If this is finally proved to be the case, it will furnish another excellent example of the power existing in bacteria of assimilating an elementary substance.

Most authorities would agree that all absorption of free nitrogen, if by means of bacteria, must be through the roots. As a matter of fact, legumes, especially when young, use nitrogen, like all other plants, derived from the soil. It has been pointed out that, unless the soil is somewhat poor in nitrogen, there appears to be but little assimilation of free nitrogen and but a poor development of root nodules.48 The free nitrogen made use of by the micro-organism is in the air contained in the interstices of the soil. For in all soils, but especially in well-drained and light soils, there is a large quantity of air. Although it is not known how the micro-organisms in legumes utilise free nitrogen and convert it into organic compounds in the tissues of the rootlet or plant, it is known that such nitrogen compounds migrate into the stem and leaves, and so make the roots really poorer in nitrogen than the foliage. But the ratio is a fluctuating one, depending chiefly on the stage of growth or maturity of the plant.

If the nodules from the rootlets of Leguminosæ be examined, the nitrogen-fixing bacteria can be readily seen. The writer has isolated these and grown them in pure culture as follows: The nodules are removed, if possible at an early stage in their growth, and placed for a few minutes in a steam steriliser. This is advisable in order to remove the various extraneous organisms attached to the outer covering of the nodule. They may then be washed in antiseptic solution, and their capsules softened by soaking. When opened with a sterile knife, thick creamy matter exudes. On microscopic examination this is found to be densely crowded with small round-ended bacilli or oval bodies, known as bacteroids. By a simple process of hardening and using the microtome, excellent sections of the nodules can be obtained which show these bacteria in situ. In the central parts of the section may be seen densely crowded colonies of the bacteria, which in some cases invade the cellular capsule of the nodule derived from the rootlet. Aërobic and anaërobic pure cultures of these bacteria were made. In some cases these cultures very closely resembled the feathery growth of the bacillus of anthrax.

4. The Saprophytic Bacteria in Soil. This group of micro-organisms is by far the most abundant as regards number. They live on the dead organic matter of the soil, and their function appears to be to break it down into simpler constitution. Specialisation is probably progressing among them, for their name is legion, and the struggle for existence keen. After we have eliminated the economic bacteria, most of which are obviously saprophytes, the group is greatly reduced. It is also needless to add that of the remnant little beyond morphology is known, for as their function is learned they are classified otherwise. It is probable, as suggested, that many of the species of common saprophytes normally existent in the soil act as auxiliary agents to denitrification and putrefaction. At present we fear they are disregarded in equal measure, and for the same reasons, as the common water bacteria. An excess of either, in soil or water, is not of itself injurious as far as we know; indeed, it is probably just the reverse. It is, however, frequently an index of value as to the amount and sometimes condition of the contained organic matter. The remarks made when considering water bacteria apply here also, viz., that an excess of saprophytes acts not only as index of increase of organic matter, but as at first auxiliary, and then detrimental, to pathogenic organisms. It will require accurate knowledge of soil bacteria generally to be able to say which saprophytic germs, if any, have no definite function beyond their own existence. It may be doubted whether the stern behests of nature permit of such organisms. However that may be, we may feel confident, though at present there are many common bacteria in soil, as also in water, the life object of which is not ascertained, that as knowledge increases and becomes more accurate this special provisional group will become gradually absorbed into other groups having a part in the economy of nature, or in the production of disease. At present the decomposition, denitrifying, nitrifying,49 and nitrogen-fixing organisms are the only saprophytes which have been rescued from the oblivion of ages, and brought more or less into daylight. It is but our lack of knowledge which requires the present division of saprophytes whose business and place in the world is unknown.

5. The Pathogenic Organisms found in Soil. In addition to these saprophytes and the economic bacteria, there are, as is now well known, some disease-producing bacteria finding their nidus in ordinary soil. The three chief members of this group are the bacillus of Tetanus (lockjaw), the bacillus of Quarter Evil, and the bacillus of Malignant Œdema.

Tetanus. The pathology of this terrible disease has during recent years been considerably elucidated. It was the custom to look upon it as "spontaneous," and arising no one knew how; now, however, after the experiments of Sternberg and Nicolaier, the disease is known to be due to a micro-organism common in the soil of certain localities, existing there either as a bacillus or in a resting stage of spores. Fortunately Tetanus is comparatively rare, and one of the peculiar biological characteristics of the bacillus is that it grows only in the absence of oxygen. This fact contributed not a little to the difficulties which were met with in securing its isolation.

Tetanus occurs in man and horses most commonly, though it may affect other animals. There is usually a wound, often an insignificant one, which may occur in any part of the body. The popular idea that a severe cut between the thumb and the index finger leads to tetanus is without scientific foundation. As a matter of fact, the wound is nearly always on one or other of the limbs, and is infected simply because they come more into contact with soil and dust than does the trunk. It is not the locality of the wound nor its size that affects the disease. A cut with a dirty knife, a gash in the foot from the prong of a gardener's fork, the bite of an insect, or even the prick of a thorn have before now set up tetanus. Wounds which are jagged, and occurring in absorptive tissues, are those most fitted to allow the entrance of the bacillus. The wound forms a local manufactory, so to speak, of the bacillus and its secreted poisons; the bacillus always remains in the wound, but the toxins may pass throughout the body, and are especially absorbed by the cells of the central nervous system, and thus give rise to the spasms which characterise the disease. Suppuration generally occurs in the wound, and in the pus thus produced may be found a great variety of bacteria, as well as the specific agent itself. After a few days or, it may be, as much as a fortnight, when the primary wound may be almost forgotten, general symptoms occur. Their appearance is often the first sign of the disease. Stiffness of the neck and facial muscles, including the muscles of the jaw, is the most prominent sign. This is rapidly followed by spasms and local convulsions, which, when affecting the respiratory or alimentary tract, may cause a fatal result. Fever and increased rate of pulse and respiration are further signs of the disease becoming general. After death, which results in the majority of cases, there is very little to show the cause of fatality. The wound is observable, and patches of congestion may be found on different parts of the nervous system, particularly the medulla (grey matter), pons, and even cerebellum. Evidence has recently been forthcoming at the Pasteur Institute to support the theory that tetanus is a nervous disease, more or less allied to rabies, and is best treated by intra-cerebral injection of antitoxin, which then has an opportunity of opposing the toxins at their favourite site. (Roux and Borrel.)

