated at room temperature. In two or three days the colonies appear upon the gelatine. They are most numerous generally in the first part of the tube. The disadvantages of FIG. 11.— Miquel's Flask. SEDGWICK'S SUGAR TUBE, in position on tripod, with siphon. SMALL HAND CENTRIFUGE. [To face page 74. BACTERIOLOGICAL EXAMINATION OF AIR 75 this process are that dried gelatine does not catch germs like the broth cultures of Pasteur or Miquel, and that many organisms are carried straight through the tube, and failing to be deposited, pass out at the aspirator exit, and thus are neither caught nor counted. The Hesse tube is generally used in practice with a pump consisting of two flasks and a double-way indiarubber tube. The flasks have a capacity for one litre of water. By a simple arrangement it is possible to secure syphon action, and hence measure with considerable exactitude the amount of air passing through the tube (Plate 5). 4. Methods of Filtration. — Frankland, Petri, Pasteur, Sedgwick, and others have suggested the adoption of methods of filtration. These depend upon catching the organisms contained in the air by filtering them through sterilised sand or sugar, and then examining these media in the ordinary way. Many different kinds of apparatus have been invented. Petri aspirates through a glass tube containing sterilised sand, which after use is distributed in Petri dishes and covered with gelatine. The principal objection to this method is the presence of the opaque particles of sand in and under the gelatine. Probably it was this which suggested the use of soluble filters like sugar. Pasteur introduced the principle, and Frank- land and others have followed it out. Sedgwick's Tube consists of a comparatively small glass tube, about a foot long. Half of it has a bore of 2*5 cm., and the other half a bore of -5 cm. It is sterilised at 150° C., after which the dry, finely granulated cane-sugar is inserted in such a way as to occupy an inch or more of the narrow part of the tube next the wide part. Next to it is placed a wool plug, and the whole is again sterilised. After sterilisation an indiarubber tube is fixed to the end of the narrow portion, and thus it is attached to the aspirator. The measured quantity (5-20 litres) of air is drawn through, and any participate matter J FIG. 12. — Sedgwick's Sugar-tube. is caught in the sugar. Warm, nutrient gelatine (10-15 c.c.) is now poured into the broad end of the tube, and by means of a sterilised stilette the sugar is pushed down into the gelatine, where it quickly dissolves. We have now in the gelatine all the micro-organisms in the air which has been drawn through the tube. After plugging with wool at both ends, the tube is rolled on ice, or under a cold-water tap, in order to fix the gelatine all round the inner wall of the tube, which is incubated at room temperature. In a day or two the colonies appear, and may be examined. Frankland used finely powdered sugar and glass wool as filtering-medium, and a tube with two constrictions. After passing sufficient air through, the tube is broken in halves and the wool and sugar are pushed by means of a sterile needle into liquefied gelatine. The sugar dissolves and the organisms are distributed in the medium. Andrewes has used a modification of this method, and the aspiration was carried out with a large brass syringe of known capacity, fitted with a two-way nozzle and cock, so that the requisite number of syringefuls could be aspirated without disturbance. * Various other methods, including Miquel's filtration method, and the methods of Laveran, and Wiirtz and Strauss, have been used, but the principal are those mentioned above. In respect of the results obtained in the examination of air bacteriologically, it may be said that they are twofold. First, a quantitative result is obtained by which we may arrive at the approximate number of bacteria and moulds. Secondly, the quality or species of organisms is determined. Reference will be made to both these points in the pages which follow. * Brit. Me.il. Jour., 1902, ii., p. 1534; and Report to London County Council. 1902. 76 BACTERIA IN THE AIR Conditions of Bacterial Contamination of Air There are, speaking in a general way, four chief external conditions affecting the occurrence of bacteria in air. They are as follow : — 1. The presence of dust and air pollution. 2. Dampness of surfaces. 3. Gravity. 4. Air currents. 1. Dust and Air Pollution. — Schwann was one of the first to point out that when a decoction of meat is effectually screened from the air, or supplied solely with calcined air, putrefaction does not set in. It is true that Helmholtz and Pasteur confirmed this, and greatly added to our knowledge of the subject, but on the whole it may be said that Schwann originated the germ-theory of air, and Lister applied it in the treatment of wounds. Lister believed that if he could surround wounds with filtered air free from dust and particulate or germ matter, the result would be as good as if the wounds were shut off from the air altogether. It was Tyndall * who first laid down the general principles upon which our knowledge of organisms in the air is based. That the dust in the air was mainly organic matter, living or dead, was a comparatively new truth; that epidemic disease was not due to " bad air " and " foul drains," but to germs conveyed in the air, was a prophecy as daring as it was novel. From these and other like investigations it came to be recognised that putrefaction begins as soon as bacteria • from the air gain an entrance to the putrefiable substance, that it progresses in direct proportion to the multiplication of these bacteria, and that it is retarded when they diminish or lose vitality. Tyndall made it clear that, both as regards quantity and quality of micro-organisms in the air there neither is nor can be any uniformity. The degree in either case will depend on air pollution and on dust particles. Bacteria may be conducted on particles of dust — "the raft theory" — but being themselves endowed with a power of flotation commensurate with their extreme smalmess and the specific lightness of their composition, dust as a vehicle is not really requisite. Nevertheless the estimation of the amount of dust present in a sample of air may be a very good index of danger. It is to Dr Aitken that we are indebted for devising a method by which we can measure dust particles in the air, even though they be invisible. His ingenious experiments, reported in the Transactions of the Royal Society of Edinburgh (vol. xxxv.), have demonstrated that by supersaturation of air the invisible dust particles may become visible. As is now well known, Dr Aitken believes that fogs, mists, * John Tyndall, F.R.S., Floating Matter of the Air, 1878. PLATE 6. AIR-PLATE EXPOSED IN LABOURER'S COTTAGE IN BUCKINGHAMSHIRE (30 minutes). Agar culture, 3 days at 22° C. (Grown and photographed by Swithinbank). [To face page 76. DUST AND AIR POLLUTION 77 and the like do not occur in dust-free air, and are due to condensation of moisture upon dust particles. And much the same has been found to be true in respect to dust and bacterial pollution. As a rule, when the former is abundant the latter is considerable. Haldane and Osborn (vide infra) found bacteria most numerous in a workplace where dust was most abundant, and their finding was merely con- firmatory of many other previous researches. On the other hand it should be remembered that, though dust forms a vehicle for bacteria, dusty air is sometimes comparatively free from bacteria. For the conditions which affect the number of bacteria in the air are various. In open fields, free from habitations, there are fewer, as would be expected, than in the vicinity of manufactures, houses, or towns. A dry, sandy soil or a dry surface of any kind will obviously favour the presence of organisms in the air. Frank- land found that fewer germs were present in the air in winter than in summer, and that when the earth was covered with snow the number was greatly reduced. Miquel and Freudenreich have declared that the number of atmospheric bacteria is greater in the morning and evening between the hours of six and eight than during the rest of the day. There is no numerical standard for bacteria in the air as there is in water. In houses and towns it would rise according to circum- stances, and frequently in dry weather reach thousands per cubic metre. When it is remembered that air possesses no pabulum far bacteria, as do water and milk, it will be understood that bacteria do not live in the air. The quality and quantity of air organisms depend entirely upon envtrCrTment and physical'iconditTOTis. In some researches which the writer made into the air of workshops in Soho in 1896, it was instructive to observe that fewer bacteria were isolated by Sedgwick's sugar-tube in premises which appeared to the naked eye polluted in a larger degree than in other premises apparently less contaminated. In the workroom of a certain skin-curer the air was densely impregnated with dust particles from the skin, yet scarcely a single bacterium was isolated. Macfadyen and Lunt have also found that the number of dust particles does not bear any relation to the number of bacteria. They found that air containing even millions of dust particles might be almost germ-free. In the polish- ing room of a well-known hat firm, in which the air appeared to the naked eye to be pure, and in which there was ample ventilation, there were found by the writer numerous bacteria belonging to four or five species of saprophytes. The public analyst for the city of Nottingham, estimated the bacterial quality of the air of the streets of that town during "the goose fair" held in the autumn. He used a modification of Hesse's apparatus, in which the gelatine is replaced by glycerine. The air was slowly drawn through and 78 BACTERIA IN THE AIR measured in the usual way. Sterilised water was then added to bring the glycerine to a known volume, the liquid thoroughly mixed, and a series of gelatine and agar plates made with quantities varying from O'l to 2 c.c. By this method a large number of bacteria were detected in this particular investigation, including Stapliyloroccus 2iyogencs aurcus and albus, the common Bacillus subtilis, and, appar- ently, B. coli communis* Carnelly, Haldane, and Anderson found 11 bacteria per litre of air in a classroom of the High School at Dundee with the boys at rest. But when the boys were instructed to stamp on the floor, and thus raise the dust, the number rose to 150 bacteria per litre. During a six years' investigation the air of the Mont Souris Park yielded, according to Miquel, an average of 455 bacteria per cubic metre. In the middle of Paris the average per cubic metre was nearly 4000. Flligge accepts 100 bacteria per cubic metre as a fair average. From this fact he estimates that " a man during a life- time of seventy years inspires about 25,000,000 bacteria, the number contained in a quarter of a litre of fresh milk."f Many authorities would place the average much below 100 per cubic metre, but even if we accept that figure it is at once clear how relatively small it is. This comparative freedom from bacteria is due to sunlight, rain, desiccation, dilution of air, moist surfaces, etc. So essentially does the bacterial content of air depend upon the facility with which certain bacteria withstand drying that Dr Eduardo Germano has addressed himself first to drying various pathogenic species and then to mixing the dried residue with sterilised dust and observing to what degree the air becomes infected.J The typhoid bacillus appears to withstand comparatively little desiccation, without losing its viru- lence. Nevertheless it is able to retain vitality in a semi -dried con- dition, and it is owing to this circumstance, in all probability, that it possesses such power of infection. The bacillus of diphtheria, on the other hand, is capable of lengthened survival outside the body, particularly when surrounded by dust. The question of its power of resistance to long drying is an unsettled point. The power of surviving a drying process is, according to Germano, possessed by the Streptococcus pyogcncs. This is not the case with the organisms of cholera or plague. Dr Germano classifies bacteria, as a result of his researches, into three groups : first, those like the bacilli of plague, typhoid, and cholera, which cannot survive drying for more than a few hours ; second, those like the bacilli of diphtheria and strepto- cocci, which can withstand it for a longer period ; thirdly, those like the tubercle bacillus, which can very readily resist drying for months * Public Health, vol. x., No. 4, p. 130 (1898). f FlUgge, Grundriss der Hygiene, 1897, pp. 161, 162. J Zvitschrift fiir Hygiene, vols. xxiv.