Bacteria / Especially as they are related to the economy of nature, to industrial processes, and to the public health

by Sir George Newman

THE QUALITATIVE ESTIMATION OF BACTERIA IN THE SOIL

We may now turn to consider the species of bacteria found in the interstices of soil. They may be classified in five main groups. The division is somewhat artificial, but convenient:

1. The Denitrifying Bacteria. A group whose function has been elucidated in recent years (largely by the investigations of Professor Warington) are held responsible for the breaking down of nitrates. With these may be associated the Decomposition or Putrefactive Bacteria, which break down complex organic products other than nitrates into simpler bodies.

A Method of Growing Cultivations in a Vacuum over Pyrogallic Solution

2. The Organisms of Nitrification. To this group belong the two chief types of nitrifying bacteria, viz., those which oxidise ammonia into nitrites, and those which change nitrites into nitrates.

3. The Nitrogen-fixing Bacteria, found mainly in the nodules on the rootlets of certain plants.

4. The Common Saprophytic Bacteria, whose function is at present but imperfectly known. Many are putrefactive germs.

5. The Pathogenic Bacteria. This division includes the three types, tetanus, malignant œdema, and quarter evil. Under this heading we shall also have to consider in some detail the intimate relation between the soil and such important bacterial diseases as tubercle and typhoid.

To enable us to appreciate the work which the "economic bacteria" perform, it will be necessary to consider shortly the place they occupy in the economy of nature. This may be perhaps most readily accomplished by studying the accompanying table (p. 145).

A SCHEME SHOWING THE PLACE AND FUNCTION OF THE ECONOMIC MICRO-ORGANISMS FOUND IN SOIL

Water		Chemical Substances [Nitrates, etc.]		Gases [CO2, H, N, O]
⬊		⬇		⬋
Plant Life
│
┌────────┬──────┬──────┬──────────┬───────────┐
Carbohydrates [albumoses, sugar, starch, etc.]	Fats	Proteids [bodies containing Nitrogen]	Vegetable Acids	Mineral Salts	Water
Animal Life
│
┌───────┬──────────┬──────────────────┐
Gases [CO2, etc.]	Water	Urea, Albuminoids, Ammonia compounds, etc.		Nitrogen in many forms locked up in the body
		PUTREFACTIVE AND DENITRIFYING BACTERIA │
	┌───────┬──────┬──────────┬───────────┐
	Free Nitrogen │ │ │ │ │ │ │ │	Gases [CO2]	Water	Ammonia and other elements of broken-down complex bodies.	[Nitrites]
				NITRIFYING BACTERIA
				│
				Nitrites[=Nitrous organism │ (Nitrosomans)]
	NITROGEN-FIXING BACTERIA			Nitrates[=Nitric organism (Nitrosomans)]
[In soil and in the nodules on the rootlets of Leguminosæ]				[In soil and available for plant life]

The threefold function of plant life is nutrition, assimilation, and reproduction: the food of plants, the digestive and storage power of plants, and the various means they adopt for multiplying and increasing their species. With the two latter we have little concern in this place. Respecting the nutrition of plant life, it is obvious that, like animals, they must feed and breathe to maintain life. Plant food is of three kinds, viz., water, chemical substances, and gas. Water is an actual necessity to the plant not only as a direct food and food-solvent, but as the vehicle of important inorganic materials. The hydrogen, too, of the organic compounds is obtained from the decomposition of the water which permeates every part of the plant, and is derived by it from the soil and from the aqueous vapour in the atmosphere. The chief chemical substances of which vegetable protoplasm is constituted are six, viz, potassium, magnesium, calcium, iron, phosphorous, and sulphur. These inorganic elements do not enter the plant as such, but combined with other substances or dissolved in water. Potassium occurs in salt form combined with various organic acids (tartaric, oxalic, etc.), calcium and magnesium as salts of lime and magnesia in combination both with organic and inorganic acids. Iron contributes largely to the formation of the green colouring matter of plants, and is also derived from the soil. Phosphorus, one of the chief constituents of seeds, generally occurs as phosphate of lime. Sulphur, which is an important constituent of albumen, is derived from the sulphates of the soil. In addition to the above, there are other elements, sometimes described as non-essential constituents of plants. Amongst these are silica (to give stiffness), sodium, chlorine, iodine, bromine, etc. All these elements contribute to the formation or quality of the protoplasm of plants.

