BACTERIA

BACTERIA

ESPECIALLY AS THEY ARE RELATED
TO THE ECONOMY OF NATURE
TO INDUSTRIAL PROCESSES
AND TO THE PUBLIC HEALTH
BY
GEORGE NEWMAN
M.D., F.R.S. (Edin.), D.P.H. (Camb.), etc.
DEMONSTRATOR OF BACTERIOLOGY IN KING'S COLLEGE, LONDON
ILLUSTRATED
NEW YORK
G. P. PUTNAM'S SONS
LONDON
JOHN MURRAY
1899

Copyright, 1899
BY
G. P. PUTNAM'S SONS
The Knickerbocker Press, New York

PREFACE

The present volume is not a record of original work, nor is it a text-book for the laboratory. Theoretical and practical text-books of Bacteriology plentifully exist both in England and America. There are two large works widely used, one by Professor Crookshank, entitled Bacteriology and Infective Diseases, the other by Dr. Sternberg, A Manual of Bacteriology. There are also, in English, a number of smaller works by Abbott, Ball, Hewlett, Klein, Macfarland, Muir and Ritchie, and Sims Woodhead. This book is of a less technical nature. It is an attempt, in response to the editor of the series, to set forth a popular scientific statement of our present knowledge of bacteria. Popular science is a somewhat dangerous quantity with which to deal. On the one hand it may become too popular, on the other too technical. It is difficult to escape the Scylla and Charybdis in such a voyage.

I am much indebted to Professor Crookshank, who, in reading the manuscript, has helped me by many valuable criticisms. My thanks are also due to Sir C. T. D. Acland, Bart., for many kind suggestions, and to Mr. E. J. Spitta, M.R.C.S., who has been good enough to take a number of excellent photo-micrographs for me. Some other illustrations have been derived from the Atlas of Bacteriology, brought out jointly by Messrs. Slater and Spitta. For these also I am glad to have an opportunity of expressing my thanks. It should be understood that the outline drawings are only of a diagrammatic nature.

GEORGE NEWMAN.

London, 1899.

CONTENTS

ILLUSTRATIONS

[Illustrations starred (*) are reproduced by permission of the Scientific Press from Drs. Spitta and Slater's Atlas of Bacteriology.]

INTRODUCTION

We live in a world that is teeming with life. From the earliest times of man that life has been studied and the observations recorded. Thus there has slowly come to be a considerable accumulation of knowledge concerning the various forms (morphology) and functions (physiology) of organised life. This we call the science of biology. It has for its object the study of organic beings, and for its end the knowledge of the laws of their organisation and activity. Slowly, too, in the midst of this gradual accumulation of facts, we begin to see incoherence becoming coherent, chaos becoming cosmos, chance and accident becoming law. Further, the contemplation and comprehension which built up the edifice of modern biology is assuming a new relationship to practical life. Biology can no longer be considered only as an academic occupation or as a theoretical pabulum upon which the leisured mind may ruminate. With rapid strides and determined face this giant of knowledge has marched into the arena of practical politics. The world is opening its eyes to a reality which it had mistaken for a vision.

This application of biology to life and its problems has in recent years been nowhere more marked than in the realm of bacteriology. This comparatively new science, associated with the great names of Pasteur, Koch, and Lister, furnishes indeed a stock illustration of the applicability of pure biology. Turn where we will, we shall find the work of the unseen hosts of bacteria daily claiming more and more attention from practical people. Thus biology, even when clothed in the form of microscopic cells, is coming to occupy a new place in the minds of men. "Its evolution," as Professor Patrick Geddes declares, "forms part of the general social evolution." Certainly its recent rapid development forms a remarkable feature in the practical science of our time. Not only in the diagnosis and treatment of disease, nor even in the various applications of preventive medicine, but in ever-increasing degree and sphere, micro-organisms are recognised as agents of utility or otherwise 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 assist in the fixation of free nitrogen, and they build up assimilable compounds. Their activity assumes innumerable phases and occupies many spheres, more frequently proving themselves beneficial than injurious. They are both economic and industrious in the best biological 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 friends. It cannot achieve everything demanded of it, nor can it furnish a cause for every disease. It is a science fuller of hope than proved and tested knowledge. We are as yet only 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 work have led to alarmist views which have not, by later investigation, been fully supported. Again, 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. But neither the worst fears of the alarmist nor the most sanguine hopes of the alchemist have been verified. Science, fortunately, does not progress at such speed, or with such kindly accommodation. It holds many things in its hands, 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 are expending public moneys in water analysis, in the examination of milk, in the inspection of cows and dairies, in the bacterial treatment of sewage, and in disinfection and other branches of public health administration. Again, the newly formed National Association for the Prevention of Tuberculosis, our increasing colonial possessions with their tropical diseases, even medical science itself, which is year by year becoming more preventive, make an increasing claim upon public opinion. The successful accomplishment and solution of these questions depend in a measure upon an educated public opinion respecting the elements of bacteriology. Recently it was urged that "the first elements of bacteriology should be shadowed forth in the primary school."[1] This course was advised owing to such knowledge being of value to those engaged in dairying. As we shall point out at a later stage, many of the undesirable changes occurring in milk are due to bacteria, even as the success of the butter and cheese industries depends on the use and control of the fermentative processes due to their action. Much of the uncertainty attending the manufacture of dairy products can only be abolished by the careful application of some knowledge of the flora of milk. In Denmark and in Scandinavia the importance of such knowledge is realised and acted upon. America, too, has not been slow to respond to these needs; but in England comparatively little has been done in this direction.[2]

Whilst there can be no doubt as to the advantage of a wider dissemination of the ascertained facts concerning bacteria, it should be borne in mind that only patient, skilled observation and experimental research in well-equipped laboratories can advance this branch of science, or indeed train bacteriologists. The lives of Darwin and of Pasteur adequately illustrate this truth. Yet it is observable that States and public bodies are slow to act upon it, and frequently in the past the most useful and substantial support for the advancement of science has been forthcoming only from private sources. As the world learns its intimate relation to science and the interdependence between its life and scientific truth, it may be expected more heartily to support science.

BACTERIA

CHAPTER I

THE BIOLOGY OF BACTERIA[3]

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 was about forty years old, Holland had already given to the world both microscope and telescope. Robert 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 and 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 instrument 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 "animalcules," 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 "animalcules." 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 infectious diseases. Spallanzani, Pasteur, and Tyndall are the three who more than others contributed to this discovery. Spallanzani was an Italian, who studied at Bologna, and was in 1754 appointed to the chair of logic at Reggio. 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 demonstrated 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 very slightly cracked, and air gained admittance, then invariably both organisms and decomposition appeared. Schwann, the founder of the cell-theory, and Schulze, both showed that if the air gaining access to the flask were either passed through highly heated tubes or drawn through strong acid the result was the same as if no air entered at all, viz., no organisms and no decomposition. The result of these investigations was that scientific men began to believe that no form of life arose de novo (abiogenesis), but had its source in previous life (biogenesis). It remained to 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 "animalculæ." He also demonstrated that these organisms existed in different 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 contamination 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 of infusion. These were boiled, after which they were allowed to cool, and might then be kept for weeks or months without putrefying or revealing the presence of germ life. Here all the conditions of the infusions were natural, except that in the air above them there was no dust.

The sum-total of result arising from all 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 body tissues of animals dead 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 compounds 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 likely 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 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 (like gelatine and agar), and Koch's "plate" method have all contributed not a little to the enormous strides of bacteriology. Owing to its relation to disease, physicians have entered keenly into the arena of bacteriological research. Hence, from a variety of causes, it has come about that the advance has been phenomenal.

We shall now take up a number of points in the biology of bacteria which call for early attention, and which are mostly the outcome of comparatively recent work on the subject.

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 diet. 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 chlorophyll, 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, among which are the bacteria, is obtained by breaking up different forms of carbohydrates. Besides 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 an elementary condition, 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 motion, are free from chlorophyll, and even feed occasionally upon products of decomposition—three physiological characters which 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 they have become more and more limited in classification and restricted in definition.