-xxvi. DAMP SURFACES 79 and yet retain their virulence. It will be obvious that from these data it is inferred that Groups 1 and 2 are rarely conveyed by the air, whereas Group 3 is frequently so conveyed. Miquel has recently demonstrated that certain soil bacteria or their spores can remain alive in dried dust in hermetically sealed tubes for as long a time as sixteen years. Even at the end of that period such soil inoculated into a guinea-pig produced tetanus. The presence of pathogenic bacteria in the air is, of course, a much rarer contamination than the ordinary saprophytes. The tubercle bacillus has been not infrequently isolated from dry dust in consumption hospitals, and in exit ventilating shafts at Brompton the bacillus has been found. From dried sputum, it has, of course, been many times isolated, even after months of desiccation. Indeed, a very large mass of experimental evidence attests the fact that the air in proximity to dried tubercular sputum or discharges may contain the specific bacillus of the disease. The bacillus of diphtheria in the same way, but in a lesser degree, may be isolated from the air, and from the nasal mucous membrane of nurses, attendants, and patients in a ward set apart for the treatment of the disease, and from the throats and nasal mucous membrane of persons who have been in contact with cases of the disease. Delalivesse, examining the air of wards at Lille, found that the contained bacteria varied more or less directly with the amount of floating matter, and depended also upon the vibration set up by persons passing through the ward and the heavy traffic in granite-paved streets adjoining. B. coli, staphylococci, and streptococci, as well as B. tuberculosis, were isolated by this observer. Other observers have found B. coli very rarely present in air (Chick, Andrewes, etc.). 2. Moisture or Dampness of Surfaces. — It is an interesting and important fact that except under special circumstances micro- organisms do not leave moist surfaces, but remain adhering to them. A clear recognition of this fact is essential to a right understanding of the pollution of air by bacteria. They cannot leave the moist surface of fluids either under evaporation or by means of air currents.* Only when there is considerable molecular disturbance, such as splashing, can microbes be transmitted to the surrounding air. This is the reason why sewer gas and all air contained within moist perimeters is almost germ-free, whereas from dry surfaces the least air current is able to raise countless numbers of organisms. This principle has been admirably illustrated in investigations made upon expired and inspired air. In a report to the Smithsonian Institute of Washington (1895) upon the composition of expired air, * Fliigge has lately attempted to demonstrate that an air current having a velocity of four metres per second can remove bacteria from surfaces of liquids by detaching drops of the liquid itself. 80 BACTERIA IN THE AIR it is concluded that "in ordinary quiet respiration no bacteria epithelial scabs or particles of dead tissue are contained in the expired air. In the act of coughing or sneezing such organisms or particles may probably be thrown out." The mucous membrane lining the cavity of the mouth and respiratory tract is a moist perimeter, from the walls of which no organisms can rise except under molecular disturbance. The popular idea that bacteria can be "given off by the breath" is therefore contrary to the laws of organismal pollution of air. The required conditions are not fulfilled, and such breath infection must be of extremely rare occurrence except in speaking, spitting, sneezing or coughing (Fltigge). Air can only become infective when impregnated with organisms arising from dried surfaces. Another series of investigations were conducted by Drs Hewlett and St Glair Thomson, and dealt with the fate of micro-organisms in inspired air and micro-organisms in the healthy nose. They estimated that from 1500 to 14,000 bacteria were inspired every hour. Yet, as we have seen, expired air contains practically none at all. It is clear, then, that the inspired bacteria are detained some- where. Lister has pointed out, from observation on a pneumo-thorax caused by a wound of the lung by a fractured rib, that bacteria may be arrested before they reach the air cells of the lung, and other observations confirm this fact, although of course there are several well-known exceptions (e.g. tubercle of the lung). Hence it is at some intermediate stage that they are detained. Hewlett and Thomson examined the mucus from the wall of the trachea, and found it germ-free. It was only when they examined the mucous mem- brane and moist vestibules and vibrissae of the nose that (they found bacteria* Here they were present in abundance. The ciliated epithelium, the mucus, and the bactericidal influence of the wandering or " phagocyte " cells, probably all contribute to their final removal.* There can be no doubt that the large number of bacteria present in the moist surfaces .of the mouth is the cause of a variety of ailments, and under certain conditions of ill-health organisms may through this channel infect the whole body. Dental caries will occur to everyone's mind as a disease probably due in part to bacteria. As a matter of fact, acids (due to acid secretion and acid fermentation) and micro-organisms are two of the chief causes of decay of teeth. Defects in the enamel, inherent or due to injury, retention of debris on and around the teeth, and certain pathological conditions of the secretion of the mouth, are predisposing causes, which afford a * Hewlett and Thomson graphically demonstrated the bactericidal power of the nasal mucous membrane by noting the early removal of Bacillus prodigiosus, which had been purposely placed on the healthy Schneiderian membrane of the nose. BACILLUS COLI 49 General Note. — Whilst the above description applies to the normal type of B. coli, it should be clearly understood that a large number of bacilli have been described which possess some, but not all, of the above characters. Eefik has described (Ann. de VInst. Pasteur, x., 1896, 242), five varying types very similar to the normal B. coli, but differing in one or more characters. Almost all forms, however, have some features in common, e.g., motility, few flagella, and characteristic growth on potato. Moreover, there are a group of organisms allied to B. coli, and often associated with it. Like it also, they are related, etiologically or otherwise, to similar pathological processes. Kefik's types are briefly as follows : — A. Ferments lactose, coagulates milk, but gives no indol reaction. B. Ferments lactose, does not coagulate milk, gives indol reaction. C. Ferments lactose, does not coagulate milk, does not give indol reaction. D. Does not ferment lactose, coagulates milk, does not give indol reaction. E.i Does not ferment lactose, does not coagulate milk, does not give indol reaction. Mervyn Gordon has made a careful study of the B. coli and its allies which he classified according to their reactions and their flagella. He differentiated 16 varieties.* Horrocks studied the cultural char- acters of 150 "varieties" of B. coli isolated partly from normal and partly from typhoid stools, f Other workers have observed an enormous variety of minor differences. The important point is the diagnosis of B. coli, and the following characters are now chiefly relied upon (see also p. 472). 1. The B. coli group is non-sporing and non- liquefying; 2. The members of the group rarely stain by Gram's method ; 3. They produce acid and gas with both glucose and lactose ; 4. They produce acid in milk and they usually also coagulate it ; 5. They produce acid and gas in bile-salt-glucose broth ; 6. They grow well at a temperature of 42° C.J Other fairly reliable features are motility, a small number of flagella, a fairly typical growth on potato, and more rapid development on all media than the typhoid bacillus. But there is not at the present time a complete unanimity of opinion as to the most reliable characters for diagnostic purposes. § * Jour, of Path. andBact., 1897, vol. iv., p. 438. f Bacteriological Examination of Water, 1901, p. 94; Jour, of Hyg., 1901, p. 202. J Roy. Com. on Sewage Disposal, Second Report, 1902, p. 101. See also Brit. Med. Jour., 1903, i. 418 (Klein), for summary of characters of B. coli. § Houston considers the following the most useful tests for B. coli: (1) Gas formation in ordinary gelatine "shake" cultures; (2) indol in broth cultures; (3) acid and clot in litmus milk - cultures ; (4) greenish-yellow fluorescence in neutral-red broth cultures ; (5) gas and acid in lactose-peptone cultures ; (6) gas, acid, and clot in peptone-lactose milk cultures ; (7) gas and acid in glucose-peptone cultures; (8) reduction of nitrate to nitrite in nitrate broth cultures; (9) strong acid in Proskauer and Capaldi's medium No. 1, and no definite production of acid D 50 BACTERIA IN WATER The significance of B. coli is of course its potential pathogenicity, and its similarity to the typhoid bacillus, but above all its relation to sewage. Roux, Rodet, and others have stated that B. coli, under certain circumstances, may assume a character not distinguishable from B. typhosus, both in its biological and cultural characteristics and in its pathogenic properties. Chantemesse, Widal, and others have held that polluted waters owe their power to produce typhoid fever to the presence of B. coli, and that possibly the organisms are transformable the one into the other. Klein and many other bacteriologists, as the result of very numerous experiments, have been unable to effect any transformation of one form into the other. Each organism has retained unimpaired its differential characters. Certain strains of B. coli are distinctly pathogenic for lower animals, and there is some ground for considering the organism a cause of disease (epidemic diarrhoea and other conditions) in man, either by itself or in association with other organisms (Delepine). In the third place, as is pointed out elsewhere, B. coli is a sewage organism, and the chief importance of its detection in water is an indication of sewage pollution and therefore of possible contamination of the water with specific bacteria. It is therefore a most reliable test of pollution. Klein and Houston have emphasised the importance of the presence of B. coli and the B. cnteritides sporogencs in water as indication of sewage pollution, and by this means a demonstration of the presence of sewage in ~ water can be carried to an incomparably higher degree than by chemical examination. Chemistry is powerless to detect pollution ' by pathogenic germs or the small amount of organic pollution which can be detected by bacteriology, which is ten in Proskauer and Capaldi's medium No. 2 ; (10) presence of motility ; (11) non-liquefaction of gelatine ; and (12) acidity in litmus whey cultures, varying from about 20-40 c.c. — Na2CO3 per 100 c.c. of culture. In dealing with sewage, effluents, and non-drinking-water streams, Houston employs the first three tests, but in dealing with drinking-water, the first five tests (Fourth Report of Royal Commission Sewage Disposal, 1904, p. 106). McWeeney relies chiefly upon '(a) the character of gelatin colony and non- liquefaction of that medium, even after a long time ; (6) non-retention of Gram's stain ; (c) fermentation of lactose with gas and acid formation ; (d) coagulation of milk within four days at 37° C. ; (e) production of yellowish-green fluorescence in neutral-red-agar-shake culture ; and (/) production of indol in liquid peptone media. (Report of Local Government Board for Ireland, 1904). Klein describes B. coli as a motile, non-spore-bearing bacillus, possessing a limited number of flagella, capable of fermenting glucose and lactose, of curdling milk with the production of acid, of forming indol in broth culture, reducing neutral red with the production of a green fluorescence^producing gas-bubbles in nutrient jelly, of forming a more or less brownish growth ran steamed potato, and of producing on the surface of gelatin a dry, translucent growth which does not liquefy the gelatin. The bacilli, under the microscope, appear asfcylindrical rods, showing more or less pronounced motility, and they do not stainjby the method of Gram (see also Appendix, pp. 466 and 472). Bacillus coli communis. Surface gelatine plate culture, O'l c.c. ot ^^ c.c. of Rugby sewage. GAS IN GELATINE SHAKE CULTURE, 24 hours at 20° C. From left to right the tubes represent TJn, Tn'rtiT, TBJTO> Tn^nr> c.c. of Nottingham crude sewage. [To face page 50. BACILLUS COLI 51 to one hundred times less than that detectable by chemistry.* It is, however, important to bear in inind that something more than the mere presence of B. coli must be ascertained. The comparative numbers present, the relative abundance, and the general character and source of the water must be considered. Waters containing no B. coli in 100 c.c. are of course of a high degree of purity.f In upland surface waters the presence of B. coli in such a small amount as 1 c.c., may be sufficient to condemn the waters. Certainly drinking- water from a deep well should contain no B. coli. The presence in a water of B. coli in conjunction with streptococci or even the spores of B. enteritidis sporogenes, or both, would of course indicate serious pollution. The differential diagnosis of B. coli from its allies or other organisms is not always a simple matter. An adherence to the characteristics set out above will generally prove safe guidance, but reliance should not be placed upon any single character or test. The tendency to adopt some rapid and easily-applied test for this organism is strongly to be deprecated, as likely to lead to error. Nothing can take the place of the careful study and sub-culture of the suspected organism in this and in all other species. At the same time, it has been found that diagnostic aid is obtained by a comparison of some of the biological characters of the colon and allied groups of bacteria. They may be divided into four divisions :— (1) The proteus group, the members of which are motile, liquefy gelatine, produce gas in glucose and sucrose but not in lactose, curdle and acidulate milk very slowly, and usually produce indol ; (2) the coli group include motile bacilli, producing gas in glucose and lactose, curdle milk rapidly, nearly always produce indol, but do not liquefy gelatine, and do not retain Gram's stain; (3) the group including B. lactis cerogenes are non-motile bacilli, which do not liquefy gelatine but which curdle and acidulate milk and ferment sugars other than, glucose; and (4) the enteritidis group contain bacilli which are motile, which only ferment glucose, and which do not liquefy gelatine or curdle milk, which is ultimately rendered alkaline. This group includes B. enteritidis of Gaertner, the para-colon and the para- typhoid bacilli. Streptococci in Water. — Houston considers the presence of strepto- cocci in water as indication of recent and dangerous pollution of water. They are absent even in large quantities of pure water and in virgin soils.j Streptococci, as a class, are delicate germs that readily lose their vitality and die when the physical conditions are unfavourable, and they comprise species highly pathogenic to human * Medical Supplement to Report of Local Government Board, 1898-99, p. 498. t See also Jour, of Hyg., 1902, p. 339 (Savage). J Report of Local Government Board, 1899-1900, p. -183. 