The gases essential to plants are four: Carbon dioxide (carbonic acid), Hydrogen, Oxygen, and Nitrogen. By the aid of the green chlorophyll corpuscles, and under the influence of sunlight, we know that leaves absorb the carbon dioxide of the atmosphere, and effect certain changes in it. The hydrogen, as we have seen, is obtained from the water. Oxygen is absorbed through the root from the interstices of the soil. Each of these contributes vitally to the existence of the plant. The fourth gas, nitrogen, which constitutes more than two thirds of the air we breathe (79 per cent. of the total volume and 77 per cent. of the total weight of the atmosphere), is, perhaps, the most important food required by plants. Yet, although this is so, the plant cannot absorb or obtain its nitrogen in the same manner in which it acquires its carbon—viz., by absorption through the leaves—nor can the plant take nitrogen into its own substance by any means as nitrogen, with the exception of the flesh-feeding plants (insectivorus). Hence, although this gas is present in the atmosphere surrounding the plant, the plant will perish if nitrogen does not exist in some combined form in the soil. Nitrates and compounds of ammonia are widely distributed in nature, and it is from these bodies that the plant obtains, by means of its roots, the necessary nitrogen.

Until comparatively recently it was held that plant life could not be maintained in a soil devoid of nitrogen or compounds thereof. But it has been found that certain classes of plants (the Leguminosæ, for example), when they are grown in a soil which is practically free from nitrogen at the commencement, do take up this gas into their tissues. One explanation of this fact is that free nitrogen becomes converted into nitrogen compounds in the soil through the influence of micro-organisms present there. Another explanation attributes this fixation of free nitrogen to micro-organisms existing in the rootlets of the plant. These two classes of organisms, known as the nitrogen-fixing organisms, will require our consideration at a later stage. Here we merely desire to make it clear that the main supply of this gas, absolutely necessary to the existence of vegetable life upon the earth, is drawn not from the nitrogen of the atmosphere, but from that contained in nitrogen compounds in the soil. The most important of these are the nitrates. Here then we have the necessary food of plants expressed in a sentence: water, gases, salts, the most important and essential gas and some of the salts being combined in nitrates.

Plant life seizes upon its required constituents, and by means of the energy furnished by the sun's rays builds these materials up into its own complex forms. Its many and varied forms fulfil a place in beautifying the world. But their contribution to the economy of nature is, by means of their products, to supply food for animal life. The products of plant life are chiefly sugar, starch, fat, and proteids. Animal life is not capable of extracting its nutriment from soil, but it must take the more complex foods which have already been built up by vegetable life. Again, the complementary functions of animal and vegetable life are seen in the absorption by plants of one of the waste materials of animals, viz., carbonic acid gas. Plants abstract from this gas carbon for their own use, and return the oxygen to the air, which in its turn is of service to animal life.

By animal activity some of these foods supplied by the vegetable kingdom are at once decomposed into carbonic acid gas and water, which goes back to nature. Much, however, is built up still further into higher and higher compounds. The proteids are converted by digestion into albumoses and peptones, ultimately entirely into peptones; these in their turn are reconverted into proteids, and become assimilated as part of the living organism. In time they become further changed into carbonic acid, sulphuric acid, water, and certain not fully oxidised products,[36] which contain the nitrogen of the original proteid. In the table these bodies have been represented by one of their chief members, viz., urea.

It is clear that there is in all animal life a double process continually going on; there is a building up (anabolism, assimilation), and there is a breaking down (katabolism, dissimilation). These processes will not balance each other throughout the whole period of animal life. We have, as possibilities, elaboration, balance, degeneration; and the products of animal life will differ in degree and in substance according to which period is in the predominance. These products we may subdivide simply into excretions during life and final materials of dissolution after death, both of which may be used more or less immediately by other forms of animal or vegetable life, or mediately after having passed to the soil. We may shortly summarise the final products of animal life as carbonic acid, water, and nitrogenous remnants. These latter will occur as urea, new albumens, compounds of ammonia, and nitrogen compounds of great complexity stored up in the tissues and body of the animal. The carbonic acid, water, and other simple substances like them will return to nature and be of immediate use to vegetable life. But otherwise the cycle cannot be completed, for the more complex bodies are of no service as such to plants or animals.