Even yet, however, we are far from a scientific classification for 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 groups of species. Again, isolated characteristics 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 company 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 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 moulds on the one hand and yeasts 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.
│
┌─────────────────┬─────────────┬───────────┐
Thallophyta. [= The lowest forms of vegetable life.No differentiation into root, stem, or leaf.] │ Protophyta. [= No sexual reproduction.]		Muscineæ		Pteridophyta.		Phanerogamia.
│
┌──────────┐
Algæ. [= Chlorophyll present.]	Fungi. [= No chlorophyll.] │
	┌─┬─┬─┬─┬─┬─┬─┬─┬─┐
		│ Schizomycetes [= multiplication by cell division or by spores] or Bacteria		(1) Coccaceæ[4]—round cells. (2) Bacteriaceæ—rods and threads.
				(3) Leptotricheæ. (4) Cladotricheæ.		Higher Bacteria

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 academic accuracy, we may accept the simple division of the family of bacteria into three chief forms, viz.:—

Lower Bacteria

(1) Round cell form—coccus. (2) Rod 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 higher and complicated functions of bacteria, but it is, as we have said, practically convenient.

Various Forms of Bacteria

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 1/25000 of an inch in 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 micrococcus (μικρος, small). Certain species of micrococci always or almost always occur in pairs, and such a combination is termed a diplococcus. Some diplococci are united by a thin capsule, which may be made apparent by special methods of staining; of others no limiting or uniting membrane can be seen with the ordinary high powers of the microscope.[5] Again, one frequently finds a species which is exactly described by saying that two micrococci 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 micrococci respond to external conditions both as regards size and shape. It must further be borne in mind that a dividing micrococcus assumes the exact appearance 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 (στρεπτος, 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 was once made to divide them into two groups, according 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 to 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 cells united to each other at their sides. The fourth form is constituted by the micrococci being arranged in masses like grapes, the staphylococcus (σταφυλις, 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 sarcina is best classified amongst the cocci, for it is composed of them, in packets of four or multiples of four, produced by division 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 sarcinæ, that 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 may readily be observed.

Sarcina

2. The Bacilli. These consist 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 Spirilla. 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 (like 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 (like 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 reproduction. In the higher bacteria we 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 Streptothrix actinomycotica and Streptothrix maduræ are the organismal cause, respectively, of Actinomycosis and Madura-foot, two diseases which have hitherto been obscure.

Pleomorphism. This term designates an irregular development of a species. Different media and external conditions bring about in protoplasm as susceptible as mycoprotein a variety of morphological phases. These may occur in succession, and represent different stages in the life-history of a bacterium, or they may be involution forms resulting from a change of environment, and occurring as "faults" in the species. In the Bacillus coli, B. typhosus, bacillus of Plague, and B. tuberculosis pleomorphism undoubtedly occurs, and is manifest in the change of shape. This is particularly marked in 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. Speaking generally, the older the culture, the more marked is the variation.

Polymorphism is a term used to define the theory which held that bacteria were one of the intermediate shapes or forms between something lower and something higher in the vegetable kingdom. Neither pleomorphism nor polymorphism is fully understood, and many bacteriologists find shelter from both in the term involution form. What we do know is that the species already named, for example, take on divers forms when placed under different conditions.

Composition. From what we have seen of the diet of micro-organisms, we shall 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, and there is no sign of a nucleus. It possesses a fortunate 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 also may appear from a like cause, which it is necessary to differentiate from spores. 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.

Normal and Pleon-Forms of the Bacillus of Tubash.

Here, then, we have the major parts in the constitution of a bacillus—its body, mycoprotein; its capsule, cellulose. But, further 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 are highly coloured, though these are generally the "innocent" bacteria, in contradistinction to the disease-producing. A pigment has been found which is designated bacterio-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 violaceus (violet); B. et M. aurantiacus (orange); B. et M. luteus (yellow); M. roseus (pink); many of the Sarcinæ; B. aureus (golden-yellow); B. fluorescens liquefaciens et 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. These are of four 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 known 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, with sometimes considerable velocity, and maintained for some seconds or even minutes. (4) A progressive darting movement, by which the bacillus passes over some considerable distance.

The conditions affecting the motion 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 chemiotaxis. These powers have been observed in bacteria by Pfeiffer and Ali-Cohen.

Bacilli, Showing Flagella

The essential condition in the motile bacilli is the presence of flagella.[6] These cilia, or hairy processes, project from the sides or from the ends of the rod, and are freely motile and elastic. Sometimes 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 vibratile 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. Micrococcus agilis (Ali-Cohen) is the only coccus which has flagella and 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 some kinds of yeast, and would be classified by some authorities under spore formation, but in practice it is so obviously a "budding" that it may be so classified. 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 spores of yeasts differs, as Hansen has pointed out, according to their 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 appearance 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 17,000,000 similar individuals; or, expressed in another way, Cohn calculated that in three days, under favourable circumstances, this rate of increase would form a mass of living organisms weighing 7300 tons, and numbering about 4772 billions. Favourable conditions do not occur, fortunately, to allow of such increase, which, of course, can only be roughly estimated. But the above figures 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 exceedingly small 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 of 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, 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 leuconostoc, 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 protoplasm to form a mother cell. An arthrospore is said to be larger, more refractile, and more resistant than an ordinary endospore. Many bacteriologists of repute have declined hitherto to definitely accept arthrospore formation as a proved fact.

Various Forms of Spore Formation and Flagella
A. Stages in formation of spore and its after development. B. Spirillum with terminal flagella.

It is important to note that spore formation in bacteria must not be considered as a method of multiplication. The general rule is undoubtedly that one bacillus produces one spore, and one spore germinates into one bacillus. It is a reproduction, not a multiplication. 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 (b) to the conditions in which it finds itself being unfavourable to its highest vegetative 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 favourable 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.

The position and size of the spore are of considerable use in differential diagnosis. The terminal spore of Bacillus tetani is well known. It is rarely seen at both ends of the bacillus, and hence when poised only at one end causes the "drumstick" appearance. In the bacillus of Quarter Evil the spore is generally towards one end of the rod rather than in the middle; in Malignant Œdema the bacillus in the blood grows out into long threads, and when such a thread sporulates the spore is also near one end. The latter further illustrates the fact that in some species the spore is of greater diameter than the mother cell, and hence dilates the bacillary capsule. The spores of anthrax are typical oval endospores. When free in the field of the microscope, spores must be distinguished 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. Roux and some other eminent bacteriologists suggest that bacteria should be classified according to their method of spore formation.

THE INFLUENCE OF EXTERNAL CONDITIONS ON GROWTH OF BACTERIA

Nutritive Medium. In the very earliest days of the study of micro-organisms it was observed that they mostly congregate where there is pabulum for their nourishment. The reason why fluids such as milk, and dead animal matter such as a carcass, and living tissues such as a man's body contain so 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 constitution frequently meet their requirements. Dead animal matter, too, yields a rich diet for some species (saprophytes). In the living tissues bacteria obtain not only nutriment, but a favourable temperature and moisture. Outside the human body it has been the endeavour of bacteriologists to provide media as like the above as possible, and containing many of the same elements of food. Thus 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,"[7] its favourable temperature, its power or otherwise of liquefaction, the curdling milk, or of gas production, its behaviour towards oxygen, its power of producing indol, pigment, and chemical 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 degeneration which may occur.

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 cc. of water). It is then filtered and sterilised. It will be understood that such an extract is acid. To provide peptone beef-broth, ten grains of peptone and five grains of common salt are added to every litre of acid beef-broth. It is rendered slightly alkaline by the addition of sodium carbonate, and is filtered and sterilised. Glycerine-broth indicates that 6 to 8 per cent. of glycerine has been added after filtration, glucose-broth 1 or 2 per cent. of grape-sugar. This latter is used for anaërobic organisms. The use of broth as a culture medium is of great value. It is undoubtedly our 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 operations. Gelatine is broth solidified by the addition of 100 grams of best French gelatine to the litre. Its advantage is twofold: it is transparent, 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 peptonising 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 melt at 25° C., like gelatine, 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 grams are added to every litre of peptone-broth. Filtration is slow and often difficult, and the result not as transparent as desirable. The former difficulty is avoided by filtering in the Koch's steamer or with a hot-water filter, the latter by the addition of the white of an egg. 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

Potato in a Roux Tube Prepared for Cultivation Glycerine is placed in the bulb of the tube 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. They may then be cut into various shapes convenient for cultivation. Upon any of these forms of solid media the characteristic 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 obtainable from those incorporated in the media; and the water which is required is obtainable from the moisture of the media.