52 BACTERIA IN WATER beings. They are present in human faeces and in crude sewage in considerable number; and as we have said, they are absent from relatively large amounts of pure waters and virgin soils, but present in abundance in water and soil recently polluted with animal dejecta. It is not claimed that all streptococci are necessarily delicate germs, or pathogenic, or of recent animal outcome. It may be that certain streptococci are comparatively hardy germs, and that others may be capable of multiplying in Nature outside the animal body. Again, there may be streptococci in Nature which do not owe their origin to excremental matter, and doubtless many of them may be non-pathogenic, although this latter circumstance is no proof that at a stage prior to their isolation they were non-virulent, nor does it impair the value of the test as an indication of recent fouling with objectionable matters. Houston found streptococci habitually present in crude sewage in TTjVo c-cv present in human faeces in one milligramme, and present in minimal quantities of soils and water recently polluted with matters of animal outcome. These results encourage the belief that the streptococcus test is one of the most delicate yet suggested for detecting recent, and therefore, presumably, specially dangerous, pollution. The question of relative abundance in connection with the strepto- coccus test also deserves consideration. For if streptococci are absent from 10 c.c. or more of pure waters and present in TTroo- c-c- °f crude sewage the distinctions as regards streptococci between water and sewage is sufficiently great to allow of considerable latitude being observed in framing a standard without seriously impairing the value of the test. What standard should be adopted is a matter of opinion, but as a rule it may be said that the presence of streptococci are to be thought of as indicating extremely recent, and B. coli less recent, but still not remote, pollution of animal sort (Houston). The presence of B. entcritidis sporogenes, however, cannot be considered to afford evidence of pollution bearing a necessary relation to the recent evacuations of animals. Streptococci and B. coli are either altogether absent or present in sparse amount in virgin soils, and may be absent even from polluted soils, unless the contamination is of comparatively recent sort. In soils recently polluted with animal matters streptococci and B. coli are of course present in abundance. B. enteritidis sporogenes may be present even in seemingly virgin soils, but in sparse proportion compared with the large number found in cultivated and polluted soils. Lastly, the presence of streptococci in any number in a water supply points not only to recent animal pollution, but also implies that the antecedent conditions — condi- tions intervening between the period of pollution of the water and the time of collection of the sample — could hardly have been of so un- PATHOGENIC BACTERIA IN WATER 53 favourable a character as to destroy the vitality of seemingly more hardy microbes — for example, the typhoid bacillus. The same cannot be said for the B. coli test, since B. coli is a more hardy germ than B. typhosus. Broadly, therefore, it will be seen that the presence of B. coli or B. enteritidis sporogenes or Streptococci in a water is presumptive evidence of sewage pollution. But that in forming an opinion it is essential to bear in mind the relative abundance of organisms per c.c. and the relative abundance of certain species. (c) Pathogenic Bacteria in Water. — The two chief types of disease-producing organisms found in water are the bacillus of typhoid fever and the bacillus of cholera. These diseases and their causal organisms are dealt with subsequently (see pp. 298 and 384). Here it will only be necessary to note one or two general facts as to the relation of pathogenic organisms to water supplies. In sterilised water, and in very highly polluted water or sewage, pathogenic bacteria do not flourish. In the former case they die of starvation, although there are experiments on record which appear not to support this view ; in the latter case they are killed by the enormous competition of common bacteria. Even in ordinary water there is a wide divergence of behaviour. Some bacteria are destroyed in a few hours ; others appear to flourish for weeks. In all cases the spores are able to resist whatever injurious properties the water may have much more persistently than the bacilli themselves. These changes in the vitality of bacteria in water, partly due to the water and partly to the other micro-organisms, bring about two character- istics which it is important to remember, viz., that pathogenic germs in water are, as a rule, scanty and intermittent. It is these features in conjunction with the enormous quantities of common water bacteria which make the search for the bacillus of typhoid fever what Klein has called " searching for a needle in a rick of hay." Not that it cannot be detected, but its detection is one of the most difficult of investiga- tions. In recent years the typhoid bacillus has been isolated from water which had given rise to cases of typhoid fever at Pierrefonds (Widal & Chantemesse), Dijon (Vaillard), Chateaudun, Cuxhaven (Dun bar), and possibly one or two other instances.* Undoubtedly a large number of epidemics have been due to typhoid infected water, but for obvious reasons (long incubation of typhoid, the fact that the bacillus only lives in water for a few days, etc.), the cases where the bacillus has been actually isolated are very few. In the Milroy Lectures for 1902, Professor Corfield gives records of between 50 and GO typhoid epidemics since 1864. We shall refer to this matter subsequently when Bacillus typhosus is under consideration. In artificial cultivation water bacteria respond very readily to * Brit. Med. Jour., 1900, ii. p. 1198. 54 BACTERIA IN WATER external conditions. Increase of alkalinity ("01 grams of sodium carbonate added to 10 c.c. of ordinary gelatine) causes the number of colonies to be five or six times greater than that revealed by using ordinary gelatine ; on the other hand, very slightly increasing the acidity of a medium as markedly diminishes the number of bacteria. Advantage is taken of this in culturing the bacillus of typhoid, which is not inhibited by an acid medium. Water may become contaminated with pathogenic bacteria in a variety of ways, as pollutions at the source, in the course, and at the periphery. Gathering grounds are frequently the source of the pollution. The Maidstone typhoid epidemic was an example. Here some of the springs supplying the town with water were con- taminated by several typhoid patients. Frequently on the gathering ground one may find a number of houses the waste and refuse of which will furnish ample surface pollution, which in its turn may readily pass into a collecting reservoir or a well. On one occa- sion the writer investigated the cause of typhoid fever in a large country house in Oxfordshire, and traced it to pollution of the private well by surface washings from the stable quarters. Leak- age of house drains into wells is not an infrequent source of contamination. The same cause is generally operative in cases of pollution of a water supply in its course from the source to the cisterns or taps at the periphery, viz., a sewer or drain leaking into the water supply. Water companies and those responsible for water supply appeal- frequently to hold the opinion that so long as there is sand filtration or subsidence reservoirs, it is unnecessary to consider the gathering ground or possible contamination during transit. But it happens that a frost may completely dislocate the efficient action of a filter, and times of flood may prevent proper sedimentation ; then our dependence for pure water is wholly upon the gathering ground and source. Hence we find water contaminated at its source by polluted wells, by sewage-infected rivers and streams, by drainage of manured fields, by innumerable excremental pollutions over the areas of the gathering grounds, and in transit by careless laying, bad construction and jointing of pipes, and close proximity of such drain pipes to the water supply. In the third place, we may get a water infected at the periphery, in the house itself. Such cases are generally due to two causes: filthy cisterns and pipes or suction. Cisterns per se are more or less indispensable where a constant service does not exist, but they should be inspected from time to time and maintained in a cleanly condition. Suction into the tap has been emphasised by Dr Vivian Poore as a cause of pollution. It is liable to occur whenever a tap is left turned on, and a vacuum is produced in the supply pipe by inter- INTERPRETATIONS OF BACTERIOLOGY 55 mission of the water supply, so that foul gas or liquid is sucked back into the house-pipe. A further point has relation to bacterially polluted water when it has gained entrance to the body. It has been known for some time past that not all waters polluted with disease germs produce disease. As we have before said, this depends upon the infective agent, its quantity and quality, and upon the human body. The body is able in many cases to resist a small dose of poison. It is, however, necessary to infection, especially in water-borne disease, that the tissues shall be in some degree disordered, weakened, or injured. For instance, the perverted action of the stomach influences the acid secretion of the gastric juice, through which bacilli might then pass uninjured. Particularly must this be so in the bacillus of cholera, which is readily killed by the normal acid reaction of the stomach. Hence, in this disease at least, it is the opinion of bacteriologists that the condition of the mucous membrane of the stomach is of primary importance. Metchnikoff has indeed demonstrated the presence of the bacillus of cholera in the intestinal excretion of apparently healthy persons, which shows that they were protected by the resistance of their tissues to the bacilli. Further light has been thrown on this question by the researches of MacFadyen, who has pointed out that suspensions of cholera bacilli in water passed through the stomach untouched, and were thus able to exert their evil influence in other parts of the alimentary canal. When, however, cholera bacilli were suspended in milk, none appeared to escape the germicidal action of the gastric juice. The explanation of this is probably the simple one that the stomach reacted with its secretion of gastric juice only to food (milk), but passed the water on into the lower and more absorptive parts of the alimentary canal. Such a condition of affairs clearly increases the danger due to water-borne germs. The Interpretation of the Finding's of Bacteriology Bacteriology is the most direct and delicate test of the safety of a water for drinking purposes. By it we obtain exact information not alone as to the constitution of a water, but as to its potentiality to cause disease. It is also a more delicate test than a chemical examination.