1. In order that this complex material should be of service in the economy of nature, and its constituents not lost, it is necessary that it should be broken down again into simpler conditions. This prodigious task is accomplished by the agency of two groups of organisms, the decomposition and denitrifying[37] bacteria. The organisms associated with decomposition processes are numerous; some denitrify as well as break down organic compounds. This group will be referred to under "Saprophytic Bacteria." The reduction by the denitrifying bacteria may be simply from nitrate to nitrite, or from nitrate to nitric or nitrous oxide gas, or indeed to nitrogen itself. In all these processes of reduction the rule is that a loss of nitrogen is involved. How that free nitrogen is brought back again and made subservient to plants and animals we shall understand at a later stage.

Professor Warington has again recently set forth the chief facts known of this decomposition process.[38] That the action in question only occurs in the presence of living organisms was first established by Mensel in 1875 in natural waters, and by Macquenne in 1882 in soils. If all living organisms are destroyed by sterilisation of the soil, denitrification cannot take place, nor can vegetable life exist. "Bacteria reduce nitrates," says Professor Warington, "by bringing about the combustion of organic matter by the oxygen of the nitrate, the temperature distinctly rising during the operation." The reduction to a nitrite is a common property of bacteria. But only a few species have the power of reducing a nitrate to gas. These few species are, however, widely distributed. In 1886 Gayon and Dupetit first isolated the bacteria capable of reducing nitrates to the simplest element, nitrogen. They obtained their species from sewage, but ten years later denitrifying bacteria were isolated from manure. That soil contains a number of these reducing organisms is known by introducing a particle of surface soil into some broth, to which has been added one per cent. of nitre. During incubation of such a tube gas is produced, and the nitrate entirely disappears.

Whenever decomposition occurs in organic substances there is a reduction of compound bodies, and in such cases the putrefying substances obtain their decomposing and denitrifying bacteria from the air. The chief conditions requisite for bringing about a loss of nitrogen by denitrification are enumerated by Professor Warington as follows: (1) the specific micro-organism; (2) the presence of a nitrate and suitable organic matter; (3) such a condition as to aëration that the supply of atmospheric oxygen shall not be in excess relatively to the supply of organic matter; (4) the usual essential conditions of bacterial growth. "Of these," he says, "the supply of organic matter is by far the most important in determining the extent to which denitrification will take place." The necessarily somewhat unstable condition facilitates its being split up by means of bacteria. The bacteria in their turn are ready to seize upon any products of animal life which will serve as their food. Thus, by reducing complex bodies to simple ones, these denitrifying organisms act as the necessary link to connect again the excretions of the animal body, or after death the animal body itself, with the soil.

In a book of this nature it has been deemed advisable not to enter into minute description of all the species of bacteria mentioned. Some of the chief are described more or less fully. We cannot, however, do more than name several of the chief organisms concerned in reducing and breaking down compounds. As we shall find in the bacteria of nitrification, so also here, the entire process is rarely, if ever, performed by one species. There is indeed a remarkable division of labour, not only between decomposition bacteria and denitrification bacteria, but between different species of the same group. Bacillus fluorescens non-liquefaciens, Mycoderma ureæ, and some of the staphylococci break down nitrates (denitrification), and also decompose other compound bodies. Amongst the group of putrefactive bacteria found in soil may be named B. coli, B. mycoides, B. mesentericus, B. liquidus, B. prodigiosus, B. ramosus, B. vermicularis, B. liquefaciens, and many members in the great family of Proteus. Some perform their function in soil, others in water, and others, again, in dead animal bodies. Dr. Buchanan Young, to whose researches in soil we have referred, has pointed out that in the upper reaches of burial soil, where these bacteria are most largely present, there is as a result no excess of organic carbon and nitrogen. Even in the lower layers of such soil it is rapidly broken down.