In the wound the bacillus is present in large numbers, but mixed up with a great variety of suppurative bacteria and extraneous organisms. It is in the form of a straight short rod with rounded ends, occurring singly or in pairs of threads, and slightly motile. It has been pointed out that by special methods of staining, flagella may be demonstrated.50 These are both lateral and terminal, thin and thick, and are shed previously to sporulation. Branching also has been described. Indeed, it would appear that, like the bacillus of tubercle, this organism has various pleomorphic forms. Next to the ordinary bacillus, filamentous forms predominate, particularly so in old cultures. Clubbed forms, not unlike the bacillus of diphtheria, may often be seen from agar cultures. Without doubt the most peculiar characteristic of this bacillus is its sporulation. The well-formed round spores occur readily at incubation temperature. They occupy a position at one or other pole of the bacillus, and have a diameter considerably greater than the rod. Thus the well-known "drumstick" form is produced. In practice the spores occur freely in the medium and in microscopical preparation. Like other spores, they are extremely resistant to heat, desiccation, and antiseptics. They can resist boiling for several minutes.

As we have seen, this bacillus is a strict anaërobe, growing only in the absence of oxygen. The favourable temperature is 37° C., and it will only grow very slowly at or below room temperature.

An excellent culture is generally obtainable in glucose gelatine. The growth occurs, of course, only in the depth of the medium, and appears as fine threads passing horizontally outwards from the track of the needle. At the top and bottom of the growth these fibrils are shorter than at the middle or somewhat below the middle. For extraction of the soluble products of the bacillus glucose broth may be used.

In some countries, and in certain localities, the bacillus of tetanus is a very common habitant of the soil, and when one thinks how frequently wounds must be more or less contaminated with such soil, the question naturally arises, How is it that the disease is, fortunately, so rare? Probably we must look to the advance of bacteriological science to answer this and similar questions at all adequately. Much has recently been done in Paris and elsewhere to emphasise the relation which other organisms have to such bacteria as those of typhoid and tetanus. When considering typhoid, we saw that in addition to the presence of the specific germ other conditions were requisite before the disease actually occurred. So in tetanus, Kitasato and others have pointed out that the presence of certain other bacteria, or of some foreign body, is necessary to the production of the disease. The common organisms of suppuration are particularly accused of increasing the virulence of the bacillus of tetanus. How these auxiliary organisms perform this function has not been fully elucidated. Probably, however, it is by damaging the tissues and weakening their resistance to such a degree as to afford a favourable multiplying ground for the tetanus. It is right to state that some authorities hold that they act by using up the surrounding oxygen, and so favouring the growth of tetanus.

Quarter Evil (or symptomatic anthrax) is a disease of animals, produced in a manner analogous to tetanus. It is characterised by a rapidly increasing swelling of the upper parts of the thigh, sacrum, etc., which, beginning locally, may attain to extraordinary size and extent. It assumes a dark colour, and crackles on being touched. There is high temperature, and secondary motor and functional disturbances. The disease ends fatally in two or three days.

Slight injuries to the surface of the skin or mucous membrane are sufficient for the introduction of the causal bacillus. This organism is, like tetanus, an anaërobe, existing in the superficial layers of the soil. From its habitat it readily gains entrance to animal tissues. It has spores, but though they are of greater diameter than the bacillus itself they are not absolutely terminal. Hence they merely swell out the capsule of the bacillus, and produce a club-shaped rod. They form gas while growing in the tissues and in artificial culture. External physical conditions have little effect upon this bacillus, and the dried and even buried flesh retains infection for a very long period of time.

The third disease-producing microbe found naturally in soil is that which produces the disease known as Malignant Œdema. Pasteur called this gangrenous septicœmia. Unlike quarter evil, malignant œdema may occur in man in cases where wounds have become septic. Animals become inoculated with this bacillus from the surface of soil, straw-dust, upper layers of garden-earth, or decomposing animal and vegetable matter.

The bacillus occurs in the blood and tissues as a long thread, composed of slender segments of irregular length. It is motile and anaërobic. The spores are larger than the diameter of the bacillus, and the organism produces gas; so much is this the case in artificial culture, that the medium itself is frequently split up.

Both malignant œdema and symptomatic anthrax are similar in some respects to anthrax itself. There are, however, a number of points for differential diagnosis. The enlargement of the spleen, the non-motility of the bacillus, the enormous numbers of bacilli throughout the body, the square ends, equal inter-bacillary spaces, aërobic growth, and characteristic staining afford ample evidence of anthrax.

The Relation of Soil generally to certain Bacterial Diseases. Recent investigations have, in effect, considerably added to our knowledge of pathogenic germs in soil; and whilst the three species enumerated above are still considered as types normally present in soil, it must not be forgotten that other virulent disease producers either live in the soil or are greatly influenced by its conditions.

Fränkel and Pasteur have both demonstrated the possible presence of anthrax. Fränkel maintained that it could not live there long, and at ten feet below the surface no growth occurred. This may have been due to the low temperature of such a depth. Pasteur held that earthworms are responsible for conveying the spores of anthrax from buried carcasses to the surface, and thus bringing about reinfection. Cholera, too, has been successfully grown in soil, except during winter. The presence of common saprophytes in the soil is prejudicial to the development of the cholera spirillum, and under ordinary circumstances it succumbs in the struggle for existence. From experiments recently conducted for the Local Government Board by Dr. Sidney Martin, evidence is forthcoming in support of the view that the bacillus of typhoid can live in certain soils. Samples of soil polluted with organic matter formed a favourable environment for living bacilli of typhoid for 456 days, whereas in sterilised soil, without organic matter, these organisms lived only twenty-three days. Tubercle also has been kept alive for several weeks in soil.