* Klein and others have shown that by bacteriological methods it is possible to detect smaller degrees of sewage pollution than by chemistry. On the other hand, it is useless to expect to learn of the exact chemical constitution of a water by bacteriological methods. Bacteriology must be interpreted by what it can * Clark and Gage state that polluted waters which might become unfit for drinking purposes are more plainly indicated by a single chemical analysis than by a single determination of B. coli. 56 BACTERIA IN WATER do and not by what it cannot; and in a general way it may be said that there are three groups of facts contained in a systematic bacteriological report of water. These findings are concerned with the number of bacteria per c.c., the presence of any organisms of contamination, and the presence of any specific organisms of disease. (1) Number of Bacteria per ex. — It would appear that in the past a great deal too much weight has been attributed to the number of bacteria per c.c. This fact is not of the first importance for two obvious reasons. In the first place there is no standard as to how many bacteria should be present in 1 c.c. of a potable water, and in the second place there is no known means by which this number can be accurately measured. In this country any number of bacteria under one hundred per c.c. is generally considered low. The metropolitan water supply, as consumed, usually contains less than twenty bacteria per c.c. Deep - well waters and spring waters frequently contain very few bacteria. Polluted or surface waters contain thousands of organisms per c.c. More than this, no standard exists. Nor would any numerical standard taken alone be of much value, for the reason that the number of bacteria in water is of comparatively little value apart from a knowledge of the species, and moreover a really accurate record of the number of bacteria per c.c. is not obtainable. Whether the organisms detected be many or few depends upon a variety of external circumstances, such as medium used for cultivation, temperature and period of incubation, length of time of cultivation before counting, or the use or not of a lens when counting. For these reasons it is evident that great reliance cannot be placed upon the number of bacteria per c.c. returned in bacteriological reports, and it is well that should be understood. The only circumstances under which such returns are valuable are (a) when used in a series of examinations of the same water supply, when such returns, if always obtained under the same conditions, are of great comparable value, and (&) when used in the examination of water before and after filtration. In these two circumstances the number of organisms per c.c. is of great value in forming an opinion as to pollution or as to failure of filtration. (2) Presence of Organisms, of Contamination. — In the general bacteriological examination of water this point is perhaps the most important. Judgment must be formed on two facts, namely, the presence of any of the "bacteria of indication," such as B. coli, B. enteritidis sporogenes, streptococci, and the para-colon types (enteritidis, Gaertner, and the chologenes type), and the relative abundance of these species. The latter point is one of importance. The chief organism of indication is B. coli, including under that ORGANISMS OF CONTAMINATION 57 term the typical bacillus and closely allied organisms. When this bacillus can be detected in a small measured quantity of water, that is to say, in 1, 2 or 3 c.c., it is assumed (a) that the organism has gained access to the water from sewage, and (I) that recently, (c) It is further assumed that certain disease-producing bacteria which occur frequently in sewage may also be present in the water, though if present at all in the water, in considerably smaller numbers than B. coli. (d) Further, judging the matter broadly, the higher the number of B. coli the heavier will have been the recent sewage pollution, and the greater the probability of the presence of disease-producing bacteria. Conversely, if B. coli is not present, one may assume with some probability of being correct, that such disease-producing bacteria as the bacillus of typhoid fever will also be absent, and that the particular sample of water under examination might safely be used for drinking purposes. There is difference of opinion as to the exact quantity of a water which must be free from a single specimen of B coli in order that it may be said that the sample is a " safe " one ; but many would in practice accept the standard 1 or 2 c.c. It has already been stated that the presence of B. coli in a water is not of importance, because this organism itself, under the ordinary conditions, is likely to be harmful, but rather because it serves as an index of sewage or surface pollution. In this connection it may be said that a single examination of a water is of practically no value when the results of the bacteriological examination are favourable ; it is only after repeated examination has shown that B. coli 'is absent from the water for a prolonged period, and after local inspection has shown that there are no possible sources of dangerous sewage con- tamination, that one is justified in giving a positive opinion as to the safety of a water. On the other hand, a single bacteriological examination with an unfavourable result will prove the actual occurrence, and suggest the possible recurrence, of sewage contamina- tion, and will necessitate renewed inspection if no obvious source of contamination is known to exist. B. coli is commonly considered as evidence of contamination by sewage, but it is possible for the bacillus to gain access to the water from other sources also. The bacillus is present in the excreta of mammals generally, and has been found in the excreta of birds, and in surface waters there will undoubtedly be a certain amount of contamination caused in this way. The question as to whether any contamination of this kind can be caused by various fishes, and other forms of aquatic life, is not fully established, though Eyre has recently found the B. coli in the excreta of fishes, as well as mammals and birds.* * Lancet, 1904, i., p. 648. 58 BACTERIA IN WATER Most bacteriologists would condemn a water containing the typical B. coli in 1 c.c. as showing signs of sewage pollution. In the case of a recent pollution the presence of B. coli affords therefore a much more delicate test of pollution than any chemical examination which can be made.* B. enteritidis sporogenes is another organism of indication as to sewage pollution, and its presence in bacillary form or as spores is now accepted as showing recent or remote contamination. The presence of streptococcus is held by many bacteriologists to be a sign of sewage contamination, although some contend that the presence of streptococci does not indicate dangerous contamination unless accompanied by B. coli. The following table (p. 59), from the Thirty-fourth Annual Eeport of the Lawrence Sta., 1903, sets forth, in less space and with more accuracy than could be recorded in many words, the relative presence of the chief organisms of contamination, and it is therefore inserted. Lastly, there are a number of organisms which appear to be fre- quently present in waters contaminated with sewage, and are rarely if ever found in pure supplies. The occurrence of such bacteria in a water should arouse suspicion as to its origin or contamination. Among this group of bacteria are B. fluorescens putridus, B. erythro- spores, B. et M. urecey B. pyocyaneus, B. lactis cyanogenus, and B. megaterium. (3) The presence of pathogenic species. — The presence of any pathogenic organisms, in however few numbers, is of course sufficient for the condemnation of a water. For instance, the presence of the bacillus of typhoid fever or the bacillus of cholera at once condemns a water. There are very few authentic records of such organisms being found, and it is therefore necessary to judge of waters by the presence of organisms of contamination. Note. — A water may be considered safe and potable (a) if it contains comparatively few organisms; (b) an absence of organisms capable of fermenting glucose or lactose media; (c) an absence of B. enteritidis sporogenes; and (d) an absence of any pathogenic species, and especially if these conditions are found to exist as a result of several examinations or of periodic examinations. A water should be condemned, as a rule, (a) if it contains a very large number of bacteria per c.c. of whatever kind; (b) if it contains B. coli communis, or B. enteritidis sporogenes or streptococci in 1 c.c. or any such small quantity; (c) if it gives the enteritidis change in milk cultures , or ferments glucose or lactose media. It should be con- demned without hesitation if it contains B. coli and B. enteritidis sporo- genes (or spores), and streptococci, or if it contains any pathogenic organism, in however small a quantity. But in condemning or * See also Fourth Report Roy. Com. Sewage Disposal, 1904, pp. 106-109. ORGANISMS OF CONTAMINATION 59 S V s ? B*P s 3 |»s :b ^ •^ s «g *» SI !^ •1 1° J 'g 'JS ^ 3 gfi! ii r^ §^ 5 4b ^^ K J X < TflfMooomoo 0 CO CO Is, •o-o OOT OOOOOJMOO : o : ^ o (M o ,,, opppppp-^copp OOOOOOOCOCOOrH OS 0 of Ost-OOoO^COt- 0 CO ^ Is, •o-o 001 rH O O O O • O CO CO ~H O CO N J ll •o-o 001 o o 'tf rH to j.-^ >o co ;o : S " . rH 'CM rH rH rH -* CO rH of J~{ o a •o-o t pc^iopp-HHopppp CO CO OS CO J •o-o OOT pvppppppp 0 rt,O ''Sw^W^SfJCJ -MCg>-i1^2gc/5gc Total Number of Sample. Per cent of Samples Posi •s-i-AV Eg J J 60 BACTERIA IN WATER approving a water supply it is important to take all the findings of chemistry, bacteriology, and topography into consideration. The whole history of the sample must be considered, and too much reliance must not be placed upon the mere presence or absence of B. coli, or any single phenomenon or reaction. No ultimate reliance should, as a rule, be placed upon any single test. Natural Purification of Water We have already noticed that rivers purify themselves as they proceed. There are many excellent examples of such self- purification. The Seine as it runs through Paris becomes highly polluted with every sort of filthy contamination. It receives daily about 250,000 c.m. of sewage. But 20 or 30 miles below the city it is found to be even purer than above the city before it received the sewage. In small rivers it is the same, provided the pollution is less in amount. The Thames and the Severn are excellent examples. Whilst authorities differ with regard to the means of self-purification which operate most effectually, all agree that in some way rivers receiving crude sewage are able in a marvellous degree to become pure again. The chief conditions influencing this phenomenon are as follow : — (a) The movement of the water. — It is probable that any beneficial result accruing from this cause is due not to any mechanical factor in the movement, but to the extra surface of water available for oxida- tion processes. Delepine has shown that the effect of agitation is an increase in the number of suspended bacteria which he attributes to the dislodgment of deposit and side adhesions. The greatest amount of purification in his experiments occurred when the rate of flow was about 8 c.m. per hour.* (b) The pressure of the water. — It is believed that the volume of water pressing down upon any given area beneath it weakens the vitality of certain microbes. In support of this theory, it is urged that the number of bacteria capable of developing is less the greater the depth from the surface. Yet it must be remembered that mud at the bottom of a river, or at the bottom of shallow sea, is teeming with living organisms, and there is no evidence to show that pressure in river water ever reaches a degree capable of affecting the life of bacteria. Delepine found that in the Manchester mains increase of pressure did not reduce the number of bacteria. f (c) Light. — We have seen how prejudicial is light to the growth of organisms in culture media. This is so, though to a less extent, in water (see p. 18). Arloing held that sunlight could not pierce * The Natural Purification of Itunning Water, Jour, of Stale Med., 1901, p. 517. f Report to the Manchester Water Works Committee, 1894. NATURAL PURIFICATION OF WATER 61 a layer of water an inch in thickness and still act inirnically on micro-organisms. But Buchuer found that the sun's rays could pass through 15 or 20 inches and yet be bactericidal. This evidence appears contradictory. On the whole, however, authorities agree that the influence of the sun's rays upon water is in some degree bactericidal and causes a diminution in the quantities of organisms after acting for some hours. Especially will this be so when the water is spread out over a wide area and is therefore shallow and stationary, or moving but slowly.* But taken as a whole it may be said that light does not exert a marked influence in water puri- fication. There is, on the other hand, evidence to prove that water in its passage through dark mains of various sizes gradually becomes deprived of a great part of its bacterial contents. (d) Vegetation in water. — Pettenkofer, in his observations upon the Iser below Munich, has shown how algse bring about a marked reduction in the organic matters present in water. Boyce has pointed out that in the river Severn, in addition to the temperature and movement being unfavourable to B. coli and presumably patho- genic bacteria, that (a) lack of pabulum, and (&) antagonism due to the fauna and flora of the river exert an unfavourable influence upon these bacteria. The organic matter so abundant when the river becomes polluted at Shrewsbury is diluted and destroyed lower down stream, and therefore the water becomes purified of bacteria living on the organic matter. Fish, birds, rats, protozoa, and forms of river life generally contribute their share to the consumption of organic pabulum. The water Ranunculus, Spkcerotilus, Lcptomitus lactcus, sewage fungi, chlorophyll containing protophytes, and river plants generally assist in the destruction of organic matter and bacteria.f (e) Dilution. — The pollutions passing into a flowing river are very soon diluted with the large quantities of comparatively pure water always forthcoming. And this, whilst it lowers the percentage of impurity, also raises the percentage of oxygenated water. Delepine has pointed out as a result of artificial experiments that dilution exerts a double effect on the bacterial content of water. In the first place it has the mechanical effect of increasing the space occupied by a definite number of bacteria, and in the second place it causes a diminution in the amount of pabulum present in a given bulk of the impure fluid. Dilution and deposition acting together exert a power- ful influence as purifiers. Clark and Gage of the Lawrence station pointed out in 1903 that the number of B. coli in a polluted river varies in inverse ratio with the dilution of the entering sewage by the river water, and is affected by the temperature, the number of B. coli being larger during the warm weather than in the cold. In * See also Spitta's work on the Spree at Berlin, Arcliw fiir Hyg., vol. xxxviii. t Roy. Com. on Sewage Disposal, Second Report, 1902, pp. 104-109. 