Micrococcus from Soil

It will be observed, from a glance at the table, that the chief results of decomposition and denitrification are as follows: free nitrogen, carbonic acid, gas and water, ammonia bodies, and sometimes nitrites. The nitrogen passes into the atmosphere, and is "lost"; the carbonic acid and water return to nature and are at once used by vegetation. The ammonia and nitrites await further changes. These further changes become necessary on account of the fact, already discussed, that plants require their nitrogen to be in the form of nitrates in order to use it. Nitrates obviously contain a considerable amount of oxygen, but ammonia contains no oxygen, and nitrites very much less than nitrates. Hence a process of oxidation is required to change the ammonia into nitrites and the nitrites into nitrates.

2. This oxidation is performed by the nitrifying micro-organisms, and the process is known as nitrification. It should be clearly understood that the process of nitrifaction may, so to speak, dovetail with the process of denitrification. No exact dividing line can be drawn between the two, although they are definite and different processes. In a carcass, for example, both processes may be going on concomitantly; so also in manure. There is no hard and fast line to be drawn in the present state of our knowledge. Other organisms beside the true nitrification bacteria may be playing a part, and it is impossible exactly to measure the action of the latter, where they began and where the preliminary attack upon the nitrogenous compounds terminated. In all cases, however, according to Professor Warington, the formation of ammonia has been found to precede the formation of nitrous or nitric acid.

It was Pasteur who (in 1862) first suggested that the production of nitric acid in soil might be due to the agency of germs, and it is to Schlösing and Müntz that the credit belongs for first demonstrating (in 1877) that the true nature of nitrification depended upon the activity of a living microorganism. Partly by Schlösing and Müntz and partly by Warington (who was then engaged in similar work at Rothamsted), it was later established (1) that the power of nitrification could be communicated to substances which did not hitherto nitrify by simply seeding them with a nitrified substance, and (2) that the process of nitrification in garden soil was entirely suspended by the vapour of chloroform or carbon disulphide. The conditions for nitrification, the limit of temperature, and the necessity of plant food, have furnished additional proof that the process is due to a living organism. These conditions are briefly as follows:

1. Food (of which phosphates are essential constituents). "The nitrifying organism can apparently feed upon organic matter, but it can also, apparently with equal ease, develop and exercise all its functions with purely inorganic food" (Warington).

Winogradsky prepared vessels and solutions carefully purified from organic matter, and these solutions he sowed with the nitrifying organism, and found that they flourished. Professor Warington has employed the acid carbonates of sodium and calcium with distinct success as ingredients of an ammoniacal solution undergoing nitrification.

2. The next condition of nitrification is the presence of oxygen. Without it the reverse process, denitrification, occurs, and instead of a building up we get a breaking down, with an evolution of nitrogen gas. The amount of oxygen present has an intimate proportion to the amount of nitrification, and with 16 to 21 per cent. of oxygen present the nitrates are more than four times as much as when the smallest quantity of oxygen is supplied. The use of tillage in promoting nitrification is doubtless in part due to the aëration of the soil thus obtained.

3. A third condition is the presence of a base with which nitric acid when formed may combine. Nitrification can take place only in a feebly alkaline medium, but an excess of alkalinity will retard the process.

4. The last essential requirement is a favourable temperature. The nitrifying organism can act at a temperature as low as 37° or 39° F. (3–4° C.), but at a higher temperature it becomes much more active. According to Schlösing and Müntz, at 54° F. (12° C.) nitrification becomes really active, and it increases as the temperature rises to 99° F. (37° C.), after which it falls. A high temperature or a strong light are prejudicial to the process.