In passing, a single remark may be made in relation to the long periods during which bacteria can retain vitality in soil. Farm soils have, as is well known, been contaminated with anthrax in the late summer or autumn, and have retained the infectious virus till the following spring, and it has even then cropped up again in the hay of the next season. In 1881 Miquel took some samples of soil at a depth of ten inches, containing six and a half million bacteria per gram. After drying for two days at 30° C., the dust was placed in hermetically sealed tubes, which were put aside in a dark corner of the laboratory for sixteen years. Upon re-examination it is reported that more than three million germs per gram were still found, amongst them the specific bacillus of tetanus. Whether or not there is any fallacy in these actual figures, there is abundant evidence in support of the fact that bacteria, non-pathogenic and pathogenic, can and do retain their vitality, and sometimes even their virulence, for almost incredibly long periods of time.

It is now some years since Sir George Buchanan, for the English Local Government Board, and Dr. Bowditch, for the United States, formulated the view that there is an intimate relationship between dampness of soil and the bacterial disease of Consumption (tuberculosis of the lungs). The matter was left at that time sub judice, but the conclusion has been drawn, and surely a legitimate one, that the dampness of the soil acted injuriously in one of two ways. It either lowered the vitality of the tissues of the individual, and so increased his susceptibility to the disease, or in some way unknown favoured the life and virulence of the bacillus. That is one fact. Secondly, Pettenkofer traced a definite relationship between the rise and fall of the ground water with pollution of the soil and enteric (typhoid) fever.51 A third series of investigations concluded in the same direction, viz., the researches of Dr. Ballard respecting summer diarrhœa. This, it is generally held, is a bacterial disease, although no single specific germ has been isolated as its cause. Ballard demonstrated that the summer rise of diarrhœa mortality does not commence until the mean temperature of the soil, recorded by the four-foot thermometer, has attained 56.4° F., and the decline of such diarrhœa coincides more or less precisely with the fall in soil temperature. This temperature (56.4° F.) is, therefore, considered as the "critical" four-foot earth temperature, that is to say, the temperature at which certain changes (putrefactive, bacterial, etc.) take place in the pores of the earth, with the consequent development of the diarrhœal poison.

After a very elaborate and prolonged investigation on behalf of the Local Government Board, Dr. Ballard formulates the causes of diarrhœa in the following conclusions:52

(a) cause of diarrhœa resides ordinarily in the superficial layers of the earth, where it is intimately associated with the life processes of some micro-organism not yet detected or isolated.

(b) That the vital manifestations of such organism are dependent, among other things, perhaps principally upon conditions of season and the presence of dead organic matter, which is its pabulum.

(c) That on occasion such micro-organism is capable of getting abroad from its primary habitat, the earth, and having become air-borne, obtains opportunity for fastening on non-living organic material, and of using such organic matter both as nidus and as pabulum in undergoing various phases of its life history.

(d) That from food, as also from contained organic matter of particular soils, such micro-organism can manufacture, by the chemical changes wrought therein through certain of its life processes, a substance which is a virulent chemical poison.

Here, then, we have a large mass of evidence from the data collected by Buchanan, Bowditch, Pettenkofer, and Ballard. But much of this work was done anterior to the time of the application of bacteriology to soil constitution. Recently the matter has received increased attention from various workers abroad, and in England from Dr. Sidney Martin, Professor Hunter Stewart, Dr. Robertson, and others. The greater part of this work we cannot here consider. But some reference must be made to Dr. Robertson's admirable researches into the growth of the bacillus of typhoid in soil. By experimental inoculation of soil with broth cultures, he was able to isolate the bacillus twelve months after, alive and virulent. He concludes that the typhoid organism is capable of growing very rapidly in certain soils, and under certain circumstances can survive from one summer to another. The rains of spring and autumn or the frosts and snows of winter do not kill them off so long as there is sufficient organic pabulum. Sunlight, the bactericidal power of which is well known, had, as would be expected, no effect except upon the bacteria directly exposed to its rays. The bacillus typhosus quickly dies out in the soil of grass-covered areas. Dr. Robertson holds that the chief channel of infection between typhoid-infected soil and man is dust. As in tubercle and anthrax, so in typhoid, dried dust or excreta containing the bacillus is the vehicle of disease.

Hitherto we have addressed ourselves to those diseases the known causal organisms of which reside, normally or abnormally, in the soil. But closely allied to these matters connected with the rôle of pathogenic bacteria in soil is the question of what has been termed the miasmatic influence of soil. The term "miasm" has had an extensive and somewhat diffuse application in medical science. It may happen in the future that typhoid will be classified strictly as a miasmatic disease. But at present, in the transition state of the science, it would hardly be justifiable to classify typhoid with a typically miasmatic disease like malaria. Yet it is clear that mention should here be made of a group of diseases of which malaria is the type, and of which the tropics generally are the native land. The bacterial etiology of the group is by no means worked out. The cause of malaria alone is not yet a closed subject. However the details of the etiology of this group finally arrange themselves, there is little doubt of two facts, viz., the diseases are probably produced by bacteria or allied protozoa, and soil plays an important part in their production.

From what has been said, it will be seen that though a considerable amount of knowledge has been obtained respecting bacteria in the soil, it may be conjectured that actually there is still a great deal to ascertain before the micro-biology of soil is in any measure complete or even intelligent. The mere mention of tetanus and typhoid in the soil, and their habits, nutriment, and products therein, not to mention the work of the economic bacteria, is to open up to the scientific mind a vast realm of possibility. It is scarcely too much to say that a fuller knowledge of the part which soil plays in the culture and propagation of bacteria may suffice to revolutionise the practice of preventive medicine. Truly, our knowledge at the moment is rather a heterogeneous collection of isolated facts and theories, some of which, at all events, require ample confirmation; still, there is a basis for the future which promises much constructive work.

CHAPTER VI

BACTERIA IN MILK, MILK PRODUCTS, AND OTHER FOODS

Injurious micro-organisms in foods are, fortunately for the consumers, usually killed by cooking. Vast numbers are, as far as we know, of no harm whatever. Alarming reports of the large numbers of bacteria which are contained in this or that food are generally as irrelevant as they are incorrect. Bacteria, as we have seen, are ubiquitous. In food we have abundance of the chief thing necessary to their life and multiplication—favourable nutriment. Hence we should expect to find in uncooked or stale food an ample supply of saprophytic bacteria. There was much wholesome truth in the assertions made some two years ago by the late Professor Kanthack, to the effect that good food as well as bad frequently contained large numbers of bacteria, and often of the same species. It is well that we should become familiarised with this idea, for its accuracy cannot be doubted, and its usefulness at the present time may not be without its beneficial effect.