62 BACTERIA IN WATER elHuents from water filters the effect upon filter ellbieney of dilution of the water in winter is less marked than the effect of high tem- perature in summer, the work of a filter in warm weather being, of course, more satisfactory than in cold. (f) Sedimentation. — Whilst Pettenkofer attributes self-purifica- tion to oxygenation and vegetation, most authorities are now agreed that it is largely brought about by the subsidence of impure matters, and by their subsequent disintegration at the bottom of the river. Sedimentation and side-adhesion to the banks in rivers and streams of solids in suspension removes a large number of bacteria in the Severn (Boyce). Sedimentation obviously is greatest in still waters. Hence lake water contains as a rule very few bacteria. "The improvement in water during subsidence is the more rapid and pro- nounced the greater the amount of suspended matter initially present" (Frankland). Tils has pointed out that the number of micro-organisms was invariably smaller in the water collected from the reservoir than in that taken from the source supplying the latter. Percy Frankland has demonstrated the same effect of sedimentation by storage as follows : — No. of Colonies in 1 c.c. of Water. 1. Intake from Thames, 25th June 1892 . 1991 2. First small storage reservoir . . 1703 3. Second small storage reservoir . . 1156 4. Large storage reservoir . . . 464 The large reservoir would of course necessitate a prolonged sub- sidence, and hence a greater diminution than in the small reservoirs. Karlinski gives the following distribution of bacteria in the Borka Lake (Herzegovina) : — Bacteria per c.c. Surface water . 4000 Five inches below surface Ten inches below surface Twelve to sixteen inches below surface Bottom when mud was stirred up 1000 600 200 6000 Delepine considers that bacteria die rapidly in the deposit, although their large numbers are evidence of the effect of sedimenta- tion. He examined some water mains after the sediment had been disturbed and also with the sediment undisturbed. The results were as follows : — Sediment undisturbed. Sediment disturbed. 1. 51 living bacteria per c.c. 334 living bacteria per c.c. 2. 356 ,, „ 3164 3. 10 „ „ 852 He concludes (1) that sedimentation is a very important factor of bacterial purification in flowing water, and (2) that the effects of NATURAL PURIFICATION OF WATER 63 sedimentation are most manifest when the flow of water is rapid enough to prevent the accumulation at any point of the products of bacterial multiplication, but not so rapid as to interfere with a comparatively rapid action of gravity.* In the case of a tidal stream the conditions are different, as recently pointed out by Foulerton.j" In such rivers the disease- producing bacteria are deposited not only on the bed of the stream, but also on the mud, or sludge, on the banks, and are uncovered by water at low tide. It now requires only the agency of a fly, feeding first on the organic matter in the sewage-contaminated mud and then on some human food, milk for instance, to convey the bacillus of typhoid fever from the river to some human being. An additional way by which a bacillus of this kind may survive after it has been discharged into a river is by its being deposited on the bed of the stream where there are shell-fish layings. It has been proved that the typhoid bacillus can survive for a considerable time in the liquor contained in the shell of the oyster or the mussel, and in this way it may escape destruction by finding itself once more inside the con- sumer of the shell-fish. Therefore, in the case of sewage discharged into a tidal river, owing to lack of dilution and sedimentation, it is a menace to the inhabitants on the banks in one or both of these ways. The exact degree of danger depends first upon the extent to which the sewage is purified before its discharge into the stream, and secondly upon the distance from the source of pollution at which con- tamination of the water by special sewage bacteria is still appreciable. This principle of sedimentation operates upon all bacteria, which are often carried down on gross particulate matter. The number of B. coli is reduced quite appreciably by storage of water (Clark and Gage). Many species remain in the mud, sand, or other deposit at the bottom of the stream or reservoir. The parasitic organisms die on account of the unfavourable environment. (g) Oxidation. — Many experiments and observations have been made to prove that large quantities of oxygen are used up daily in oxygenating the Thames water. Oxygenated water will come up with the tide and down with the fresh water from above London. There will also be oxygen absorption going on upon the surface of the water, and from these three sources enough oxygen is obtained to oxidise impurities and produce what is really an " effluent." In many smaller streams the opportunity for oxidation is afforded by weirs and falls. Probably all these factors play a part in the self-purification of rivers, but we may take it that oxidation, dilution, and sedimentation are three of the principal agencies. The test of purification is in the * Jour. ofStat. Med., 1901, p. 518. t Report on Pollution of Tidal Ouse, 1903, p. 11. 64 BACTERIA IN WATER number and character of the bacteria at different stages of the river (e.g., see Table of Bacteria in Severn, p. 38). Jordan has pointed out the peculiar value of the reduction of B. coli.* We may here refer in passing to the facts obtainable from the late Sir Edward Frankland's report on Metropolitan water supply in 1894, as they will afford a connecting link between natural purifica- tion and artificial purification. First, judged by the relatively low proportion of carbon to nitrogen, the organic matter present in the water was, as usual, found to be chiefly of vegetable origin. Secondly, an immense destruction of bacteria was effected by storage in subsidence reservoirs. Thirdly, the bacterial quality of the water might differ widely from its chemical qualities. It is, of course, a much finer index of pollution. These three facts are of primary importance in the interpretation of water reports, and it will be well to bear them in mind. Sir E. Frankland also referred to the physical conditions affecting microbial life in river waters, and, as in previous reports, to the importance of changes of temperature, the effect of sunlight, and rate of flow. Eespecting the relative proportion of these factors, he wrote : " The number of microbes in Thames water is determined mainly by the flow of the river, or, in other words, by the rainfall, and but slightly, if at all, by either the presence or absence of sunshine, or a high or low temperature. With regard to the effect of sunshine, the interesting researches of Dr Marshall Ward leave no doubt that this agent is a powerful germicide, but it is probable that the germicidal effect is greatly diminished, if not entirely prevented, when the solar rays have to pass through even a comparatively thin stratum of water before they reach the living organisms." Subsequent investigations have confirmed the im- portance of these broad principles, and from which it is clear that evidence favours the effect of sedimentation and dilution. These two factors in conjunction with filtration are, practically speaking, the methods of artificial water purification, to which reference will now be made. Artificial Purification of Water Sedimentation and Precipitation. — In nature we see this factor in operation in lakes and reservoirs. For example, the water supply of Glasgow is the untreated overflow from Loch Katrine. Purification has been brought about by means of subsidence of impurities. Nothing further is needed. Much of the purification obtained in reservoirs supplying large towns is due to the same factor. Artificially we find it is this factor which is the mechanical purifier of biological impurity in such methods as Clark's process. By this mode "temporary hardness," or that due to soluble * Jour. ofHyg., 1901, p. 293. DUST AND AIR POLLUTION 81 suitable nidus for putrefactive bacteria. The large quantities of bacteria which a decayed tooth contains are easily demonstrated. From the two series of experiments which we have now con- sidered we may gather the following facts : — (a) That air may contain great numbers of bacteria which may be readily inspired. (&) That in health those inspired do not, as a rule, pass beyond the moist surface of the nasal and buccal cavities, except in persons who practice oral instead of nasal respiration. (c) That in the nose and mouth there are various influences of a bactericidal nature at work in defence of the individual. (d) That expired air in normal quiet breathing contains, as a rule, no bacteria whatever. The practical application of these things is a simple one. To, keep air free from bacteria, the surroundings must be moist. Strong acids and disinfectants are not required. Moisture alone will be effectual. Two or three examples at once occur to the mind. Anthrax spores are conveyed from time to time from dried infected hides and skins to the hands or bodies of workers in warehouses in Bradford, Bermondsey, Finsbury, and other places. If the surround- ings are moist and the hides moist, anthrax spores and other bacteria do not remain free in the air. As a matter of actual experience, it has been found that handling dried hair or dried skins leads to more anthrax infection than handling the same articles in a moist condition.* Again, the bacilli (or " spores ") of tuberculosis present in sputum in great abundance cannot infect the air until and unless the sputum dries. So long as the expectorated matter remains on the pavement or handkerchief wet, the surrounding air will derive from it no bacilli of tubercle. But when in the course of time the sputum dries, then the least current of air will at once infect itself with the dried spores or bacilli. It should, however, be remembered that the " cough-spray " and microscopic particles of saliva emitted in shout- ing, heavy breathing through the mouth, etc., have been shown by Fliigge and others to carry the bacilli of tubercle. Such conveyance may, of course, prove a channel of infection between diseased and healthy persons. The typhoid bacillus, too, occupies the same position. Only when the excrement dries can the contained bacteria infect the air. It is of course well known that the common channel of infection in typhoid fever is, not the air, whereas the reverse holds true of tuberculosis. But if it happens that the excrement of patients suffering from typhoid dries, the air may become infected ; if, on the other hand, it passes in a moist state into the sewer, even though untreated with disinfectants, all will be well as regards the surrounding air. * Annual Reports of Medical Inspector of Factories and Workshops, 1902 and 1903. F 82 BACTERIA IN THE AIR A still more remarkable illustration of the effect of a moist perimeter upon the contained bacteria is to be found in sewer air. For long it has been known that air polluted by sewage emanations is capable of giving rise to various degrees of ill-health. These chiefly affect two parts of the body; one is the throat and the other the intestine. Irritation and inflammation may be set up in both or either by sewer air. Such conditions are in all probability produced by a lowering of the resistance and vitality of the tissues, and not by a conveyance of bacteria in sewer air or by any stimulating effect upon bacteria exercised by sewer air. What evidence we have is against such factors. Several series of investigations have been made into the bacteriology of sewer air, amongst others by Uffel- mann, Carnelly and Haldane, and Laws and Andrewes. From their Jabours we may formulate four simple conclusions : — 1. The air of sewers contains very few micro-organisms indeed, sometimes not more than two organisms per litre (Haldane), and generally fewer than the outside air (Laws and Andrewes). 2. There is not, as a rule, intimate relationship between the microbes contained in sewer air and those contained in sewage. Indeed, there is a marked difference which forms a contrast as striking as it is at first sight unexpected. The organisms isolated from sewer air are those commonly present in the open air. Micrococci and moulds predominate, whereas in sewage moulds and micrococci are rare, and bacilli are most numerous. Liquefying bacteria, too, which are common in sewage, are extremely rare in sewer air. Bacillus coli communis, which occurs in sewage from 20,000 to 200,000 per c.c., is altogether absent from sewer air. 3. As a rule it may be said that only when there is splashing in the sewage, or when bubbles are bursting (Carnelly and Haldane), is it possible for sewage to part with its contained bacteria to the air of the sewer. But under these conditions it may part with a considerable number. 