Sir George Newman

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. [=Nochlorophyll.]
	+-----------------+
		Schizomycetes [=multiplication by cell division or by spores] or Bacteria	"	(1) CoccaceÆ4—round cells. (2) BacteriaceÆ—rods and threads.
				(3)LeptotricheÆ. (4)CladotricheÆ.	"	HigherBacteria

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.Micrococcus	2.Diplococcus	3.Streptococcus
4.Staphylococcus	5.Leuconostoc,showingArthrospores
6.Merismopedia	7.Sarcina	8.Bacilli
	9. Spirillum

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 (?????, 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 (st?ept??, 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 (staf????, 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.

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.

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.

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 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.

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.

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.

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.

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.

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 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 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 foetidus. 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.