Nevertheless, it is well we should know the bacterial flora of good and bad foods for at least two reasons. First, there is no doubt whatever that a considerable number of cases of poisoning can be traced every year to food containing harmful bacteria or their products. To several of the more notorious cases we shall have occasion to refer in passing. Secondly, we may approach the study of the bacteriology of foods with some hope that therein light will be found upon some important habits and effects of microbes. There can be little doubt that food-bacteria afford an example of association and antagonism of organisms to which reference has already been made. Any information that can be gleaned to illumine these abstruse questions would be very welcome at the present time. But there is a still further, and possibly an equally important, point to bear in mind, namely, the economic value of microbes in food. In a short account like the present it will be impossible to enter into hypotheses of pathology, but we shall at least be able to consider some of these interesting experiments which have been conducted in the sphere of beneficial bacteria.

The injurious effects of organisms contained in foods has been elucidated by the excellent work of the late Dr. Ballard. From the careful study of a number of epidemics due to food poisoning, this patient observer was able, without the aid of modern bacteriology, to arrive at a simple principle which must not be forgotten. Food poisoning is due either to bacteria themselves or to their products, which are contained in the substance of the food. In cases of the first kind, bacteria gaining entrance to the human alimentary canal, set up their specific changes and produce their toxins, and by so doing in course of time bring about a diseased condition, with its consequent symptoms. On the other hand, if the products, sometimes called ptomaines, are ingested as such, the symptoms set up by their action in the body tissues appear earlier. From these facts Dr. Ballard deduced the simple principle that if there is no incubation period or, at all events, a comparatively short space of time between eating the poisoned food and the advent of disease, the agents of the disease are products of bacteria. If, on the other hand, there is an incubation period, the agents are probably bacteria.

It is necessary to mention two other facts. Dr. Cautley53 has recently been engaged in isolating from poisoned foods the different species of bacteria present. It would appear that these are limited, as a rule, to two or three kinds. As regards disease, the organisms of suppuration are the most common. Liquefying or fermentative bacteria are frequently present, the Proteus family being well represented. In addition there are, according to circumstances, a number of common saprophytes. Now, as we have pointed out, these organisms may act injuriously by some kind of cooperation, or they may by themselves be harmless, and pathological conditions be due to the occasional introduction of pathogenic species.

The other fact, requiring recognition from anyone who proposes to study the bacteriology of foods, is that a certain appreciable amount of the responsibility for food poisoning rests with the tissues of the individual ingesting the food. There is ample evidence in support of the fact that not all the persons partaking of infected food suffer equally, and occasionally some escape altogether. We know little or nothing of the causes of such modification in the effect produced. It may be due to other organisms, or chemical substances already in the alimentary canal of the individual, or it may be due to some insusceptibility or resistance of the tissues. Be that as it may, it is a matter which must not be neglected in estimating the effects of food contaminated with bacteria or their products.

Milk. There are few liquids in general use which contain such enormous numbers of germs as milk. To begin with, milk is in every physical way admirably adapted to be a favourable medium for bacteria. It is constituted of all the chief elements of the food upon which bacteria live. It is frequently at a temperature favourable to their growth. It is par excellence an absorptive fluid. A dish of ordinary water and a dish of newly drawn milk laid side by side, and under similar conditions of temperature, will rapidly demonstrate the difference in degree of absorptivity between the two fluids. Yet, whilst this general fact is true, we must emphasise at the outset the possibility and practicability of securing absolutely pure sterile milk. Recently some milking was carried out under strict antiseptic precautions, with the above sterile result. The udder was thoroughly cleansed, the hands of the milker washed with corrosive sublimate and then pure water, the vessels which were to receive the milk had been carefully sterilised, and the whole process was carried out in strict cleanliness. The result was that the sample of milk remained sweet and good and contained no germs. It should be stated that the first flow of milk, washing out the milk-ducts of the udder, was rejected. This fact of the sterility of cleanly drawn milk is not a new one, and has been established by many bacteriologists. Milk, then, is normally a sterile secretion. How does it gain its enormously rich flora of bacteria?

Sources of Pollution of Milk. These are various, and depend upon many minor circumstances and conditions. For all practical purposes there are three chief opportunities between the cow and the consumer when milk may become contaminated with bacteria:

1. At the time of milking.

2. During transit to the town, or dairy, or consumer.

3. After its arrival.

Pollution at the Time of Milking arises from the animal, the milker, or unclean methods of milking. It is now well known that in tuberculosis of the cow affecting the udder the milk itself shows the presence of the bacillus of tubercle. In a precisely similar manner all bacterial diseases of the cow which affect the milk-secreting apparatus must inevitably add their quota of bacteria to the milk. To this matter we shall have occasion to refer again. There is a further contamination from the animal when it is kept unclean, for it happens that the unclean coat of a cow will more materially influence the number of micro-organisms in the milk than the popularly supposed fermenting food which the animal may eat. It is from this external source rather than from the diet that organisms occur in the milk. The hairy coat offers many facilities for harbouring dust and dirt. The mud and filth of every kind that may be habitually seen on the hinder quarters of cattle all contribute largely to polluted milk. Nor is this surprising. Such filth at or near the temperature of the blood is an almost perfect environment for many of the putrefactive bacteria.

The milker is also a source of risk. His hands, as well as the clothes he is wearing, can and do readily convey both innocent and pathogenic germs to the milk. Clothed in dust-laden garments, and frequently characterised by dirty hands, the milker may easily act as an excellent purveyor of germs. Not a few cases are also on record where it appears that milkers have conveyed germs of disease from some case of infectious disease, such as scarlet fever, in their homes. But under the more efficient registration of such disease which has recently characterised many dairy companies, the danger of infection from this source has been reduced to a minimum. The habit of moistening the hands with a few drops of milk previous to milking is one to be strongly deprecated.