4. Pathogenic organisms and those nearly allied to them are found in sewage, but are absent in sewer air. Uffelmann isolated the Staphylococcus pyogenes aureus (one of the organisms of sup- puration), but such a species is exceptional in sewer air. Hence, though sewer air is popularly held responsible for directly conveying virulent micro-organisms of various diseases, there is up to the present no evidence of a substantial nature in support of such views. In 1894, Laws and Andrewes found an average of 2,781,650 bacteria per c.c. in fresh sewage, and in older sewage from 3,400,000 per c.c., to 11,216,000, and they pointed out that temperature and dilution of sewage were determining factors in the number of bacteria present. They consider that sewage may become a medium for the dissemina- tion of the typhoid bacillus, and that sewage-polluted soil may possibly BACTERIA IN SEWER AIR 83 give up germs to the subsoil air, but they are satisfied that the air of sewers themselves does not play any part in the conveyance of the typhoid bacillus.* In passing, mention may be made of some interesting observations recorded by Mr S. G. Shattock on the effect of sewer air upon the toxicity of lowly virulent bacilli of diphtheria. Some direct relation- ship, it has been surmised, exists between breathing sewer air and "catching" diphtheria. Clearly, it cannot be that the sewer air contains the bacillus. But some have supposed that the sewer air has had a detrimental effect by increasing the virulent properties of bacilli already in the human tissues. Two cultivations of lowly virulent B. diphtheria were therefore grown by Mr Shattock in flasks upon a favourable medium over which was drawn sewer air. This was continued for two months in the one case, and five weeks in the other. Yet no increased virulence was secured.f Such experiments require ample confirmation, but even now it may be said that sewer air does not necessarily have a favouring influence upon the virulence of the bacilli of diphtheria. Such experiments do not affect the contrary question of the possibility of sewer air depressing the vitality of the individual, and so allowing even lowly virulent bacilli to do mischief. Of such depression caused by breathing sewer air there is clinical proof, and although sewer-men do not appear to be affected, persons freshly breathing sewer air may be. It should be noted that the bacilli of diphtheria are capable of lengthened survival outside the body, and are readily disseminated by very feeble air - currents. The condition necessary for their existence outside the body for any period above two or three days is moisture. Dried diptheria bacilli soon lose their vitality. It is possible, owing to this fact, that the disease is not as commonly con- veyed by air as, for example, tubercle. 3. The Influence of Gravity upon bacteria in the air may be observed in various ways, in addition to its action within a limited area like a sewer or a room. Miquel found in some investigations in Paris that, whereas on the Eue de Eivoli 750 germs were present in a cubic metre, yet at the summit of the Pantheon only 28 were found in the same quantity of air. Frankland found that air at the top of Primrose Hill contained 9 organisms per ten litres, and air at the bottom 24. On the spire of Norwich Cathedral (310 feet), ten litres of air yielded 7 organisms, on the tower (180 feet) 9, and on the ground 18. At the level of the golden gallery of St Paul's Cathedral he found in every ten litres 11 bacteria, at the stone gallery 34, and in St Paul's Churchyard 70. As Tyndall has pointed out, * Report to the London County Council on the Result of Investigation on the Micro- organisms of Salvage, by J. Parry Laws and F. W. Andrewes, 1894, p. 14. f Pathological Society of London, Transactions, 1897. 84 BACTERIA IN THE AIR even ultra-microscopic cells obey the law of gravitation. This is equally true in the limited areas of a laboratory or warehouse, and in the open air. At high altitudes, the air may be looked upon as practically germ-free, although here again the lighter spores of the mould fungi may cause them to be carried by air currents to a very great height. In the recent researches of Dr Jean Binot of the Pasteur Institute,* 100 litres of air taken at the summit of Mont Blanc did not contain a single, microbe, and the total number of organisms varied between 4 and 11 per metre cube (1000 litres). An examination of the air of the interior of M. Janssen's Observatory, situated on the highest point of Mont Blanc, and taken in two different rooms, gave, on the other hand, 540 and 260 organisms per metre cube. The gradual increase of the number of organisms as descent to lower level takes place is of interest. Thus 6 per metre cube were found in the Grand Plateau, 8 at the Grand Mulet, and 14 at the Plon de 1* Aiguille. Upon the Mer de Glace 23 organisms were found, and 49 at Montan- vert. Graham Smith found that at the top of the Clock Tower of the Houses of Parliament in London there was only about one-third of the number of bacteria found at the ground level. f 4. Air Currents. — Miquel, Pasteur, Cornet, and other workers have shown that the presence of micro-organisms in air depends in part upon air currents, winds, etc. In the month of August, with the wind from the south, i.e. blowing from the country citywards, the number of organisms was found by Miquel to be 40 in the Mont Souris Pare around the Observatory, while at the same moment a record of 14,800 was obtained in the 4th Arrondissement, which may be taken as the centre of Paris, and comprises the surroundings of Notre Dame and of the Hotel de Ville. In the month of June, on the other hand, with the wind blowing from the N.E., i.e. across the city towards Mont Souris, the numbers were, in the 4th Arrondissement, 10,000 per metre cube, and in the Park of Mont Souris itself, 1180 per metre cube. The seasonal variations of the organisms present in the air are also worthy of note, and depend chiefly upon dust and air currents. The following table shows the mean over a period of ten years in the air taken at Mont Souris : — Average per metre cube. Season. Bacteria. Moulds. Winter . Spring . Summer Autumn 170 327 480 195 175 145 210 235 * Communication a VAcad6mie des Sciences de Paris, 17 Mars 1902. f Jour, of Hyg., 1903, p. 513. INFLUENCE OF CARBONIC ACID GAS 85 Similar experiments have been carried out by Frankland, Fliigge, Delalivesse, Neisser, Chick, Andrewes, and others.* The last named conducted some experiments in London streets in 1902, and reported his results to the Pathological Society. He found the number of organisms varied greatly, but no pathogenic species were detected. The four species he isolated were staphylococci, sarcinse, strepto- thricesB, and moulds. Carnelly, Haldane, and Anderson found the ratio of organisms in the air increased according to whether the air was examined on still damp days, windy damp days, still dry days, and windy dry days, and in brief this expresses the findings of most investigators. Some new light has been thrown upon the subject of pathogenic organisms in air by Neisser in his investigations concerning the amount and rate of air currents necessary to convey certain species through the atmosphere. He states that the bacteria causing diphtheria, typhoid fever, plague, cholera, and pneumonia, and possibly the common Streptococcus pyogenes, are incapable of being carried by the molecules of atmospheric dust which the ordinary insensible currents of air can support, whilst Bacillus anthracis, B. pyocyaneus, and the bacillus of tubercle are capable of being aerially conveyed. This work will require further confirmation before being entirely accepted. Finally, some mention may be made of the relationship alleged to exist between the presence of a considerable degree of carbonic acid gas in an atmosphere and the number of bacteria contained in the same atmosphere. As far as may be judged, it would appear that the relationship is but slight. But to illustrate the subject as well as other points of importance in the bacteriology of air, four separate investigations may be mentioned. (i.) Haldane and Osborn, in their inquiry into the ventilation of factories and workshops, made a number of bacteriological examina- tions.f The determinations of bacteria were made by a slightly modified form of Frankland's method. The air was drawn through a sterilised plug of glass wool by means of a brass syringe of known capacity. The glass tubes containing the glass wool plugs were each enclosed in a separate outside sterilised glass tube, with an asbestos plug. In taking the sample of air the inside tube was attached directly to the pump by means of a short piece of stout rubber tubing. The plug was afterwards transferred with the necessary precautions to a shallow, flat-bottomed flask, containing a small quantity of liquefied gelatine, which was shaken so as to disintegrate and spread the glass * See also Jour, of Sanitary Institute (Oct. 1902), vol. xxiii., pt. iii., p. 209-236. The Dust Problem, by Sir J. Crichton-Browne, F.R.S. t First Report of the Departmental Committee appointed to inquire into the Ventilation of Factories and Workshops, 1902. J. S. Haldane, M.D., F.R.S., and E. H. Osborn. 86 BACTERIA IN THE AIR wool. The gelatine having set, the flask was incubated at 20 C. till no further colonies of bacteria or moulds developed. Some of the chief results were as follows : — Cub. Content. CO2 per 10,000" parts. Bacteria and Moulds per Litre of Air. Inside. Outside Air. Bacteria. Moulds. Tailor, Whitechapel. . . ., 67,500 35'8 3-5 17 22 •>1 953 9 '2 3 '5 8 1 •> 750 4'6 3-5 16 2 IS 636 lO'O 3'5 9 8 9 800 7'4 3-5 9 0 London, E. . ... 27,265 14-6 3-5 10 2 London, E.G. 26,460 14-6 3-5 25 3 Capmaker, London, E. . 4,296 23-0 3-5 9 2 Dressmaker, London, W. 21,600 13-2 3-5 8 0 Boot Workshop, London, E. . 8,688 8-8 3-5 25 6 Railway Works, Wilts. . 93,786 4-6 3-5 20 2 Chocolate Factory, Bermondsey Newspaper Printer, Lond. E. C. 12,000 24,098 6'2 16-5 3'5 3-5 8 9 0 0 » »» » 45,259 15-2 3'5 6 6 »» »» » 23,562 25'4 3-5 10 2 Ropemaker, Chatham * . ... 20 6 »» »» • 82 8 »» »» • ... 850 18 * The ventilation of this large room was considerable, but having the effect of keeping dust in suspension rather than expelling it from the room. Three tests made here were all in the same work- place, differing only in degree of dust present. (ii.) In 1902 the writer made some observations in Finsbury on the number of bacteria to be found in the air of underground bakehouses. Four were selected, and the degree of carbonic acid gas was estimated by Pettenkofer's method, and examinations were made as follow of the bacteria pollution. In each of these bakehouses, whilst work was going on, three agar-plates (of 9 '6 inches area each) were exposed for thirty minutes. One plate was placed on the floor, one on the table or trough where the bread was being made, and one on a shelf near the ceiling. After exposure for thirty minutes the plates were re-covered and incubated at blood-heat (37° C.), for exactly twejity- two hours. All the plates then showed abundant growth. Doubtless if the plates had been incubated for forty-eight hours, or three or four days, there would have been a greater growth of colonies, and it is probable also that if some of the plates had been placed at room temperature certain bacteria would have grown which did not appear at blood-heat in twenty -two hours. It is not suggested that these plates provide an adequate record of the bacteria present in the air of these bakehouses. The object was merely to obtain a comparative f>LATE 7. AIR-PLATES EXPOSED IN BAKEHOUSES (30 minutes). (i.) Above-ground Bakehouse (Z.) Agar culture, 22 hours at 37° C. (ii.) Under-ground Bakehouse (C.) Agar culture, 22 hours at 37° C [To face page 86. BACTERIA IN BAKEHOUSE AIR 87 idea of the air of underground bakehouses and above-ground bake- houses in Finsbury. Accordingly, the whole of the 30 plates used in this examination were treated exactly the same in every way, the medium, exposure, and temperature and period of incubation being precisely similar. The results, therefore, whilst of little value as a complete examination of the air, are useful and reliable for comparison with each other. The results were as follow : — Carbonic Acid Gas Average No. of in parts Bacteria per 10,000 (Col well). on each Plate. Underground Bakehouse B 12'0 800 »» >» C 17-5 680 »» »» D 16-9 600 E 13'6 600 Above-ground Bakehouse Outside Air in street Z (C) 4-9 4-5 200 160 Inside the bakehouses there was also an interesting distribution of bacteria as follows : — No. of Bacteria per Plate on Shelf. No. of Bacteria per Plate on Table. No. of Bacteria per Plate on Floor. Underground Bakehouse C Above-ground Shop of C Above-ground Bakehouse Z 490 130 150 720* 150 170* 850 720 300 Illustrations of these two plates are attached (Plate 7). From these figures it will be seen (a) that underground bakehouse air contained at least four times more bacteria than street air around it ; (5) at least three times more bacteria than the air of the shop over it ; and (c) at least three times more bacteria than the above- ground bakehouse. The general result of the investigations was that the air of the typical underground bakehouses examined (1) contained 14*8 volumes per 10,000 of carbonic acid gas, C02 (as compared with 4*9 in above-ground bakehouses and 4*3 in the streets of Finsbury) ; (2) that it contained between 10 and 24 per cent, less moisture than outside air surrounding the bakehouses ; and (3) that it contained at least four times more bacteria than surrounding street air, and three 88 BACTERIA IN THE AIR times more bacteria than the air of a typical above-ground bake- house.