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 inoculating 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 anaërobic cultures will be described at a later stage. When the medium has been inoculated the culture is placed at a temperature which will be favourable. Two standards of temperature are in use in bacteriological laboratories. The one is called room temperature, and varies from 18° C.-20° C.; the other is blood-heat, and varies from 35° C.-38° C. It is true, some species will grow below 18° C., and others above 38° C. The pathogenic (disease-producing) bacteria thrive best at 37° C., and the non-pathogenic at the ordinary temperature of the room. The different degrees of temperature are regulated by means of incubators. For the low temperatures gelatine is chosen; as a medium for the higher temperatures agar.

Staphylococcus Pyogenes Aureus
× 1000

Incubator
(Temperature of blood-heat, registered by thermometer, and regulated by thermo-regulator)

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 moister 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 other species is an exception to this rule, for they prefer a dry medium.

Culture Media Ready for Inoculation

Temperature. Most bacteria grow well at room temperature, but they will grow more luxuriantly and speedily at blood-heat. The optimum temperature is generally that of the natural habitat of the organism. In exceptional cases growth will occur as low as 5° C. or as high as 70° C. Indeed, some have been cooled to-20° C. and-30° C., and yet retained their vitality,[8] whereas some few can grow at 60–70° C. These latter are termed thermophilic bacteria. The average thermal death-point is at or about 50° C.

Inoculating Needles
Plantinum wire fused into glass handles

Light acts as an inhibitory or even germicidal agent. This fact was first established by Downes and Blunt in a memoir to the Royal Society in 1877. They found by exposing cultures to different degrees of sunlight that thus 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 of the rays of the spectrum which acted inimically the blue and violet rays acted most bactericidally, 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, and found that sunlight inhibited the growth temporarily. A large number of experimenters in Europe and England have worked at this fascinating subject since 1877, and though many of their results appear contradictory, we may be satisfied to adopt 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 irrespective 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 increases this action.

(5) The sunlight acts prejudicially upon the culture medium, and thereby complicates the investigation and after-growth.

(6) The time occupied in the bactericidal action depends upon the heat of the sun and the intrinsic vitality of the organism.

(7) With regard to the action of light upon pathogenic organisms, some results have recently been obtained with Bacillus typhosus. 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.

Pasteur's Large Incubator for Cultivation at Room Temperature

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 protective 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. It has been found that the electric light has but little action upon bacteria, though that which it has is similar to sunlight. Recent experiments with the Röntgen rays have given negative 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 powers of phosphorescence. Pflüger 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.

Desiccation. A later opportunity will occur for consideration of the effect of drying upon bacteria. Here it is only necessary to say that, other things being equal, drying diminishes virulence and lessens growth.

Oxygen. 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, aërobic, or whether they flourished most without it, anaërobic. Some have the faculty of growing with or without the presence of oxygen, and are designated as facultative aërobes or anaërobes. As regards the cultivation of anaërobic germs, it is only necessary to say here that hydrogen, nitrogen, or carbonic acid gas may be used in place of oxygen, or they may be grown in a medium containing some substance which will absorb the oxygen.

Modes of Bacterial Action. In considering the specific action of micro-organisms, it is desirable, 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 and upon which it lives. There is a third or intermediate group, known as "facultative," owing to their ability to act as parasites or saprophytes, as the exigencies of their life-history may demand.

Method of Producing Hydrogen by Kipp's Apparatus for Cultivation of Anaërobes (See page [139])

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 themselves. 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.

Anaërobic Culture
(Buckner's Tube)
with Pyrogallic
Solution in Bulb. 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 find different receptions 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 we shall further have to keep clear in our minds that their action is double and complex, and not single or simple. In the first place, we have an infection of the body due 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 versâ. 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 all bacteria—is due to their poisonous products, or toxins, 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 the 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: the tissues or medium, the bacteria, and the products of the bacteria; and in all organismal processes these three elements act and react upon each other.

A word may be said here respecting the much-discussed question of species in bacteria. A species may be defined as "a group of individuals which, however many characters they share with other individuals, agree in presenting one or more characters of a peculiar and hereditary kind with some certain degree of distinctness."[9] Now, as regards bacteria, there is no doubt that separate species occur and tend to remain as separate species. It is true, there are many variations, due in large measure to the medium in which the organisms are growing,—variations of age, adaptation, nutrition, etc.,—yet the different species tend to remain distinct. Involution forms occur frequently, and degeneration invariably modifies the normal appearance. But because of the occurrence of these morphological and even pathological differences it must not be argued that the demarcation of species is wholly arbitrary.

Means of Sterilisation. As this term occurs frequently in even a book of this untechnical 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 some passing allusion to it.

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 perfection. Hence in the investigation involved in bacteriological research heat is the common sterilising agent. A temperature of 70° C. (158° F.) will kill all bacilli; even 58° C. will kill most kinds. Boiling at 100° C. (212° F.) for three minutes will kill anthrax spores, and boiling for thirty to sixty minutes will kill all bacilli and all spores. This difference in the thermal 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 anthrax, which can then grow into a pure culture of anthrax bacilli. Fractional 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 sterilisation. The following methods are those generally used in the laboratory. For dry heat (which is never so injurious to organisms as moist heat)[10]: (a) the Bunsen burner, in the flame of which platinum needles, etc., are sterilised; (b) hot-air chamber, in which flasks and test-tubes are heated to a temperature of 150–170°

Koch's Steam Steriliser C. for half an hour. For moist heat: (c) boiling, for knives and instruments; (d) Koch's steam steriliser, by means of which a crate is slung in a metal cylinder, at the bottom of which the 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 nutriment media upon which bacteria are cultivated 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.-58° C.

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 somewhat differently under differing 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 bacteriology 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 regular function 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 may 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 virulence 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 rôle. 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 pneumoniæ and streptococcus, and association amongst the various suppurative organisms, we cannot doubt that there is an explanation to be found here of many hitherto unsolved 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 Leptotricheæ (Crenothrix, Beggiatoa, Leptothrix, etc.) and the Cladotricheæ (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. 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 considering 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 carcass 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 population, 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 per se that hindered his 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 pyogenes fœtidus. B. anthracis is, in the body, opposed by either B. pyocyaneus or Streptococcus erysipelatis, and yet it is aided in its growth by B. prodigiosus. B. aceti is, under certain circumstances, antagonistic to B. coli communis.

In several of the most recent of the admirable reports of Sir Richard Thorne issued from the Medical Department of the Local Government Board, we have the record of a series of experiments performed by Dr. Klein into this question of the antagonism 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 more in detail at a later stage. There is also in many species, as Dr. Klein has 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 intracellular 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.

Reference 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 white microbe which fills the water-ducts. Wilting vines are full of the same sticky germs. Desiccation and sunlight have a strongly prejudicial effect upon these organisms. 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.

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 produced. 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 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 a too alkaline or too acid medium. A fourth, but rarely necessary, 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.

[CHAPTER II]

BACTERIA IN WATER

In entering upon a consideration of such a common article of use as water, we shall do well to describe in some detail the process by which we systematically investigate the bacteriology of a water, or, indeed, of any similar fluid suspected of bacterial pollution.

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 substance or conditions we wish to examine. Some authorities advise that it is necessary to allow the tap to run for some minutes previous to collecting the sample; but if we desire to examine for lead chemically or for micro-organisms in the pipes biologically, then such a proceeding would be injudicious.[11] Hence 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 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.

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, and in point of fact, as regards distance, this may be so. 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 must 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. 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. Even in almost pure water, at the ordinary temperature of a room, Frankland found organisms multiplied as follows:—

Another series of observations revealed the same sort of rapid increase of bacteria. On the date of collection the micro-organisms per cc. in a deep-well water (in April) were seven. After one day's standing at room temperature the number had reached twenty-one per cc. After three days under the same conditions it was 495,000 per cc. At blood-heat the increase would, of course, be much greater, as a higher temperature is more favourable to multiplication. But this would depend upon the degree of impurity in the water, a pure water decreasing in number 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.

Furthermore, it is desirable to remember that organisms, in an ordinary water, do not continue to increase indefinitely. There is a limit to all things, even to numbers in bacteriology. Cramer, of Zurich, examined the water of the Lake after it had been standing for different periods, with the following results:—

The writer's own experience is entirely in agreement with this cessation of multiplication at or about the end of a week, and the later decline.

Method of Examination. At the outset of a systematic study of a water it is well to observe its physical characters. The colour, if any, should be noted. Suspended matter and deposit may indicate organic or inorganic pollution. If abundant or conspicuous, a microscopic examination of the sediment may be made. The reaction, whether acid, neutral, or alkaline, must be tested, and the exact temperature taken. Any and every fact will help us, perhaps not so much to determine the contents of the water as to interpret rightly the facts we deduce from the entire examination.