Professor Russell recounts a simple experiment which clearly demonstrates these simple but effective sources of pollution:

The influence of the barn air, and the cleanliness or otherwise of the barn, is obviously great in this matter. As we have seen, moist surfaces retain any bacteria lodged upon them; but in a dry barn, where molecular disturbance is the rule rather than the exception, it is not surprising that the air is heavily laden with microbic life. Here again many improvements have been made by sanitary cleanliness in various well-known dairies. Still there is much more to be done in this direction to ensure that the drawn milk is not polluted by a microbe-impregnated atmosphere.

The risks in transit differ according to many circumstances. Probably the commonest source of contamination is in the use of unclean utensils and milk-cans. Any unnecessary delay in transit affords increased opportunity for multiplication; particularly is this the case in the summer months, for at such times all the conditions are favourable to an enormous increase of any extraneous germs which may have gained admittance at the time of milking. Thus we have (1) the milk itself affording an excellent medium and supplying ideal pabulum for bacteria, (2) a more or less lengthened railway journey or period of transit giving ample time for multiplication, (3) the favourable temperature of summer heat. We shall refer again to the rate of multiplication of germs in milk.

Lastly, many are the advantages given to bacteria when milk has reached its commercial destination. In milk-shops and in the home there are not a few risks to be added on to the already imposing category. Water is occasionally, if not frequently, added to milk to increase its volume. Such water of itself will make its own contribution to the flora of the milk, unless indeed, which is unlikely, the water has been recently and thoroughly boiled before addition to the milk. Again, it is impossible to suppose that in small homes—perhaps of only one room—where the milk stands for several hours, pollution is avoidable. From a hundred different sources such milk runs the risk of being polluted.

Before proceeding, a word must be said respecting the first milk which flows from the udder in the process of milking, and which is known as the fore-milk. This portion of the milk is always rich in bacterial life on account of the fact that it has remained in the milk-ducts since the last milking. However thorough the manipulation, there will always be a residue remaining in the ducts, which will, and does, afford a suitable nidus and incubator for organisms. The latter obtain their entrance through the imperfectly closed teat of the udder, and pass readily into the milk-duct, sometimes even reaching the udder itself and setting up inflammation (mastitis). Professor Russell states that he has found 2800 germs in the fore-milk in a sample of which the average was only 330 per cc. Schultz found 83,000 micro-organisms per cc. in the fore-milk, and only 9000 in the mid-milk. As a matter of fact, most of this large number belong to the lactic-acid fermentation group, and the fore-milk rarely contains more than two or three species, and still more rarely any disease-producing bacteria. Still, they occur in such enormous numbers that their addition to the ordinary milk very materially alters its quality. Bolley and Hall, of North Dakota, report sixteen species of bacteria in the fore-milk, twelve of which produced an acid reaction. Dr. Veranus Moore, of the United States Department of Agriculture,55 concludes from a large mass of data that freshly drawn fore-milk contains a variable but generally enormous number of bacteria, but only several species, the last milk containing, as compared with the fore-milk, very few micro-organisms. The bacteria which become localised in the milk-ducts, and which are necessarily carried into the milk, are for the greater part rapidly acid-producing organisms, i. e., they ferment milk-sugar, forming acids. They do not produce gas. Still their presence renders it necessary to "pasteurise" as soon as possible. Dr. Moore holds that much of the intestinal trouble occurring in infants fed with ordinarily "pasteurised" milk arises from acids produced by these bacteria between the drawing of the milk and the pasteurisation.

The Number of Bacteria in Milk. From all that has been said respecting the sources of pollution and the favourable nidus which milk affords for bacteria, it is not surprising that a very large number of germs are almost always present in milk. The quantitative estimation of milk appears more alarming than the qualitative. It is true some diseases are conveyed by bacteria in milk, but on the whole most of the species are non-pathogenic. Nor need the numbers, though serious, too greatly alarm us, for, as we shall see at a later stage, disease is a complicated condition, and due to other agencies and conditions than merely the bacteria, which may be the vera causa. In addition to the fact that the high numbers have but a limited significance, we must also remember that there is no uniformity whatever in these numbers. The conditions which chiefly control them are (1) temperature, (2) time.

The Influence of Temperature. We have already noticed, when considering the general conditions affecting bacteria, how potent an agent in their growth is the surrounding temperature. Generally speaking, temperature at or about blood-heat favours bacterial growth. Freudenreich has drawn up the following table which graphically sets forth the effect of temperature upon bacteria in milk:

This instructive table claims some observations. It will be noticed that at 59° F. there is very little multiplication. That may be accepted as a rule. At 77° F. the multiplication, though not particularly rapid at the outset, results finally, at the end of the twenty-four hours, in the maximum quantity. These were probably common species of saprophytic bacteria, which increase readily at a comparatively low temperature. During the subsequent hours, after the twenty-four, we should expect a decline rather than an increase in 62,000, owing to the keen competition consequent upon the limitation of the pabulum. From a consideration of these figures we conclude that a warm temperature, somewhat below blood-heat, is most favourable to multiplication of bacteria in milk; that the common saprophytic organisms multiply the most rapidly; that, in the course of time, competition kills off a large number.

Let us take another example, from Professor Conn:

These almost incredibly large figures illustrate much the same points, particularly the rapid multiplication at blood-heat, and the later rise at 77° C.

The Influence of Time is not less marked than that of temperature, as the following table will show:

Freudenreich gives another example, as follows:

Concerning these figures little comment is necessary. But here again, we may remember that this rapid multiplication continues only up to a certain point, after which competition brings about a marked reduction.

The effect of temperature and time has been illustrated by Dr. Buchanan Young's recent researches, laid before the Royal Society of Edinburgh. He estimated that in the Edinburgh milk supply three hours after milking there were 24,000 micro-organisms per cc. in winter; 44,000 in spring; 173,000 in late summer and autumn. Again, he found that five hours after milking there were 41,000 micro-organisms per cc. in country milk, and more than 350,000 micro-organisms per cc. in town milk. Many London milks would exceed 500,000 per cc.56

There is no standard or uniformity in the numerical estimation of bacteria in milk. A host of observers have recorded widely varying returns due to the widely varying circumstances under which the milk has been collected, removed, stored, and examined. Nor is it possible to establish any standard which may be accepted as a normal or healthy number of bacteria, as is done in water examination. Bitter has suggested 50,000 micro-organisms per cc. as a maximum limit for milk intended for human consumption.