We are now in a position to consider shortly some of the characters of these nitrification bacteria. They may readily be divided into two chief groups, not in consideration of their form or biological characteristics, but on account of the duties which they perform. Just as we observed that there were few denitrifying organisms which could break down ammonia compounds to nitrogen gas, so is it also true that there are few nitrifying bacteria which can build up from ammonia to the nitrates. Nature has provided that this shall be accomplished in two stages, viz., a first stage from ammonia bodies to nitrites, and a second stage from nitrites to nitrates. The agent of the former is termed the nitrous organism, the latter the nitric organism. Both are contributing to the final production of nitrates which can be used by plant life.[39]

The Nitrous Organism (Nitrosomonas). Prior to Koch's gelatine method the isolation of this bacterium proved an exceedingly difficult task. But even the adoption of this isolating method seemed to give no better results, and for an excellent reason: the nitrifying organisms will not grow on gelatine. To Winogradsky and Percy Frankland belongs the credit of separately isolating the nitrous organism on the surface of gelatinous silica containing the necessary inorganic food. Professor Warington, in his lectures under the Lawes Agricultural Trust, has described this important germ as follows:

"The organism as found in suspension in a freshly nitrified solution consists largely of nearly spherical corpuscles, varying extremely in size. The largest of these corpuscles barely reaches a diameter of one-thousandth of a millimetre, and some are so minute as to be hardly discernible in photographs. The larger ones are frequently not strictly circular, and are sometimes seen in the act of dividing.

"Besides the form just described, there is another, not universally present in solutions, in which the length is considerably greater than its breadth. The shape varies, being occasionally a regular oval, but sometimes largest at one end, and sometimes with the ends truncated. The circular organisms are probably the youngest.

"This organism grows in broth, diluted milk, and other solutions without producing turbidity. When acting on ammonia it produces only nitrites. It is without action on potassium nitrite. It is, in fact, the nitrous organism which, as we have previously seen, may be separated from soil by successive cultivations in ammonium carbonate solution."

The elongated forms appear to be a sign of arrested growth. Normally the organ is about 1.8 µ long, or nearly three times as long as the nitric organism. It possesses a gelatinous capsule. "The motile cells, stained by Löffler's method, are seen to have a flagellum in the form of a spiral." When grown on silica the nitrous organism appears in the same two forms—zooglea and free cells—as when cultivated in a fluid. It commences to show growth in about four days, and is at its maximum on about the tenth day. Winogradsky found that there were considerable differences in the morphology of the organism according to the soil from which it was taken. One of the Java soils he investigated contained a nitrous organism having a spiral flagellum of thirty micromillimetres; but its movement was slow.

As we have already seen, the most astonishing property of this organism is its ability to grow and perform its specific function in solutions absolutely devoid of organic matter. Some authorities hold that it acquires its necessary carbon from carbonic acid. The mode of culturing it is as follows:

To sterilised flasks add 100 cc. of a solution made of one gram of ammonium sulphate, one gram of potassium sulphate, and 1000 cc. of pure water. To this add one gram of basic magnesium carbonate which has been previously sterilised by boiling. Now inoculate the flask with a small portion of the soil under investigation, and after four or five days sub-culture on the same medium in fresh flasks, and let this be repeated half a dozen times. Now, as this inorganic medium is unfavourable to ordinary bacteria of soil, it is clear that after several sub-cultures the nitrous organism will be isolated in pure culture.

Winogradsky employs for culturing upon solid media a mineral gelatine. A solution of from 3 to 4 per cent. of silicic acid in distilled water is placed in flasks. By the addition of the following salts to such a solution gelatinisation occurs:

(a)		Ammonium sulphate	0.4 gram
		Magnesium sulphate	0.05 "
		Calcium chloride	A trace
(b)		Potassium phosphate	0.1 gram
		Sodium carbonate	0.6, 0.9 "
		Distilled water	100 cc.

The sulphates and chloride are mixed in 50 cc. of distilled water, and the latter substance in the remaining 50 cc. in separate flasks. After sterilisation and cooling these are all mixed and added in small quantities to the silicic acid.

Upon this medium it is possible to sub-culture a pure growth from the film at the bottom of the flasks in which the nitrous organism is first isolated.