Levelling Apparatus for Koch's Plate

At the beginning of the bacteriological work the water should be examined by means of the gelatine plate method. This consists in drawing up into a fine sterilised pipette a small quantity of the water and introducing it thereby into a test-tube of melted gelatine at a temperature below 40° C.[13] It will depend upon the apparent quality of the water as to the exact quantity introduced into the gelatine; about .5 or .1 of a cubic centimetre is a common figure. The stopper is then quickly replaced in the test-tube, and the contents gently mixed more or less equally to distribute the one-tenth cubic centimetre throughout the melted gelatine. A sterilised sheet of glass (4 inches by 3) designated a Koch's plate is now taken and placed upon the stage of a levelling apparatus, which holds iced water in a glass jar under the stage. The gelatine is now poured out over the glass plate, and by means of a sterilised rod stroked into a thin, even film all over the glass. It is then covered with a bell-jar and left at rest to set. The level stage prevents the gelatine running over the edge of the plate; the iced water under the stage expedites the setting of the gelatine into a fixed film. When it is thus set the plate is placed upon a small stand in a moist chamber, and the whole apparatus removed to the room temperature incubator. A moist chamber is a glass dish, in which some filter paper, soaked with corrosive sublimate, is inserted, and the dish covered with a bell-jar. By this means the risks of pollution are minimized, and moisture maintained. In all cases at least two plates must be prepared of the same sample of water, and it is often advisable to make several. They may be made with different media for different purposes, and with different quantities of water, though the same method of procedure is adopted. In a highly polluted water extremely small quantities would be taken, and, vice versâ, in pure water a large quantity.

Moist Chamber in which Koch's Plates are Incubated

When we come to discuss the relation of disease organisms to water, particularly those causing typhoid fever, we shall learn that they are both scarce and intermittent. This point has been dwelt upon frequently by Dr. Klein, and it is clear that such a state of things greatly enhances the difficulties in detecting such bacteria, and he has proposed a simple procedure by which the difficulty of finding the Bacillus typhosus in a large body of water may be met.

Hot Air Steriliser
For the Sterilization of Glass Apparatus, etc.

One or two thousand cubic centimetres of the water under examination are passed through a sterilised Berkefeld filter by means of siphon action or an air-pump. The candle of the filter retains on its outer surface all, or nearly all, the particulate matter contained in the water. The matter thus retained on this outer surface is brushed by means of a sterile brush into 10 or 20 cc. of sterilised water. Thus we have all the organisms contained in two litres of the water reduced into 10 cc. of water. From this, so to speak, concentrated emulsion of the bacteria of the original water, phenol-gelatine plates or Eisner plates (both acid media) may be readily made. In this way we not only catch many bacteria which would evade us if we were content with the examination merely of a few drops of the water, but we eliminate by means of the acid those common water bacteria, like Bacillus fluorescens liquefaciens, which so greatly confuse the issue.

In the course of two or three days the film of gelatine on the plate becomes covered with colonies of germs, and the next step is to examine these quantitatively and qualitatively. We may here insert a simple scheme by which this may be most fully and easily accomplished:—

1. Naked-Eye Observation of the Colonies. By this means at the very outset certain facts may be obtained, viz., the size, elevation, configuration, margin, colour, grouping, number, and kinds of colonies, all of which facts are of importance, and assist in final diagnosis. Moreover, in the case of gelatine plates (it is otherwise in agar) one is able to observe whether or not there is present what is termed liquefaction of the gelatine. Some organisms produce in their development a peptonizing ferment which breaks down gelatine into a fluid condition. Many have not this power, and hence the characteristic is used as a diagnostic feature.

2. Microscopic Examination of Colonies, which confirms or corrects that which has been observed by the naked eye. Fortunately some micro-organisms when growing in colonies produce cultivation features which are peculiar to themselves (especially is this so when growing in test-tube cultures), and in the early stages of such growths a low power of the microscope or magnifying glass facilitates observation.

3. Make cover-glass preparations: (a) unstained—"the hanging drop"; (b) stained—single stains, like gentian-violet, methyl blue, fuchsin, carbol fuchsin, etc.; double stains—Gram's method, Ziehl-Neelsen's method, etc.

The Hanging Drop

This third part of the investigation is obviously to prepare specimens for the microscope. "The hanging drop" is a simple plan for securing the organisms for microscopic examination in a more or less natural condition. A hollow ground slide, which is a slide with a shallow depression in it, is taken, and a small ring of vaseline placed round the edge of the depression. Upon the under side of a clean cover-glass is placed a drop of pure water, and this is inoculated with the smallest possible particle taken from one of the colonies of the gelatine plate on the end of a sterilised platinum wire. The cover-glass is then placed upon the ring of vaseline, and the drop hangs into the space of the depression. Thus is obtained a view of the organisms in a freely moving condition, if they happen to be motile bacteria. As a matter of practice the hollow slide may be dispensed with, and an ordinary slide used.

Drying Stage for Fixing Films

With regard to staining, it will be undesirable here to dwell at length upon the large number of methods which have been adopted. The "single stain" may be shortly mentioned. It is as follows: A clean cover-glass is taken (cleaned with nitric acid and alcohol, or bichromate of potash and alcohol), and a drop of pure sterilised water placed upon it. This is inoculated with the particle of a colony on the end of a platinum needle, and a scum is produced. The film is now "fixed" by slowly drying it over a flame. When the scum is thus dried, a drop of the selected stain (say gentian-violet) is placed over the scum and allowed to remain for varying periods: sarcinæ about thirty seconds; for many of the bacilli three or four minutes. It is then washed off with clean water, dried, and mounted in Canada balsam. The organisms will now appear under the microscope as violet in colour, and will thus be clearly seen.

The "double staining" is adopted when we desire to stain the organisms one colour and the tissue in which they are situated a contrast colour. Some of the details of these methods are mentioned in the Appendix.

4. Sub-culture. The plate method was really introduced by Koch in order to facilitate isolation of species. In a flask it is impossible to isolate individual species, but when the growth is spread over a comparatively large area, like a plate, it is possible to separate the colonies, and this being done by means of a platinum wire, the colonies may be replanted in fresh media; that is to say, a sub-culture may be made, each organism cultivated on its favourite soil, and its manner of life closely watched. We have already mentioned the chief media which are used in the laboratory, and in an investigation many of these would be used, and thus pure cultures would be obtained. Let us suppose that a water contains six kinds of bacteria. On the plate these six kinds would show themselves by their own peculiar growth. Each would then be isolated and placed in a separate tube, on a favourite medium, and at a suitable temperature. Thus each would be a pure culture; i. e., one and only one, species would be present in each of the six tubes. By this simple means an organism can be, we say, cultivated, in the same sort of way as in floriculture. From day to day we can observe the habits of each of our six species, and probably at an early stage of their separated existences we should be able to diagnose what species of bacteria we had found in the water. If not, further microscopic examination could be made, and, if necessary, secondary or tertiary sub-cultures.

5. Inoculation of Animals. It may be necessary to observe the action of supposed pathogenic organisms upon animals. This is obviously a last resource, and any abuse of such a process is strictly limited by law. As a matter of fact, an immense amount of bacteriological investigation can be carried on without inoculating animals; but, strictly speaking, as regards many of the pathogenic bacteria, this test is the most reliable of all. Nor would any responsible bacteriologist be justified in certifying a water as healthy for consumption by a large community if he was in doubt as to the disease-producing action of certain contained organisms.

Types of Liquefaction of Gelatine

By working through some such scheme as the above we are able to detect what quantity and species of organisms, saprophytic or parasitic, a water or similar fluid contains. For, observe what information we have gained. We have learned the form (whether bacillus, micrococcus, or spirillum), size, consistence, motility, method of grouping, and staining reactions of each micro-organism; the characters of its culture, colour, composition, presence or absence of liquefication or gas formation, its rate of growth, smell, or reaction; and lastly, when necessary, the effect that it has upon living tissues. Here, then, are ample data for arriving at a satisfactory conclusion respecting the qualitative estimation of the suspected water.

As to to the quantitative examination, that is fulfilled by counting the number of colonies which appear, say by the third and fourth day, upon the gelatine plates. Each colony has arisen, it is assumed, from one individual, so that if we count the colonies, though we do not thereby know how many organisms we have upon our plate, we do know approximately how many organisms there were when the plate was first poured out, which are the figures we require, and which can at once be multiplied and returned as so many organisms per cubic centimetre. There is, unfortunately, at present no exact standard to which all bacteriologists may refer.