But owing to differences of nomenclature and classification, in addition to differences in mode of examination at present existing in various countries, it is impossible to state even approximately how many bacteria and how many species of bacteria have been isolated from milk. Until some common international standard is established mathematical computations are practically worthless. They are needlessly alarming and sensational. And it should be remembered that great reliance cannot be placed upon these numerical estimations. They vary from day to day, and even hour to hour. Furthermore, vast numbers of bacteria are economic in the best sense of the term, and the bacteria of milk are chiefly those of a fermentative kind, and not disease-producers.

Kinds of Bacteria in Milk. It is clear from the foregoing that the only valuable estimation of bacteria in milk is a qualitative one. The kinds commonly found may be classified thus:

1. Non-pathogenic; fermenting and various unclassified micro-organisms.

2. Pathogenic; tuberculosis, typhoid, cholera, scarlet fever, diphtheria, and suppurative diseases have all been spread by the agency of milk.

1. The Fermentation Bacteria

At the most we can make a merely provisional classification of these processes. Many of them are intimately related. Of others, again, our knowledge is at present very limited. It may be advisable, before proceeding, to consider shortly what are the constituents of milk upon which living ferments of various kinds exert their action. A tabulation of the chief constituents would be as follows:

Another mode of expressing average milk constitution would be thus:

It is probably too obvious to need remark that milks vary in standard, but the above figures may be taken as authentic averages.

Milk-sugar, or Lactose (C₁₂H₂₄O₁₂). This is an important and constant constituent of milk. It forms the chief substance in solution in whey or serum. Milk-sugar approximates to dextrose in its action on polarised light. By boiling with sulphuric acid it is converted into dextrose and galactose.

Fat occurs in milk as suspended globules, and by churning may be made into butter.

The Proteids include casein, albumen, lactoprotein, and a small quantity of globulin. These are the nitrogenous bodies.

Mineral Matter. The ash of milk, obtained by careful ignition of the solids, contains calcium, magnesium, potassium, sodium, phosphoric acid, sulphuric acid, chlorine, and iron, phosphoric acid and lime being present in the largest amounts.

(1) Lactic Acid Fermentation. If milk is left undisturbed, it is well known that eventually it becomes sour. The casein is coagulated, and falls to the bottom of the vessel; the whey or serum rises to the top. In fact, a coagulation analogous to the clotting of blood has taken place. In addition to this, the whole has acquired an acid taste. Now, this double change is not due to any one of the constituents we have named above. It is, in short, a fermentation set up by a living ferment introduced from without. The constituent most affected by the ferment is the milk-sugar, which is broken down into lactic acid, carbonic acid gas, and other products.

For many years it has been known that sour milk contained bacteria. Pasteur first described the Bacillus acidi lactici, which Lister isolated and obtained in pure culture. Hueppe contributed still further to what was known of this bacillus, and pointed out that there were a large number of varieties, rather than one species, to be included under the term B. acidi lactici. We have already seen that these bacilli do not as a rule liquefy gelatine, form spores, are non-motile, and are easily killed by heat.

When a certain quantity of lactic acid has been formed the fermentation ceases. It will recommence if the liquid be neutralised with carbonate of lime, or pepsine added. Since Pasteur's discovery of a causal bacillus for this fermentation, other investigators have added a number of bacteria to the lactic acid family. Some of these in pure culture have been used in dairy industry to add to the butter a pure sour taste, a more or less aromatic odour, and a higher degree of preservation.

(2) Butyric Acid Fermentation. This form of fermentation is also one which we have previously considered.

Both in lactic and butyric fermentation we must recognise that in the decomposition of milk-sugar there are almost always a number of minor products occurring. Some of the chief of these are gases. Hydrogen, carbonic acid, nitrogen, and methane occur, and cause a characteristic effect which is frequently deleterious to the flavour of the milk and its products. Most of the gas-producing ferments are members of the lactic acid group, and are sometimes classified in a group by themselves. In cheese-making the gases create the pin-holes and air-spaces occasionally seen.

(3) Curdling Fermentations without Acid Production. Of these there are several, caused by different bacteria. What happens is that the milk coagulates, as we have described, but no acid is produced, the whey being sweet to the taste rather than otherwise. Digestion of casein may or may not take place.

We must now mention several fermentations about which little is known. They are designated by terms denoting the outward condition of the milk, without giving any information respecting the real physiological alteration which has occurred.

(4) Bitter Fermentation. Some bitter conditions of milk are due to irregularity of diet in the cow. Similar changes occur in conjunction with some of the acid fermentations. Weigmann and Conn have, however, shown that there is a specific bitterness in milk due to bacteria which appear to produce no other change. Hueppe suggests that it may be due in part to a proteid decomposition resulting in bitter peptones. There seems to be some evidence for supposing that the bitter bacteria produce very resistant spores, which enable them to withstand treatment under which the lactic acid succumbs.

(5) Slimy Fermentation. This graphic but inelegant word is used to denote an increased viscosity in milk, and its tendency when being poured to become ropy and fall in strings. Such a condition deprives the milk of its use in the making of certain cheeses, whilst in other cases it favours the process. In Holland, for example, in the manufacture of Edam cheese, this "slimy" fermentation is desired. Tættemœlk, a popular beverage in Norway, is made from milk that has been infected with the leaves of the common butter wort, Pinguicula vulgaris, from which Weigmann separated a bacillus possessing the power of setting up slimy fermentation. There are, perhaps, as many as a dozen species of bacteria which have in a greater or less degree the power of setting up this kind of fermentation. In 1882 Schmidt isolated the Micrococcus viscosus, which occurs in chains and rosaries, affecting the milk-sugar. It grows at blood-heat, and is not easily destroyed by cold. Its effect on various sugars is the same. The M. Freudenreichii, the specific micro-organism of "ropiness" in milk, is a large, non-motile, liquefying coccus, which can produce its result in milk within five hours. On account of its resistance to drying, it is difficult to eradicate when once it makes its appearance in a dairy. The organism used in making Edam cheese is the Streptococcus Hollandicus, and in hot milk it can produce ropiness in one day. A number of bacilli have been detected by several observers and classified as slime fermentation bacteria. The Bacillus lactis pituitosi, a slightly curved, non-liquefying rod, which is said to produce a characteristic odour, in addition to causing ropiness, brings about some acidity. B. lactis viscosus is slow in starting its fermentation, but maintains its action for as long as a month. Many of the above organisms, with others, produce "slimy" fermentation in alcoholic beverages as well as in milk.