The Nitric Organism. It was soon learned that the nitrous organism, even when obtainable in large quantities and in pure culture, was not able entirely to complete the nitrifying process. As early as 1881 Professor Warington had observed that some of his cultures, though capable of changing nitrites into nitrates, had no power of oxidising ammonia. These he had obtained from advanced sub-cultures of the nitrous organism, and somewhat later Winogradsky isolated and described this companion of the nitrous organism. It develops freely in solutions to which no organic matter has been added; indeed, much organic matter will prevent its growing. He isolated it from soils from various parts of the world on the following media:

Water	1000.0
Potassium phosphate	1.0
Magnesium sulphate	0.5
Calcium chloride	A trace
Sodium chloride	2.0

About 20 cc. of this solution is placed in a flat-bottom flask, and a little freshly washed magnesium carbonate is added. The flask is closed with cotton wool, and the whole is sterilised. To each flask 2 cc. of a 2 per cent. solution of ammonium sulphate is subsequently added. The temperature for incubation is 30° C. Winogradsky concluded that the oxidation of nitrites to nitrates was brought about by a specific organism independently of the nitrous organism. He successfully isolated it in silica jelly. He believes the organism, like its companion, derives its nutriment solely from inorganic matter, but this is not finally established.

The form of the nitric organism (or nitromonas, as it was once termed) is allied to the nitrous organism. The cells are elongated, rarely oval, but sometimes pear-shaped. They are more than half a micromillimetre in length, and somewhat less in thickness. The cells have a gelatinous membrane. Like the other nitrifying bacteria, its development and action are favoured by the presence of the acid carbonates of calcium and sodium. Of the latter, six grams per litre or even a smaller quantity gives good results. The sulphate of calcium can be used, but the organism prefers the carbonates. The differences between these two bacteria are small, with the exception of their chemical action. The nitric organism has no action upon ammonia, and the presence of any considerable amount of ammonium carbonate hinders its development and prevents its action on a nitrite.[40]

We may here summarise the general facts respecting nitrification. Winogradsky proposes to term the group nitro-bacteria, and to classify thus:

Nitrous organisms	=		Nitrosomonas, containing at least two species, viz., the European and the Java.
			Nitrosococcus.
Nitric organism	=		Nitrobacter.

Nitrification occurs in two stages, each stage performed by a distinct organism. By one (nitrosomonas) ammonia is converted into nitrite; by the other (nitrobacter) the nitrite is converted into nitrate.[41] Both organisms are widely and abundantly distributed in the superficial soils. They act together and in conjunction, and for one common purpose. They are separable by employing favourable media.

"If we employ a suitable inorganic solution containing potassium nitrite, but no ammonia, we shall presently obtain the nitric organism alone, the nitrous organism feeding on ammonia being excluded. If, on the other hand, we employ an ammonium carbonate solution of sufficient strength, we have selected conditions very unfavourable to the growth of the nitric organism, and a few cultivations leave the nitrous organism alone in possession of the field" (Warington).

Nitrous Organism × 1000	Nitric Organism × 1000
Nitrogen Fixing Organism from Secretion of Root-Nodules × 1000

A word upon the natural distribution of these nitrifying bacteria before we leave them. They belong to the soil, river water, and sewage. They are also said to be frequently present in well water. From some experiments at Rothamsted it appears that the organisms occur mostly in the first twelve inches, and in subsoils of clay down to three or four feet. In sandy soils nitrification may probably occur at a greater depth. These facts should be borne in mind when arranging for the purification of sewage by intermittent filtration.

We have now given some consideration to the chief events in the life-cycle of nature depicted in the table. There is but one further process in which bacteria play a part, and which requires some mention. It will have been noticed that at certain stages in the cycle there is more or less appreciable "loss" of free nitrogen. In the process of decomposition brought about by the denitrifying bacteria, a very considerable portion of the nitrogen is dissipated into the air in the form of a free gas. This is the last stage of all proteid decomposition, so that wherever putrefaction is going on there is a continual "loss" of an element essential to life. Thus it would appear at first sight that the sum-total of nitrogen food must be diminishing.

But there are other ways also in which nitrogen is being set free. In the ordinary processes of vegetation there is a gradual draining of the soil and a passing of nitrogen into the sea; the products of decomposition pass from the soil by this drainage, and are "lost" as far as the soil is concerned. Many of the methods of sewage disposal are in reality depriving the land of the return of nitrogen which is its necessity. Again, nitrogen is freed in explosions of gunpowder, nitroglycerine, and dynamite, for whatever purpose they are used. Hence the great putrefactive "loss" of nitrogen, with its subsidiary losses, contributes to reduce this essential element of all life, and if there were no method of bringing it back again to the soil, it would seem that plant life, and therefore animal life, would speedily terminate.