Miquel and Crookshank have suggested standards which allow "very pure water" to contain up to 100 micro-organisms per cc. Pure water must not contain more than 1000, and water containing up to 100,000 bacteria per cc. is contaminated with surface water or sewage. Macé gives the following table:

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 so far impossible to arrive at one 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 estimation of bacteria in water. In Germany and England it is the custom to adopt a stricter limit. Koch in 1893 fixed 100 bacteria per cc. as the maximum number of bacteria which should be present in a properly filtered water. Hence the following has been recognised more or less as the standard:

The personal view of the writer after some experience of water examination would favour a standard of "under 500" being a potable water, if the 500 were of a nature indicating neither sewage pollution nor disease. 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 rapidly liquefying bacteria associated with sewage or surface pollution would, even though present in fewer numbers than a standard, condemn a water. Thus it will be seen that it is impossible to judge alone by the numbers unless they are obviously enormously high.

Wolfhügel's Counter

When we are counting colonies upon a Koch's plate, Wolfhügel's counter may be used. This is a thin plate of glass a size larger than Koch's plates, and upon it are scratched squares, each square being divided into nine smaller squares. The Wolfhügel plate is superimposed upon the Koch's plate, and the colonies counted in one little square or set of squares and multiplied.

Petri's Dish

By using flat, shallow, circular glass dishes, generally known as Petri's dishes, instead of Koch's plates, much manipulation and time is saved, and, on the whole, less risk of pollution occurs. Moreover, these are easily carried about and transferred from place to place. When counting colonies in a Petri's dish it is sufficient to divide the circle into eight equal divisions, and counting the colonies in the average divisions, multiply and reduce to the common denominator of one cc. For example, if the colonies of the plate appear to be distributed fairly uniformly we count those in one of the divisions. They reach, we will suppose, the figure of 60; 60 × 8=480 micro-organisms in the amount taken from the suspected water and added to the melted gelatine from which the plate was made. This amount was .25 cc. Therefore we estimate the number of micro-organisms in the suspected water as 60 × 8=480 × 4= 1920 m.-o. per cc., which is over standard by about 1500. A water might then be condemned upon its quantitative examination alone or qualitative alone, or both. If the quantity were even that of an artesian well, say 4–10 m.-o. per cc., but those four or ten were all Bacillus typhosus, it would clearly be condemned on its quality, though quantitatively it was an almost pure water. If, on the contrary, the water contained 10,000 m.-o. per cc., and none of them disease-producing, it would still be condemned on the ground that so large a number of organisms indicated some kind of organic pollution to supply pabulum for so many organisms to live in one cc. of the water. It is not the number per se which condemns. The large number condemns because it indicates probable pollution with surface water or sewage in order to supply pabulum for so many bacteria per cc.

It should always be remembered that a chemical report and a bacteriological report should assist each other. 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 inspection and investigation of the source of the water, the origin of the organic matter or contamination. Hence a water report should contain not only a record of physical characters, of chemical constituents, and of the presence or absence of micro-organisms, injurious and otherwise, but it should also contain information 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 from watching its history, that is, not from one examination only, but from a series of observations. A water yielding a steady standard of bacterial contents is a much more satisfactory water, from every point of view, than one which is unstable, one month possessing 500 bacteria per cc. and another month 5000. It is obvious that rainfall and drought, soil and trade effluents, will have their influence in materially affecting the bacterial condition of a water.

It is perhaps scarcely necessary to add that we have not in the above account of the examination of water included all, or nearly all, the various methods adopted for acquiring a knowledge of the bacterial contents of the water. Many of these are of too detailed and technical a nature to enter into here. Three points, however, we may touch upon. In the first place, as we have said, the particulate matter out of a large body of water should be concentrated in a small quantity. Accordingly it has become the custom to pass 2000 or 3000 cc. of the suspected water through a Berkefeld filter. When this has been accomplished, by means of a

Berkefeld Filter
In Position for Filtration of Water to be Examined. sterile brush the particulate matter on the candle of the filter is brushed off into 10 or 15 cc. of sterilised water. This simple arrangement is analogous to the use of gravity or centrifugal methods of securing the solid matter in milk. The smaller quantity of water is then readily examined, and scanty germs more readily detected. A second point elaborating the scheme of water examination is the choice of media for sub-culturing. Mere examination on gelatine is not sufficient. Even in making the primary plate cultivations it is well to vary the media—agar, carbol-gelatine, Elsner, etc. But when colonies have appeared upon these plates it is important to sub-culture with accuracy and good judgment upon all or any media—gelatine, agar, broth, potato, milk, blood serum, glucose agar, glycerine agar, etc.—that will reveal the real characters of the bacteria present. A method proposed by Professor Sheridan Delépine is to place some of the suspected water in sterilised test-tubes without further treatment, and incubate at 37° C. for twelve or eighteen hours, and then plate out and estimate the number of bacteria as in the ordinary course. "In polluted water, containing an excess of organic matter," he says, "an extremely rapid multiplication of bacteria is observed. In unpolluted water, containing only water bacteria and a very small amount of organic matter, very little or no multiplication takes place, and the growth of the water bacteria liquefying gelatine is checked to a remarkable extent." Thirdly, by none of these methods should we be able to isolate anaërobic bacteria, and to furnish a complete report these also must receive careful attention.

Apparatus for Filtering Water to Facilitate its Bacteriological Examination

The Bacteriology of Water. In many natural waters there will be found varied contents even in regard to flora alone: algæ, diatoms, spirogyræ, desmids, and all sorts of vegetable detritus. Many of these organisms are held responsible for divers 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 upon the amount of organic pabulum present, and upon certain physical conditions of the water. As we have already seen, bacteria multiply with enormous rapidity. In some species multiplication does not appear to depend on the presence of much organic matter, and, indeed, some can live and multiply in sterilised water: Micrococcus 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 competition of other species monopolising the pabulum. Probably at least one hundred different species of non-pathogenic organisms have been isolated from water. Some species are constantly occurring, and are present in almost all natural waters. Amongst such are B. liquefaciens, B. fluorescens liq., B. fluorescens non-liquefaciens, B. termo, B. aquatilis, B. ubiquitus, and not a few micrococci, etc. Percy Frankland[14] collected water from various quarters at various times and seasons, and some of his results may here be added:

RIVER THAMES WATER COLLECTED AT HAMPTON

Number of Micro-organisms Obtained from 1 cc. of Water.

Again, another example:

RIVER LEA WATER COLLECTED AT CHINGFORD

Number of Micro-organisms Obtained from 1 cc. of Water.

"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."—Frankland.

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:

Professor Percy Frankland also points out how the river Dee affords another example, even more perfect, of pollution and restoration repeated several times until the river becomes almost bacterially pure.

We cannot here enter more fully into the many conditions of a water which affect its bacterial content than to say that it varies considerably with its source, at different seasons, and under different climatic conditions. An enormous increase will occur if the sediment is disturbed, and conversely sedimentation and subsidence during storage will greatly diminish the numbers of bacteria. Sand filtration, plus a "nitrifying layer," will remove more than 90 per cent. of the bacteria. Sea-water contains comparatively few bacteria, and the deeper the water and the farther it is from shore so much less will be the bacterial pollution.

THE CHIEF DISEASE ORGANISMS FOUND IN WATER

We will now consider several of the more important disease-producing bacteria found in water.

Bacillus Typhosus (Eberth-Gaffky). In 1880–81 Eberth announced the discovery of this bacillus in cases of clinical enteric fever. In 1884 it was first cultivated outside the body by Gaffky. Since then other organisms have been held responsible for the causation of enteric (or typhoid) fever. In 1885 the B. coli communis was recognised, and it has been a matter of great debate amongst bacteriologists as to how far these two organisms are the same species, and the typhoid germ merely a higher evolution of the B. coli. The differentiating signs between them will be referred to shortly. Bacteriologists generally regard the Eberth-Gaffky bacillus as the specific cause of the disease, though complete proof is still wanting.

Bacteria of Typhoid Fever

Microscopic Characters (in pure culture). Rods, 2–4 µ long, .5 µ broad, having round ends. Sometimes threads are observable, being 10 µ in length. In the field of the microscope the bacilli differ in length from each other, but are all the same thickness approximately. Round and oval cells constantly occur even in pure culture, and many of these shorter forms of typhoid are identical in morphology with some of the many forms of Bacillus coli. There are no spores. Motility is marked; indeed, in young culture it is the most active pathogenic germ we know. The small forms dart about with extreme rapidity; the longer forms move in a vermicular manner. Its powers of movement are due to some five to twenty flagella of varying length, some of them being much longer than the bacillus itself, though, owing to the swelling of the bacillus under flagellum-staining methods, it is difficult to gauge this exactly. The flagella are terminal and lateral, and are elastic and wavy. Their active contraction produces an evident current in the field of the microscope.