* Dr Scott Tebb has also made a somewhat parallel examination of the air of London streets as compared with the railway tube of the City and South London railway.f As the result of a large number of investigations carried out in a similar way to the writer's examinations in bakehouses, the following figures were arrived at : — No. of Air of UU2 per 10,000 parts. Micro-organisms per Plate. The open streets .... 3'8 459 Platforms in Railway Tube . Railway Carriages in Tube 7'9 . 11-6 114 218 (iii.) Thirdly, some of the results of the investigations of Graham Smith into the condition of the atmosphere of the House of Commons may be mentioned.^ He used a modification of Frankland's method of filtering the air to be examined (4'5 litres in each case) through glass wool. An air-pump and a rubber tube of 10 feet in length were used for drawing the air through, and gelatine was used as the medium, the cultures being incubated at 20° C. for five days or longer. The results may be expressed in tabular form in three series : — Experiments on Outside Air, 1 8th July. No. of No. of Moulds Position. Bacteria and Moulds only per litre. per litre. 1. Terrace (ground level) . 2. ,, (10 feet from ground) 4'2 2'9 I'l 1-1 3. „ (20 feet from ground) 3'3 2'0 4. Clock Tower (half-way up) . 5. „ (top) . 6. Peers' Inner Court 1-5 1-3 4-2 0-2 0-6 0'6 7. Star Court 6-0 1'3 Similar experiments were performed in the House itself during a * Report on Bakehouses in Finsbury (Newman), 1902, p. 51. t Report of Public Analyst of Southwark on Condition of Air on City and South London Railway, 1903. W. Scott Tebb, M.D. J Jour. o/Hyg., 1903, pp. 498-513. OF HOUSE OF COMMONS 89 sitting. The air as it entered the House contained 2 '6 bacteria and moulds per litre. Experiments in Debating Chamber, 21st July. Bacteria and Moulds per litre. Position of Examination. Early Series. Early Series. Late Series. Late Series. Average of all Experiments. 7 P.M. 7.15 P.M. 10.30 P.M. 10.45 P.M. 1. Government Side (third seat) . 10-6 5-2 7-0 6'0 7-2 2. „ „ (back seat) . 3. Opposition Side (third seat) . 5-1 4-4 6-2 4-4 5'2 5*2 5-7 4-8 5'4 4. Equalising Chamber 8-0 9'2 8-4 7-7 8-3 (Air before it passes into Debating Chamber.) 5. Roof 8-8 8'2 7-0 6-4 7'6 A third series of examinations was made by Graham Smith of the air in certain committee rooms, etc., as follows : — Experiments in Committee, Dining, and Smoking Booms. No. of No. of Moulds Position of Examination. Bacteria and Moulds only per litre. per litre. 1. Committee Room 9, fans working, 150 persons present, 1.45 P.M. .... 13-3 4-0 2. Committee Room 9, fans working, 150 persons present, 1.45 P.M. . 20-9 4-6 3. Committee Room 1, fans not working, 41 persons present, 1.30 P.M 35-5 5'3 4. Committee Room 1, fans not working, 41 persons present, 1.30 P.M. .... 5. Central Dining-room, 36 persons present, 33'7 4-2 8 P.M. 41-3 8-4 6. Central Dining-room, 36 persons present, 8 P.M. 44-2 12'0 7. Members' Smoking-room, 24 persons present, 9 P.M 30-6 10-6 8. Members' Smoking-room, 24 persons present, 9 P.M. 8-6 4-4 Separate investigation as to the degree of C02 present in the Debating Chamber of the House of Commons revealed between 3-4 parts per 10,000. Dr Graham Smith, as a result of his investigations, arrived at the following conclusions : — 1. The number of micro-organisms in the open space surrounding the House of Parliament is comparatively small (4*2 per litre). 90 BACTERIA IN THE AIR 2. The air in the debating chamber is from a bacteriological point of view remarkably pure (5*8 per litre as average of eleven experiments). 3. The number of bacteria found in the committee, dining, and smoking rooms was several times greater than in the chamber (32 '3 per litre as average of six experiments). 4. No organisms pathogenic to man were isolated, and only a few which were pathogenic to animals. (iv.) Fourthly, in 1902 Andre wes furnished a report to the London County Council on the micro-organisms present in the air of the tube of the Central London Kail way.* The method he employed was in principle that of Frankland, viz., the aspiration by means of a brass syringe (capacity 425 c.c.) of a known volume of air (5 litres), through a plug of glass wool and finely-powdered cane-sugar. The latter retains the micro-organisms, which can be subsequently distributed through a suitable cultivating medium (such as gelatine) in a Petri dish. The gelatine plate-cultures were incubated at 20° C. for four days, when the colonies were counted, examined, and sub-cultured. Special control experiments were made, and search was also made for the presence of anaerobic organisms. The twelve series of experiments yielded results which may be abstracted and tabulated as follow : — a ^ "a i s-o2 4 01 t>> ^ i j§ ^ m •s 1? a O si* 1 •a O g Q g •I 1*1 'o * H d 1 d' o S°o" ^o'oS d O 1 i j? to O 1. Platform . 11.30a.m. 66 30-3 •09 •0019 14 13 2. Lift . 11.45 a.m. 61 30'5 •109 •0028 20 19 3. Carriage (smoking) 4. Tunnel 11.45a.m. 11.45 a.m. 70 67 29-5 30-2 •108 •082 •0026 •0010 51 10 10 8 5. Carriage (non-smoking) 6. Platform . 2.45 p.m. 5.10 p.m. 68 67 30-5 30-1 •111 •103 •0027 •0012 13 30 14 11 7. Platform . 11.40 a.m. 68 30-4 •094 •0010 106 6 8. Carriage (non-smoking) 9. Tunnel 11.20 a.m. 11.15a.m. 72 70 30-2 30-2 •134 •104 •0016 •0018 90 10 13 4 10. Lift .... 11.0 a.m. 66 29-9 •152 •0042 64 9 11. Staircase, Passage 11.0 a.ra. 64 29-8 •078 •0013 13 3 12. Staircase, Passage 11.0 a.m. 68 30-0 •102 •0023 17 7 * Examination of the Atmosphere of the Central London Railway, London County Council, 1902. No. 615. F. W. Andrewes, M.D., F.R.C.P. OF CENTRAL LONDON RAILWAY 91 By way of summary, it may be said that Andrewes found that micro-organisms were present in the air of the Central London Railway in a somewhat greater proportion (as 13 to 10) than in the fresh air outside. The number was high in proportion to the concentration of human traffic, being highest in carriages, platforms, and lifts. The tube air does not compare unfavourably with that known to exist in ordinary dwelling-rooms. No pathogenic germs were discovered, though the number of organisms capable of growing at body temperature was greater in the tube air than in the outside air, and the number of organisms in the tube air was found generally proportional to the degree of chemical contamination, but this rule was subject to striking exceptions. It is evident that bacterial contamination of air, though, as a rule, parallel to chemical con- tamination, may yet vary quite independently as the result of special conditions, such as air currents, which indicates that chemical examination alone cannot always be taken as a trustworthy guide to the contamination of air. The species of bacteria which Andrewes found in the railway air were in the main identical with those occurring in fresh air, and included Staplylococcus cereus flavus et albus, Micrococcus candicans, M. flavus, M. citreus, M. ladis, M. allicans tardissimus, Sarcina lutea, S. flava, S. alba, Bacillus luteus, B. lactis innocuus, Streptothrix Forsteri, S. chromogencs, S. albido-flava, Torula alba, and Saccharomyccs cerevisice. Interpretation of Reports on Bacterial Content of Air In the present position of our knowledge of the bacteriology of air, reports are only of comparative value. Mere numbers of sapro- phytic bacteria in air are not of great service. Up to the present it has not been possible to isolate pathogenic organisms, though such must inevitably occur in air under certain circumstances, though even then probably only in very small numbers. To detect pathogenic organisms, it will probably be necessary to examine large volumes of air, and by methods which will eliminate the common saprophytes. The truth is that the foundations of our knowledge concerning the bacterial flora of the air are only beginning to be laid, and until we can detect, by bacteriological examination, organisms of disease, the bacteriology of air can only be a subject of relative importance. CHAPTEK IV BACTERIA AND FERMENTATION Early Work — Kinds of Fermentation: (1) Alcoholic Fermentation, Ascospores, Pure Cultures, Films ; (2) Acetous Fermentation ; (3) Lactic Acid Fermenta- tion ; (4) Butyric Fermentation ; (5) Ammoniacal Fermentation — Diseases of Wine and Beer: Turbidity, Ropiness, Bitterness, etc. — Industrial Applica- tions of Bacterial Ferments. IT was Pasteur who, in 1857, first propounded the true cause and process of fermentation. The breaking down of sugar into alcohol and carbonic acid gas had been known, of course, for a long period. Since the time of Spallanzani (1776) the putrefactive changes in liquids and organic matter had been prevented by boiling and subse- quently sealing the flask or vessel containing the fluid. Moreover, this successful preventive practice had been in some measure correctly interpreted as due to the exclusion of the atmosphere, but wrongly credited to the exclusion of the oxygen of the air. It was not until the beginning of the present century that authorities modi- fied their view and declared in favour of yeast cells as the agents in the production of fermentation. That this process was due to oxygen per se was disproved by Schwann, who showed that so long as the oxygen admitted to the flask of fermentable fluid was sterilised no fermentation occurred. It was thus obvious that it was not the atmosphere or the oxygen of the atmosphere, but some fermenting agent borne into the flask by the admission of unsterilised air. It was but a step further to establish this hypothesis by adding unsterilised air plus some antiseptic substance which would destroy the fermenting agent. Arsenic was found by Schwann to have this germicidal property. Hence Schwann supported Latour's theory WORK OF PASTEUR 93 that fermentation was due to something borne in by the air, and that this something was yeast. Passing over a number of counter-experiments of Helmholtz and others, we come to the work of Liebig. He viewed the transforma- tion of sugar into alcohol and carbonic acid gas simply and solely as a non-vital chemical process, depending upon the dead yeast com- municating its own decomposition to surrounding elements in contact with it. Liebig insisted that all albuminoid bodies were unstable, and if left to themselves would fall to pieces — i.e. ferment — without the aid of living organisms, or any initiative force greater than dead yeast cells. It was at this juncture that Pasteur intervened to dispel the obscurities and contradictory theories which had been propounded. As in all the conclusions arrived at by Pasteur, so in those relat- ing to fermentation, there were a number of different experiments which were performed by him to elucidate the same point. We will choose one of many in relation to fermentation. If a sugary solution of carbonate of lime is left to itself, it begins after a time to effervesce, carbonic acid is evolved, and lactic acid is formed ; and this latter decomposes the carbonate of lime to form lactate of lime. This lactic acid is formed, so to speak, at the expense of the sugar, which little by little disappears. Pasteur demonstrated the cause of this trans- formation of sugar into lactic acid to be a thin layer of organic matter consisting of extremely small moving organisms. If these be withheld or destroyed in the fermenting fluid, fermentation will cease. If a trace of this grey material be introduced into sterile milk or sterile solution of sugar, the same process is set up, and lactic acid fermentation occurs. Pasteur examined the elements of this organic layer by aid of the microscope, and found it to consist of small short rods of protoplasm quite distinct from the yeast cells which previous investigators had detected in alcoholic fermentation. One series of experiments was accomplished with yeast cells and these bacteria, a second series with living yeast cells only, a third series with bacteria only, and the con- clusions at which Pasteur arrived as the result of these labours he expressed in the following words : — " As for the interpretation of the group of new facts which I have met with in the course of these researches, I am confident that who- ever shall judge them with impartiality will recognise that the alcoholic fermentation is an act correlated to the life and to the organisation of these corpuscles, and not to their death or their putre- faction, any more than it will appear as a case of contact action in which the transformation of the sugar is accomplished in the presence of the ferment without the latter giving or taking anything from it." 94 BACTERIA AND FERMENTATION Pasteur occupied six years (1857-1863) in the further elucidation of his discovery of the potency of these hitherto unrecognised agents, and the establishment of the fact that "organic liquids do not alter until a living germ is introduced into them, and living germs exist everywhere." It must not be supposed that to Pasteur is due the whole credit of the knowledge acquired respecting the cause of fermentation. He did not first discover these living organisms ; he did not first study them and describe them ; he was not even the first to suggest that they were the cause of the processes of fermenta- tion or disease. But nevertheless it was Pasteur who " first placed the subject upon a firm foundation by proving with rigid experiment some of the suggestions made by others." Kinds of Fermentation Although fermentation is nearly always due to a living agent, as proved by Pasteur, the process is conveniently divided into two kinds.