(6) Soapy Milk. This is still another form of fermentation, the etiology of which has been elucidated by Weigmann. The Bacillus saponacei imparts to milk a peculiar soapy flavour. It was detected in the straw of the bedding and hay of the fodder, and from such sources may infect the milk. There is little or no coagulation.

(7) Chromogenic Changes. We have already remarked that colour is the natural and apparently only product of many of the innocent bacteria. They put out their strength, so to speak, in the production of bright colours. The chief colours produced by germs in milk are as follows:

Red Milk. Bacillus prodigiosus, in the presence of oxygen, causes a redness, particularly on the surface of milk. It was the work of this bacillus that caused "the bleeding host," which was one of the superstitions of the Middle Ages. B. lactis erythrogenes produces a red colour only in the dark, and in milk that is not strongly acid in reaction. When grown in the light this organism produces a yellow colour. There is a red sarcina (Sarcina rosea) which also has the faculty of producing red pigment. One of the yeasts is another example.

It must not be forgotten that redness in milk may actually be due to the presence of blood from the udder of the cow. In such a case the blood and milk will be inextricably mixed together, and not in patches or a pellicle.

Blue Milk is due to the growth of Bacillus cyanogenus. This is an actively motile rod, the presence of which does not materially affect the milk, but causes the milk products to be of poor quality.

Yellow Milk. Bacillus synxanthus is held responsible for curdling the milk, and then at a later stage, in redissolving the curd, produces a yellow pigment.

Violet and Green Pigments in milk are also the work of various bacteria.

2. Various Unclassified Bacteria

In milk this is a comparatively small group, for it happens that those bacteria in milk which cannot be classified as fermentative or pathogenic are few. The almost ubiquitous Bacillus coli communis occurs here as elsewhere, and might be grouped with the gaseous fermentative organisms on account of its extraordinary power of producing gas and breaking up the medium (whether agar or cheese) in which it is growing. What its exact rôle is in milk it would be difficult to say. It may act, as it frequently does elsewhere, by association in various fermentations. Some authorities hold that its presence in excessive numbers may cause epidemic diarrhœa in infants.

Several years ago a commission was appointed by the British Medical Journal to inquire into the quality of the milk sold in some of the poorer districts of London. Every sample was found to contain Bacillus coli, and it was declared that this particular microbe constituted 90 per cent. of all the organisms found in the milk.57 We record this statement, but accept it with some misgiving. The diagnosis of B. coli four or five years ago was not such a strict matter as to-day. Still, undoubtedly, this particular organism is not uncommonly found in milk, and its source is unclean dairying. In the same investigation Proteus

vulgaris, B. fluorescens, and many liquefying bacteria were frequently found. Their presence in milk means contamination with putrefying matter, surface water, or a foul atmosphere.

A number of water bacteria find their way into milk in the practice of adulteration, and foul byres afford ample opportunity for aërial pollution.

Another unclassified group occasionally present in milk is represented by moulds, particularly Oidium lactis, the mould which causes a white fur, possessing a sour odour. It is allied to the Mycoderma albicans (O. albicans), which also occurs in milk, and causes the whitish-grey patches on the mucous membrane of the mouths of infants (thrush). These and many more are occasionally present in milk.

3. The Disease-Producing Power of Milk

The general use of milk as an article of diet, especially by the younger and least resistant portion of mankind, very much increases the importance of the question as to how far it acts as a vehicle of disease. Recently considerable attention has been drawn to the matter, though it is now a number of years since milk was proved to be a channel for the conveyance of infectious diseases. During the last twenty years particular and conclusive evidence has been deduced to show that milch cows may themselves afford a large measure of infection. The recent extensive work in tuberculosis by the Royal Commission has done much to obtain new light on the conveyance of that disease by milk and meat. The enormous strides in the knowledge of diphtheria and other germ diseases have also placed us in a better position respecting their conveyance by milk. Generally speaking, for reasons already given, milk affords an ideal medium for bacteria, and its adaptibility therefore for conveying disease is undoubted. We may now suitably turn to speak shortly of the outstanding facts of the chief diseases carried by milk.

Tuberculosis. It is well known that this disease is not a rare one amongst cattle. The problem of infective milk is, however, simplified at the outset by recognising the now well-established fact that the milk of tuberculous cows is only certainly able to produce tuberculosis in the consumers when the tuberculous disease affects the udder. This is not necessarily a condition of advanced tuberculosis. The udder may become affected at a comparatively early stage. But to make the milk infective the udder must be tubercular, and milk from such an udder possesses a most extraordinary degree of virulence. When the udder itself is thus the seat of disease, not only the derived milk, but the skimmed milk, butter-milk, and even butter, all contain tuberculous material actively injurious if consumed. Furthermore, tubercular disease of the udder spreads in extent and degree with extreme rapidity. From these facts it will be obvious that it is of first-rate importance to be able to diagnose udder disease. This is not always possible in the early stage. The signs upon which most reliance may be placed are the enlargement of the lymph-glands lying above the posterior region of the udder; the serous, yellowish milk which later on discharges small coagula; the partial or total lack of milk from one quarter of the udder (following upon excessive secretion); the hard, diffuse nodular swelling and induration of a part or the whole wall of the udder; and the detection in the milk of tubercle bacilli. The whole organ may increase in weight as well as size, and on post-mortem examination show an increase of connective tissue, a number of large nodules of tubercle, and a scattering of small granular bodies, known as "miliary" tubercles. Tuberculin may be used as an additional test. The udder is affected in about two per cent. of tuberculous cows.