Cultures. This organism may be isolated from ulcerated Peyer's patches in the intestine, from the liver, the spleen, and the mesenteric glands. Owing to the mixture of bacteria found elsewhere, it is generally best to isolate it from the spleen. The whole spleen is removed, and a portion of its capsule seared with a hot iron to destroy superficial organisms. With a sterilised knife a small cut is made into the substance of the organ, and by means of a sterilised platinum wire a little of the pulp is removed and traced over the surface of agar. Agar reveals a growth in about twenty-four hours at 37° C., which is the favourite temperature. A greyish, moist, irregular growth appears, but it is invariably attached to the track of the inoculating needle. On gelatine the growth is much the same, but its irregular edge is, if anything, more apparent. There is no liquefaction and no gas formation. On plates of gelatine the colonies appear large and spreading, with jagged edges. The whole colony appears raised and almost limpet-shaped, with delicate lines passing over its surface. There is an appearance under a low power of transparent iridescence. The growth on potato is termed "invisible," and is of the nature of a potato-coloured pellicle, which looks moist, and may at a late stage become a light brown in colour, particularly if the potato is alkaline. Milk is a favourable medium, and is rendered slightly acid. No coagulation takes place. Broth is rendered turbid.

Micro-pathology. Typhoid fever is an infiltration and coagulation, necrosis, and ulceration of the Peyer's patches in the small intestine of man. The mesenteric glands show the same features, except that there is no ulceration. The spleen is enlarged, and contains the germs of the disease in almost a pure culture. The bacillus is present in the intestinal contents and excreta, particularly so when the Peyer's glands have commenced ulceration. In the blood of the general circulation the bacillus is not demonstrable, except in very rare instances. Typhoid fever is not, like anthrax, a blood disease.

COMPARATIVE FEATURES OF BACILLUS TYPHOSUS AND B. COLI

The two species, Bacillus typhosus and B. coli, agree in possessing the following characters: no spores, no liquefaction of gelatine; both grow well on phenolated gelatine, and in Parietti's broth; both act similarly upon animals, though typhoid fever is not a specific disease of animals.

The Bacillus typhosus, though a somewhat susceptible bacillus, can when dried retain its vitality for weeks. In sewage it is very difficult indeed to detect, and is soon crowded out. Dr. Andrews and Mr. Parry Laws, in their bacterial researches into sewage for the London County Council,[15] found that when they examined specially infected typhoid sewage it was only with extreme difficulty they isolated Eberth's bacillus. In ordinary sewage it is clear such difficulty would be greatly enhanced.

B. Coli Communis

We have pointed out elsewhere the relation between soil and typhoid. In water, even though we know it is a vehicle of the disease, the Bacillus typhosus has been only very rarely detected. The difficulties in separating the bacillus from waters (like that at Maidstone, for example), which appear definitely to have been the vehicle of the disease, are manifold. To begin with, the enormous dilution must be borne in mind, a comparatively small amount of contamination being introduced into large quantities of water. Secondly, the huge group of the B. coli species considerably complicates the issues, for it copiously accompanies the typhoid, and is always able to outgrow it. Further, we must bear in mind a point that is systematically neglected, namely, that the bacteriological examination of a water which is suspected of having conveyed the disease is from a variety of circumstances conducted too late to detect the causal bacteria. The incubation period of typhoid we may take at fourteen days. Let us suppose a town water supply is polluted with some typhoid excreta on the 1st of January. Until the 14th of January there may be no knowledge whatever of the state of affairs. Two or three days are required for notification of cases. Several more days elapse generally before bacteriological evidence is demanded. Hence arises the anomalous position of the bacteriologist who sets to work to examine a water suspected of typhoid pollution three weeks previously. There can be no doubt that these difficulties are very real ones. The solution to the problem will be found in Dr. Klein's dictum that "a water in which sewage organisms have been detected in large numbers should be regarded with suspicion"[16] as the vehicle of typhoid, even though no typhoid bacilli were discoverable. The chief of these sewage bacteria are believed to be Proteus vulgaris, B. coli, P. zenkeri, and B. enteritidis, and they are all nearly related to B. typhosus. The presence of the B. coli in limited numbers is not sufficient to indicate sewage pollution, seeing that it is so widely distributed. But in large numbers, and in company with the other named species, it is almost certain evidence of sewage-polluted water.

It may occur to the general reader that, as the typhoid bacillus is not extremely rare, drinking water may frequently act as a vehicle to carry the disease to man. But, to appreciate the position, it is desirable to bear in mind the following facts: the typhoid bacillus is only found in the human excrement of patients suffering from the disease; it is short-lived; in ordinary waters there exist organisms which can exert an influence in diminishing its vitality; exposure to direct sunlight destroys it; and it has a tendency to be carried down-stream, or in still waters settle at the bottom by subsidence. Even when all the conditions are fulfilled, it must not be forgotten that a certain definite dose of the bacillus is required to be taken, and that by a susceptible person. Into these latter questions of how bacteria produce disease we shall have an opportunity of inquiring at a later stage.

We must now mention several of the special media and tests used in the separation of Bacillus typhosus and B. coli.

1. The Indol Reaction. Indol and skatol are amongst the final products of digestion in the lower intestine. They are formed by the growth, or fermentation set up by the growth, of certain organisms. Indol may be recognised on account of the fact that with nitrous acid it produces a dull red colour. The method of testing is as follows. The suspected organism is grown in pure culture in broth, and incubated for forty-eight hours at 37° C. Two cc. of a 4 per cent. solution of potassium nitrite are added to 100 cc. of distilled water, and about 1 cc. of this is added to the test-tube of broth culture. Now a few drops of concentrated sulphuric acid (unless quite pure, hydrochloric should be used) are run down the side of the tube. A pale pink to dull red colour appears almost at once, and may be accentuated by placing the culture in the blood-heat incubator for half an hour. Much dextrose (derived from the meat of the broth) inhibits the reaction. Bacillus typhosus does not produce indol, and therefore does not react to the test; B. coli and the bacillus of Asiatic cholera do produce indol, and react accordingly. It should be pointed out, however, that the bacillus of cholera also produces nitrites. Hence the addition of acid only to a peptone culture of cholera yields the "red reaction" of indol.

2. Carbolised Gelatine. To ordinary gelatine .05 per cent. of phenol is added. This inhibits many common water bacteria.

3. "_Shake Cultures._" To 10 cc. of melted gelatine a small quantity of the suspected organism is added. The test-tube is then shaken and incubated at 22° C. If the organism is Bacillus coli, the next day reveals a large number of gas-bubbles.

4. Elsner's Medium. This special potassium-iodide-potato-gelatine medium is used for the examination of typhoid excreta. It is made as follows: 500 grams of potato gratings are added to 1000 cc. of water; stand in cool place for twelve hours, and filter through muslin; add 150 grams of gelatine; sterilise and add enough deci-normal caustic soda until only faintly acid; add white of egg; sterilise and filter. Before use add half a gram of potassium iodide to every 50 cc. Upon this acid medium common water bacteria will not grow, but Bacillus typhosus and B. coli flourish.

5. Parietti's Formula consists of—phenol, five grams; hydrochloric acid, four grams; distilled water, 100 cc. To 10 cc. of broth 0.1–0.3 cc. of this solution is added. The tube is then incubated in order to see if it is sterile. If that is so, a few drops of the suspected water are added, and the tube reincubated at 37° C. for twenty-four hours. If the water contains the B. typhosus or B. coli, the tube will show a turbid growth.

6. Widal's Reaction. Mix a loopful of blood from a patient suspected of typhoid fever with a loopful of young typhoid broth culture in a hanging drop on a hollow ground slide. Cover with a cover glass and examine under 1/6-inch objective. If the patient is really suffering from typhoid, there will appear in the hanging drop two marked characteristics, viz., agglutination and immotility. This aggregation, together with loss of motility, is believed to be due to the inhibitory action of certain bacillary products in the blood of patients suffering from the disease. The test may be applied in various ways, and its successful issue depends upon one or two small points in technique into which we cannot enter here, but which the reader will find dealt with in the appendix.