* (1) When the action is direct, and the chemical changes involved in the process occur only in the presence of the cell, the latter is spoken of as an organised ferment ; (2) when the action is indirect, and the changes are the result of the presence of a soluble material secreted by the cell, acting apart from the cell, this soluble substance is termed an unorganised soluble ferment, or enzyme. The organised ferments are bacteria or vegetable cells allied to the bacteria ; the unorganised ferments, or enzymes, are ferments found in the secretions of specialised cells of the higher plants and animals. It will be sufficient to illustrate the enzymes by a few of the more familiar examples, such as the digestive agents in human assimilation. This function is performed, in some cases, by the enzyme combining with the substance on which it is acting and then by decomposition yielding the new " digested " substance and regenerating the enzyme ; in other cases, the enzyme, by its molecular movement, sets up molecular movement in the substance it is digesting, and thus changes its condition. These digestive enzymes are as follow : in the saliva, ptyalin, which changes starch into sugar ; in the gastric juice of the stomach, pepsin, which digests the proteids of the food and changes them into more soluble forms ; the pancreatic ferments, amylopsin, trypsin, and steapsin, capable of attacking all classes of food stuffs ; and the intestinal ferments, which have not yet been separated in pure condition. In addition to these, there are ferments in bitter almonds, mustard, etc. Concerning these unorganised ferments we have little further to say. Perhaps the commonest of them all is diastase, which occurs in malt, and to which some reference will be made later. Its function is to convert the starch, which occurs in * E. A, Schafer, F.R.S., Text-book of Physiology, vol. i., p. 312, CONDITIONS OF FERMENTATION 95 barley, into sugar. These unorganised ferments act most rapidly at a high temperature.* We may preface our consideration of the organised ferments by an axiom by which Professor Frankland sums up the vitalistic theory of fermentation, which was supported by the researches of Pasteur : " No fermentation without organisms, in every fermentation a particular organism" From these words it is to be inferred that there is no one particular organism or vegetable cell to be designated the micro- organism of fermentation, but that there are a number of fermenta- tions each started by some specific form of agent. It is true that the chemical changes, induced by organised ferments, depend on the life processes of micro-organisms which feed upon the sugar or other substance in solution, and excrete the product of the fermentation. Fermentation always consists of a process of breaking down of complex bodies, like sugar, into simpler ones, like alcohol and carbonic acid. Of such fermentations we may mention at least five : the alcoholic, by which alcohol is produced ; the acetous, by which wine absorbs oxygen from the air and becomes vinegar ; the lactic, which sours milk ; the butyric, which out of various sugars and organic acids produces butyric acid; and the ammoniacal, which is the putrefactive breaking down of compounds of nitrogen into ammonia. We shall have occasion to refer at some length to this process when considering denitrifying organisms in the soil. There are four chief conditions common to all these five kinds of organised fermentation. They are as follow : — 1. The presence of the special living agent or organism of the particular fermentation under consideration. This, as Pasteur pointed out, differs in each case. 2. A sufficiency of pabulum (nutriment) and moisture to favour the growth of the micro-organism. 3. A temperature at or about blood-heat (35-38° C., 98'5° F.). 4. The absence from the solution or substance of any obnoxious or inimical substances which would destroy or retard the action of the living organism and agent. Many of the products of fermenta- tion are themselves antiseptics, as in the case of alcohol; hence alcoholic fermentation always arrests itself at a certain point. The causal micro-organisms of particular fermentations are of various kinds, belonging, according to botanical classification, to * The unorganised ferments are frequently otherwise classified than as above, according to function. The chief are these : — amylotytic, those which change starch and glycogen (amyloses) into sugars, e.g., ptyalin, diastase, amylopsin (organisms of the subtilis group and the micrococcus of mastitis are said to produce amylolytic ferments) ; proteotytic, those which change proteids into proteoses and peptones, e.g. , trypsin, pepsin ; inversive, those which change maltose, sucrose, and lactose into glucose, e.g. , invertin (various species of bacteria produce inversive ferments) ; coayulative, 1:hose which change soluble proteids into insoluble, e.g., rennet ; steato- lytlc, those which split up fats into fatty acids and glycerine, e.g., steapsin. 9G BACTERIA AND FERMENTATION various different subdivisions of the non-flowering portion of the vegetable kingdom. A large part of fermentation is based upon the growth of a class of microscopic plants termed yeasts. These differ from the bacteria in but few particulars, mainly in their method of reproduction by budding (instead of dividing or sporulating, like the bacteria). Their chemical action is closely allied to that of the bacteria. Secondly, there are special fermentations and modifications of yeast fermentation due to bacteria. Thirdly, a group of somewhat more highly specialised vegetable cells, known as moulds, make a perceptible contribution in this direction. According to Hansen, these latter, so far as they are really alcoholic ferments, induce fermentation, that is, inversion of sugar, not only in solutions of dextrose, but also in maltose. Mucor raeemosus is the only member that is capable of inverting a cane-sugar solution ; Mucor erectus is the most active fermenter, yielding 8 per cent, by volume of alcohol in ordinary beer wort. Both of these will be referred to as they occur in considering the five important fermentations already mentioned. The general microscopic appearance of yeast cells may be shortly stated as follows : They are round or oval cells, and by budding become " daughter " yeast cells. Each consists of a cellulose membrane and clear homogeneous contents. As they perform their function of fermentation, vacuoles, fat-globules, and granules make their appearance in the enclosed plasma. Just as in many vegetable cells a nucleus was detected by Schmitz by means of special methods of staining, so Hansen has found the nucleus in old-yeast cells from " films " without any special staining. 1. Alcoholic Fermentation Cause, yeast ; medium, sugar solutions ; result, alcohol and carbonic acid. It was Caignard-Latour who first demonstrated that yeast cells, by their growth and multiplication, set up a chemical change in sugar solutions which resulted in the transference of the oxygen in the sugar compound from the hydrogen to the carbon atoms, that is to say, in the evolution of carbonic acid gas and the production, as a result, of alcohol. Expressed in chemical formula, the change is as follows : — C6H12O6 (plus the fermenting agent) = 2 C2H0O + 2 CO2. A natural sugar, like grape-sugar, present in the fruit of the vine, is thus fermented. The alcohol remains in the liquid ; the carbonic acid escapes as bubbles of gas into the surrounding air. If we go a step further back, to cane-sugar (which possesses the same elements as grape-sugar, but in different proportions), dissolve it in water, ALCOHOLIC FERMENTATION 97 and mix it with yeast, we get exactly the same result, except that the first stage of the fermentation would be the changing of the cane- sugar into grape-sugar, which is accomplished by a soluble ferment secreted by the yeast cells themselves. If now we go yet one step further back, to starch, the same sort of action occurs. When starch is boiled with a dilute acid it is changed into a gum-like substance, dextrin, and subsequently into maltose, which latter, when mixed with these living yeast cells, is fermented, and results in the evolution of carbonic acid gas and the production of alcohol. In the manu- facture of fermented drinks from cereal grains containing starch there is, therefore, a double chemical process : first, the change of starch into sugar by means of conversion, a chemical change obtained by the action of sulphuric or some other acid, or by the influence of diastase; and secondly, the change of the sugar into alcohol and carbonic acid gas by the process of fermentation, an organic change brought about by the living yeast cells. In all these three forms of alcoholic fermentation the principal features are the same, viz., the sugar disappears ; the carbonic acid gas escapes into the air ; the alcohol remains behind. Though it is true that the sugar disappears, it would be truer still to say that it reappears as alcohol. Sugar and alcohol are built up of precisely the same elements: carbon, hydrogen, and oxygen. They differ from each other in the proportion of these elements. It is obvious, there- fore, that fermentation is really only a change of position, a breaking down of one compound into two simpler compounds. And this redistribution of the molecules of the compound results in the production of some heat. Hence, we must add heat to the results of the work of the yeasts.* It will be necessary subsequently to consider a remarkable faculty which bacteria possess of producing products inimical to their own growth. In some degree this is true of the yeasts, for when they have set up fermentation in a saccharine fluid there comes a time when the presence of the resulting alcohol is injurious to further action on their part. It has become indeed a poison, and, as we have already mentioned, a necessary condition for the action of a ferment is the absence of poisonous substances. This limit of fermentation is reached when the fermenting fluid contains 13 or 14 per cent, of alcohol. The Biology of Yeast. — Having briefly discussed the "medium" and the results, we may now turn to the other side of the matter, and enumerate some of the chief forms of the yeast plant. Jorgensen " When alcohol is pure and contains no water it is termed absolute alcohol. If, however, it is mixed with 16 per cent, of water, it is called rectified spirit, and when mixed with more than half its volume of water (56*8 pej? cent.) it is known as proof spirit. */ G 98 BACTERIA AND FERMENTATION gives more than a score of different members of this family of Saccharomycetes* But before mentioning some of the chief of these, it will be desirable to consider a number of properties common to the genus. The yeast cell is a round or oval body of the nature of a fungus, composed of granular protoplasm surrounded by a definite envelope, or capsule. It reproduces itself, as a general rule, by budding, or gemmation. At one end of the cell a slight swelling or protuberance appears, which slowly enlarges. Ulti- mately there is a constriction, and the bud becomes partly and at last completely separated from the parent cell. In many cases the capsules of the daughter cell and the parent cell adhere, thus forming a chain of budding cells. The character of the cell and its method of reproduction do not depend merely upon the particular species alone, but are also dependent upon external circumstances. There are differences in the behaviour of species towards different media at various temperatures, towards the carbohydrates (especially FIG. 13. — Diagram of Ascospore Formation. FIG. 14. — Gypsum Block. maltose), and in the chemical changes which they bring about in nutrient liquids. In connection with these variations Professor Hansen has pointed out that, whilst some species can be made use of in fermentation industries, others cannot, and some even produce " diseases " in beer.f One of the most remarkable evidences of the adaptability of the yeasts to their surroundings^ and a specific characteristic, occurs in what is termed ascospore formation. If a yeast cell finds itself lacking nourishment or in an unfavourable medium, it reproduces itself not by budding, but by forming spores out of its own intrinsic substance, and within its own capsule. To obtain this kind of spore formation Hansen used small gypsum blocks as the medium on which to grow his yeast cells. Well-baked plaster-of-Paris is mixed with distilled water, and made into a liquid paste. The moulds are made by pouring this paste into cardboard dishes, where it hardens * Micro-organisms and Fermentation. f E. C. Hansen, Studies in Fermentation (Copenhagen), p. 98. PLATE 8. SacchciTomyces cerevisice. Film preparation, x 1000. ASCOSPORE FORMATION IN YEAST. The capsule of the parent cell around the spores is invisible, x 1000. V I