There are a variety of causes in addition to the vera causa, the presence of the bacillus of tubercle, which make the disease common amongst cattle. Constitution, temperament, age, work, food, and prolonged lactation are the individual features which act as predisposing conditions; they may act by favouring the propagation of the bacillus or by weakening the resistance of the tissues. To this category must further be added conditions of environment. Bad stabling, dark, ill-ventilated stalls, high temperature, prolonged and close contact with other cows, all tend in the same direction.

Though there can be no doubt as to the virulence of tuberculous milk, it may be remembered with satisfaction that only about two per cent. of tuberculous cows have unmistakably tubercular milk. Even of this tubercular milk, unless it is very rich in bacilli and is ingested in large quantities, the risks are practically small or even absent. Practically the danger from drinking raw milk exists only for persons who use it as their sole or principal food, that is to say, young children and certain invalids. With adults in normal health the danger is greatly minimised, as the healthy digestive tract is relatively insusceptible. Moreover, dairy milk is almost invariably mixed milk; that is to say, if there is a tubercular cow in a herd yielding tubercle bacilli in her milk, the addition of the milk of the rest of the herd so effectually dilutes the whole as to render it almost innocuous.

But if for practical purposes we look upon all milk derived from tubercular udders as highly infective, we may adopt a comparatively simple and efficient remedy. To avoid all danger it is sufficient to bring the milk to a boil for a few minutes before it is consumed; in fact, the temperature of 85° C. (160° F.) prolonged for five minutes kills all bacilli. The common idea that boiled milk is indigestible, and that the boiling causes it to lose much of its nutritive value, is largely groundless.

Milk may become tubercular through the carelessness or dirty habits of the milker. Such a common practice as moistening the hands with saliva previously to milking may, in cases of tubercular milkers, effectually contaminate the milk. Again, it may become polluted by dried tubercular excreta getting into it. Such conveyances must be of rare occurrence, yet their possibility should not be forgotten.

An infant suckled by a tuberculous mother would run similarly serious risks of becoming infected with the disease.

In Liverpool, Dr. E. W. Hope, the Medical Officer of Health, has organised an admirable system of examination by skilled bacteriologists to find to what degree the Liverpool milk supply is contaminated with tubercle. The final result of this pioneer work, which ought really to be undertaken by every great corporation responsible to the citizens for a pure water and pure milk supply, is to the effect that in Liverpool 5.2 per cent. of the samples of milk taken from the city shippons contains tubercle bacilli. As regards the milk sent in from the country, the return is that 13.4 per cent. is contaminated with the bacillus of tubercle.

Such results are very significant, and indicate the importance of all large corporations obtaining the service of systematic and periodic bacteriological examination of the milk supply. Nor are the results surprising, for when we remember the habits of the tubercle bacillus we cannot conceive a more favourable nurture ground than the typical byre. "Nothing worse than the insanitary conditions of the life of the average dairy cow," says Sir George Brown, late of the Board of Agriculture, "can be imagined." It will be obvious that the above facts make it incumbent upon responsible authorities to see that not a stone is left unturned to enforce cleanliness in all dairy work, isolation of diseased cows, and strict treatment of all infected milk.

Typhoid Fever. Jaccoud in France and Hart in England have shown that enteric fever (typhoid) is not infrequently spread by milk. An epidemic affecting 386 persons in Stamford, Conn., U.S.A., was traced to milk, 97 per cent. of the cases coming from one single milk supply. Dr. McNail recently recorded an outbreak of twenty-two cases of enteric, due to a polluted milk supply.

Within the last twelve months much attention has been drawn to a milk source of typhoid infection by the epidemic of typhoid at Bristol. Dr. D. S. Davies has pointed out that a brook received the sewage of thirty-seven houses, the overflow of a cesspool serving twenty-two more, the washings from fields over which the drainage of several others was distributed, and the direct sewage from at least one other, and then flowed directly through a certain farm. The water of this stream supplied the farm pump, and the water itself, it is scarcely necessary to add, was highly charged with putrescent organic matter and micro-organisms. This water was used for washing the milk-cans from this particular farm, otherwise the dairy arrangements were efficient. Part of the milk was distributed to fifty-seven houses in Clifton; in forty-one of them cases of typhoid occurred. Another part of the milk was sold over the counter; twenty households so obtaining it were attacked with typhoid fever, and a number of further infections and complications arose. This evidence would appear to support the fact that milk may act in the same way, though not in such a high degree, as water in the conveyance of typhoid fever.

It may be pointed out that specific typhoid is not a disease of animals; consequently no danger need be apprehended from milk if it is properly cared for after it comes from the cow. Typhoid milk is almost invariably due to the addition of typhoid-infected water, either by way of adulteration or in the process of washing out the milk-cans. Cases have, however, been recorded in which there has been direct transmission to the milk from a person convalescing from the disease, and also indirect transmission by a milker serving also in the capacity of nurse to a patient in his own family.

Though the typhoid bacillus appears not to have the power of multiplying in milk, it has the faculty of existing and thriving in milk, even when it has curdled or soured, for a considerable time, and may thus infect milk products like butter and cheese. But infection by milk products may be eliminated as of too rare occurrence to deserve attention. The bacillus does not coagulate the milk like its ally the Bacillus coli communis, which is a much more frequent and less injurious inhabitant of milk.

Cholera. The cholera bacillus, as we have already pointed out, is unable to live in an acid medium. Hence its life in milk is a limited one, and generally depends on some alkaline change in the milk. Heim found that cholera bacilli would live in raw milk from one to four days, depending upon the temperature. D. D. Cunningham, from the results of a large number of investigations in India, concludes that the rapidly developing acid fermentations normally or usually setting in, connected with the rapid multiplication of other common bacteria and moulds, tend to arrest the multiplication of cholera bacilli, and eventually to destroy their vitality. Boiling milk appears, on the contrary, to increase the suitability of milk as a nidus for cholera bacilli, partly by its germicidal effect upon the acid-producing microbes, and partly because it removes from the milk the enormous numbers of common bacteria, which in raw milk cause such keen competition that the cholera bacillus finds existence impossible.