7. Flagella-staining. Special methods must be adopted for staining the flagella of Bacillus typhosus and B. coli. The cover glasses should be absolutely clean, the cultures young (say eighteen hours old), and a diluted emulsion with distilled water must be made in a watch-glass in order to get bacilli discrete and isolated enough. Van Ermengem's Method is as follows:—Place a loopful of the emulsion on a clean cover glass and dry it in the air, fixing it lastly by passing it once or twice through the flame of a Bunsen burner. Place films for thirty minutes in a solution of one part boric acid (2 per cent.) and two parts of tannin (15.25 per cent.), which also contains four or five drops of glacial acetic acid to every 100 cc. of the mixture. Wash in distilled water and alcohol. Then place for five to ten seconds in a 25.5 per cent. solution of silver nitrate. Immediately thereafter, and without washing, treat the cover glass to the following solution for two or three seconds: gallic acid, five grams; tannin, three grams; fused potassium acetate, ten grams; distilled water, 350 cc. After this place in a fresh capsule of silver nitrate until the film begins to turn black. Wash in distilled water, dry, and mount. The process contracts the bacilli somewhat, but the flagella stain well.

The Bacillus coli communis occupies such an important place in all bacteriological investigation that a few words descriptive of it are necessary in this place. The "colon bacillus," as it is termed, appears to be almost ubiquitous in distribution. The idea once held that it belonged exclusively to the alimentary canal or sewage is now discarded. It is one of the most widely distributed organisms in nature, though, as its name implies, its habitat is in the intestinal tract of man and animals. It is an aërobic, non-sporulating, non-liquefying bacillus, about .4 µ in thickness, and twice that measurement in length; hence it often appears oval or egg-shaped. Its motility is in varying degree, occasionally being as active as B. typhosus, but generally much less so. It possesses lateral flagella. On gelatine plates at 20° C. B. coli produces non-liquefying, greyish-white, round colonies; in a stroke culture on the same medium, a luxuriant greyish band, much broader and less restricted to the track of the needle than B. typhosus. In depth of medium or "shake" cultures there is an abundant formation of bubbles of gas (methane or carbon dioxide) in the medium. On potato it produces a light yellow, greasy growth, which must be distinguished from the growth of B. fluorescens liquefaciens, B. pyocyaneus, and several other species on the same medium. If the potato is old or alkaline, the yellow colour may not appear. Milk is curdled solid in from twenty-four to forty-eight hours, and a large amount of lactic acid produced. In broth it produces a uniform turbidity, with later on some sediment and a slight pellicle. It gives the reaction to indol.

It is now the practice to speak of the family of Bacillus coli rather than the individual. The family is a very large one, and shows throughout but few common characters. The morphology readily changes in response to medium, temperature, age, etc. Fermentation of sugar, coagulation of milk, or indeed the indol reaction cannot always be used as final tests as to whether or not the organism is B. coli, for unfortunately some members of the family do not show each of these three features. Most varieties, however, appear to show some motility, a small number of flagella, a typical growth on potato, and develop more rapidly on all media than B. typhosus. These characters, plus one or more of the three features above named, are diagnostic data upon which reliance may be placed.

Cholera. This word is used to cover more a group of diseases rather than one specific well-restricted disease. In recent years it has become customary to speak of Asiatic cholera and British cholera, as if indeed they were two quite different diseases. But, as a matter of fact, we know too little as yet concerning either form to dogmatise on the matter. Until 1884 practically nothing was known about the etiology of cholera. In that year, however, Koch greatly added to our knowledge by isolating a spirillum from the intestine and in the dejecta of persons suffering from the disease.

Cholera has its home in the delta of the Ganges. From this endemic area it spreads in epidemics to various parts of the world, often following lines of communication. It is a disease which is characterised by acute intestinal irritation, manifesting itself by profuse diarrhœa and general systemic collapse, with cramps, cardiac depression, and subnormal temperature. The incubation period varies from only a few hours to several days. In the intestine, and setting up its pathological condition, are the specific bacteria; in the general circulation their toxic products, bringing about the systemic changes. Cholera is generally conveyed by means of water.

The spirillum of Asiatic cholera (Koch, 1884) generally appears, in the body and in artificial culture, broken into elements known as "commas." These are curved rods with round ends, showing an almost equal diameter throughout, and sometimes united in pairs or even a chain (spirillum). The latter rarely occur in the intestine, but may be seen in fluid cultures. The common site for Koch's comma is in the intestinal wall, crowding the lumina of the intestinal glands, situated between the epithelium and the basement membrane, abundant in the detached flakes of mucous membrane, and free in the contents of the intestine. They do not occur in the blood, nor are they distributed in the organs of the body.

The Comma-Shaped Bacilli of Cholera

The bacilli are actively motile, and possess at least one terminal flagellum. The organism is aërobic, and liquefies gelatine. It stains readily with the ordinary aniline dyes. It does not produce spores, though certain refractile bodies inside the protoplasm of the bacillus in old cultures have been regarded as such. The virulence of the bacillus is readily attenuated, and both the virulence and morphology appear to show in different localities and under different conditions of artificial cultivation a large variety of what are termed involution forms. Unless the organism is constantly being sub-cultured, it will die. Acid, even the .2 per cent. present in the gastric juice, readily kills it. Desiccation, 55° C. for ten minutes, and weak chemicals have the same effect. The bacilli, however, have comparatively high powers of resistance to cold. Unless examined by the microscope in a fresh and young stage, it is difficult to differentiate Koch's comma from many other curved bacilli.

Its cultivation characters are not always distinctive. Microscopically the young colonies in gelatine appear as cream-coloured, irregularly round, and granular. Liquefaction sets in on the second day, producing a somewhat marked "pitting" of the medium, which soon becomes reduced to fluid. In the depth of gelatine the growth is very characteristic. An abundant, white, thick growth exactly follows the track of the needle, here and there often showing a break in continuity. Liquefaction sets in on the second day, and produces a distinctive "bubble" at the surface. The liquefied gelatine does not fall from the sides of the tube, as in the Finkler-Prior comma of cholera nostras, but occurs inside the border where the gelatine joins the glass. In the course of a week or two all the gelatine may be reduced to fluid. On agar Koch's comma produces with rapidity a thick, greyish, irregular growth. On potato, especially if slightly alkaline, an abundant brownish layer is formed. Broth and peptone water are excellent media. In milk it rapidly multiplies, curdling the medium, with production of acid. Unlike Bacillus coli, it does not form gas, but, like B. coli, it produces large quantities of indol and a reduction of nitrates to nitrites. Hence the indol test may be applied by simply adding to the peptone culture several drops of strong sulphuric acid, when in the course of several hours, if not at once, there will be produced a pink colour, the "cholera red reaction." Although it readily loses virulence, and its resistance is little, the comma bacillus retains its vitality for considerable periods in moist cultures, upon moist linen, or in moist soil. In cholera stools kept at ordinary room temperature the cholera bacillus will soon be outgrown by the putrefactive bacteria. The same is true of sewage water.

The lower animals do not suffer from any disease at all similar to Asiatic cholera, and hence it is impossible to fulfil the postulate of Koch dealing with animal inoculation. In this respect it is like typhoid. It is, however, provisionally accepted that Koch's bacillus is the cause of the disease. The four or five other bacteria which have from time to time been put forward as the cause of cholera have comparatively little evidence in their support. It is less from these, and more from several spirilla occurring in natural waters, that difficulties of diagnosis arise.

Some hold that, however many comma bacilli be introduced into the alimentary canal, they will not produce the disease unless there is some injury or disease of the wall of the intestine. It need hardly be added that cholera acts, like other pathogenic bacteria, by the production of toxins. Brieger separated cadaverin and putrescin and other bodies from cholera cultures, and other workers have separated a tox-albumen.

Methods of Diagnosis of Cholera:

1. The nature of the evacuations and the appearance of the mucous membrane of the intestine afford striking evidence in favour of a positive diagnosis. Nevertheless it is upon a minute examination of the flakes and pieces of detached epithelium that reliance must be placed. In these flakes will be found in cholera abundance of bacilli having the size, shape, and distribution of the specific comma of cholera. The size and shape have been already touched upon. The distribution is frequently in parallel lines, giving an appearance which Koch described as the "fish-in-stream arrangement." This distribution of comma bacilli in the flakes of watery stools is, when present, so characteristic of Asiatic cholera that it alone is sufficient for a definite diagnosis. But unfortunately it is not always present, and then search for other characters must be made.

2. The appearance of cultivation on gelatine, to which reference has been made, is of diagnostic value.

3. The "cholera red reaction." It is necessary that the culture be pure for successful reaction.

4. Isolation from water is, according to Dr. Klein, best accomplished as follows: A large volume of water (100–500 cc.) is placed in a sterile flask, and to it is added so much of a sterile stock fluid containing 10 per cent. peptone, 5 per cent. sodium chloride, as will make the total water in the flask contain 1 per cent. peptone and .5 per cent. salt. Then the flask is incubated at 37° C. If there have been cholera vibrios in the water, however few, it will be found after twenty-four hours' incubation that the top layer contains actively motile vibrios, which can now be isolated readily by gelatine-plate culture.

5. To demonstrate in a rapid manner the presence of cholera bacilli in evacuations, even when present in small numbers, a small quantity must be taken up by means of a platinum wire and placed in a solution containing 1 per cent. of pure peptone and .5 per cent. sodium chloride (Dunham). This is incubated as in the case of the water, and in twelve hours is filled with a turbid growth, which when examined by means of the hanging drop shows characteristic bacilli.

NATURAL PURIFICATION OF WATER

We have already noticed that rivers purify themselves as they proceed. There are many excellent examples of this self-purification. The Seine as it runs through Paris becomes highly polluted with every sort of filthy contamination. But twenty or thirty miles below the city it is found to be even purer than above the city before it received the city sewage. In small rivers it is the same, provided the pollution is less in amount. Whilst authorities differ with regard to the mode of self-purification, all agree that in some way rivers receiving crude sewage are able in a marvellous degree to become pure again.

The conditions influencing this phenomenon are as follows:

(a) The Movement of the Water. It is probable, however, 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 oxidation processes.

(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 the sea, is teeming with living organisms.

(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. Arloing held that sunlight could not pierce a layer of water an inch in thickness and still act inimically on micro-organisms. But Buchner found that the sun's rays could pass through fifteen or twenty 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 distinctly bactericidal and causes a marked diminution in the quantity of organisms after acting for some hours. Especially will this be so when the water is spread over a wide area and is therefore shallow and stationary, or moving but slowly.

(d) Vegetation in Water. Pettenkofer, in his observations upon the Iser below Munich, has shown how algæ bring about a marked reduction in the organic matters present in water.

(e) Dilution. There can be no doubt in anyone's mind that the pollutions passing into a flowing river are very soon diluted with the large quantities of comparatively pure water always forthcoming. But this, whilst it would lower the percentage of impurity, cannot remove impurities.

(f) Sedimentation. Whilst Pettenkofer attributes self-purification 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 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 pronounced 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:

The large reservoir would of course necessitate a prolonged subsidence, and hence a greater diminution than in the small reservoirs. Many like examples might be cited, but a typical one such as the above will suffice.

(g) Oxidation. Many experiments and observations have been made to prove that large quantities of oxygen are used up daily in oxygenising 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.

We may here digress to refer in passing to the facts obtainable from Sir Edward Frankland's report on Metropolitan water supply in 1894, as they will afford a connecting link between self purification and artificial purification. Judged by the relatively low proportion of carbon to nitrogen, the organic matter present in the water was, as usual, found to be chiefly, if not entirely, of vegetable origin. An immense destruction of bacteria was found to be effected by storage in subsidence reservoirs. The bacterial quality of the water might differ widely from its chemical qualities. 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 refers to the physical conditions affecting microbial life in river waters. The importance of changes of temperature, the effect of sunlight, and rate of flow had been referred to in previous reports. Respecting the relative proportion of these factors, he adds:

"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 a comparatively thin stratum of water before they reach the living organisms."

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, with which we are now in a position to deal.

ARTIFICIAL PURIFICATION OF WATER

Sedimentation and Precipitation. Naturally, we see this factor in action in lakes or 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. Artificially, we find it is this factor which is the mechancial purifier of biological impurity in such methods as Clark's process. By this mode "temporary hardness," or that due to soluble bicarbonate of lime, is converted into insoluble normal carbonate of lime by the addition of a suitable quantity of lime-water. 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 CO₂ to form the insoluble carbonate, which falls as a sediment:

CaCO₃ + CO₂ + CaH₂O₂ (lime-water) = 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 300 or 400 m.-o. per cc. are reduced to 4 or 5 per cc. We shall refer to this same action when we come to speak of bacterial purification of sewage. Alum has been frequently used to purify waters which contain much suspended matter. Five or six grains of alum are added to each gallon of water, with 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, therefore, 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, and affords time for the action of light and the competitive suicidal behaviour of the common water bacteria. Yet, after all, 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 the present 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. 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, like "turbid," "cloudy," etc. It is needless to say that this difference in estimation was due to the introduction by Koch of the gelatine-plate method of examination. As a result of Percy Frankland's work, he formulated the following conclusions as regards the chief factors influencing the number of microbes passing through the filter.

It depends upon:

(1) The Storage Capacity for Unfiltered Water. This, of course, has reference to the advantages, which we have noticed above, of securing a large collection of water previous to filtration for subsidence, etc.

(2) The Thickness of Fine Sand through which Filtration is Carried on. An argument needing no further support, for it is clear, other things being equal, the more sand water passes through the greater the opportunity of leaving its impurities behind.

(3) Rate of Filtration. The slower filtration will be generally the more complete in its results.

(4) Renewal of Filter-Beds. After a certain time the filter-bed becomes worn out and inefficient; 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 1893, Koch brought out his monograph upon Water Filtration and Cholera, and his work had a deservedly great influence upon the whole question. He shows 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 Hamburg. 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-bed which really removed micro-organisms effectively was the slimy organic layer upon the surface. 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 disturbed until it requires removal owing to its impermeability. A filter-bed, as is well known, consists of say three feet of sand and one foot of coarse gravel. The water to be filtered is collected into large reservoirs, where subsidence by gravitation occurs. Thence it is led by suitable channels to the surface of the filter-bed. Having passed through the three or four feet of the bed, it is collected in a storage reservoir and awaits distribution. 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. Chemically also the action is twofold. 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 were 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 harm in drinking water. From what has been said it will be seen that the action of a filter-bed is of a complicated nature. There is (1) subsidence of the grosser particles of impurity in the water; (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. Hence it is wise to allow a new filter-bed to act for a short period (say twenty-four to forty-eight hours) before the filtered water is used for domestic purposes, in order to allow 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 cc., 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. If it be true that efficient sand filtration is a safeguard against pathogenic germs like typhoid and cholera, 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. Filtered water containing more than 100 micro-organisms of any kind per cc. is below the standard in purity, and should on no account 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 places where it was found that a well yielded infected water. Such pollution in a well may be due to 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 with gravel to its highest water level, 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 effect of the remedies we have been discussing upon the number of bacteria is demonstrated in the results which Sir Edward Frankland arrived at in his investigation of London waters.[17]

Mean of Monthly Examinations for the Year

Name of Company.	Source of Supply	M.-o. per cc.				Average per cent. of Micro-organisms Removed by Filtration.
		At Source.	After Storage.	After Filtration.
The Chelsea Co.	Thames at Hampton	16,138	1067		34		98.96
West Middlesex Co.	"	16,138	1788		58		99.40
Southwark & Vauxhall Co.	"	16,138	....		80		97.72
Grand Junction Co.	"	16,138	2500		623 100 96		98.46
Lambeth Co.	"	16,138	7820		75		99.50

The teaching of these figures could, with great ease, be reproduced again and again if such was necessary; but these will suffice 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 here be said, 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 maintained for five 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 filters on the market with, in many cases, but little modification from each other. 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 Dr. Sims Woodhead and Dr. Cartwright Wood 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 to (b) the growing through of the micro-organisms. The conditions which chiefly influence the growth of bacteria through a filter appear to be the temperature, the intermittent 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, from their searching and most able investigation, concluded that the Pasteur-Chamberland

Pasteur-Chamberland Filter
Attached to Water Supply candle filters (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." Pure cholera bacillus in suspension (5000 bacilli to every cc.) and typhoid bacillus in suspension (8000 per cc.) 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 fault of not being a "continuous" steriliser. A certain Parisian filter ("Porcelaine d'Amiante"), made of unglazed porcelain, rendered 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 nominally 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 probably does more harm than good. It appears to add nitrogen and phosphates to water, which are both nutritive substances on which bacteria grow. Moreover it readily absorbs impurities 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 days.