It is at this juncture, and to perform this vital function, that the nitrogen-fixing bacteria play their wonderful part: they bring back the free nitrogen and fix it in the soil. Excepting a small quantity of combined nitrogen coming down in rain and in minor aqueous deposits from the atmosphere, the great source of the nitrogen of vegetation is the store in the soil and subsoil, whether derived from previous accumulations or from recent supplies by manure.

Sir William Crookes has recently[42] pointed out the vast importance of using all the available nitrogen in the service of wheat production. The distillation of coal in the process of gas-making yields a certain amount of its nitrogen in the form of sulphate of ammonia, and this, like other nitrogenous manures, might be used to give back to the soil some of the nitrogen drained from it. But such manuring cannot keep pace, according to Sir W. Crookes, with the present loss of fixed nitrogen from the soil. We have already referred to several ways in which "loss" of nitrogen occurs. To these may well be added the enormous loss occurring in the waste of sewage when it is passed into the sea. As the President of the British Association pointed out,[43] the more widely this wasteful system is extended, recklessly returning to the sea what we have taken from the land, the more surely and quickly will the finite stocks of nitrogen, locked up in the soils of the world, become exhausted. Let us remember that the plant creates nothing in this direction; there is nothing in wheat which is not absorbed from the soil, and unless the abstracted nitrogen is returned to the soil, its fertility must be ultimately exhausted. When we apply to the land sodium nitrate, sulphate of ammonia, guano, and similar manurial substances, we are drawing on the earth's capital, and our drafts will not be perpetually responded to.[44] We know that a virgin soil cropped for several years loses its productive powers, and without artificial aid becomes unfertile. For example, through this exhaustion forty bushels of wheat per acre have dwindled to seven. Rotation of crops is an attempt to meet the problem, and the four-course rotation of turnips, barley, clover, and wheat witnesses to the fact that practice has been ahead of science in this matter.

The store of nitrogen in the atmosphere is practically unlimited, but it is fixed and rendered assimilable only by cosmic processes of extreme slowness. We may shortly glance at these, for it is upon these processes, plus a return to the soil of sewage, that we must depend in the future for storing nitrogen as nitrates.

1. Some combined nitrogen is absorbed by the soil or plant from the air, for example, fungi, lichens, and some algæ, and the absorption is in the form of ammonia and nitric acid. This is admittedly a small quantity.

2. Some free nitrogen is fixed within the soil by the agency of porous and alkaline bodies.

3. Some, again, may be assimilated by the higher chlorophyllous plants themselves, independently of bacteria (Frank).

4. Electricity fixes, and may in the future be made to fix more, nitrogen. If a strong inductive current be passed between terminals, the nitrogen from the air enters into combination with the oxygen, producing nitrous and nitric acids.

5. Abundant evidence has now been produced in support of the fact that there is considerable fixation by means of bacteria.

Bacterial life in several ways is able to reclaim from the atmosphere this free nitrogen, which would otherwise be lost. The first method to which reference may be made is that involving symbiosis. This term signifies "a living together" of two different forms of life, generally for a specific purpose. It may be to mutual advantage, a living for one another, or it may be, by means of an interchange of metabolism or products, finally to produce or obtain some remote chemical result. It is convenient to restrict the term symbiosis to complementary partnerships such as exist between algoid and fungoid elements in lichens, or between unicellular algæ and Radiolarians,[45] or between bacteria and higher plants. The partnerships between hermit crabs and sea-anemones and the like are sometimes defined by the term commensalism (joint diet). Symbiosis and commensalism must be distinguished from parasitism, which indicates that all the advantage is on the side of the parasite, and nothing but loss on the side of the host. The distinction between symbiosis and commensalism cannot be rigid, but between these conditions which are advantageous to the partners and parasitism, there is an obvious and radical difference. Association of organisms together for increase of virulence and function should be distinguished from symbiosis, and mere

Rootlet of Pea with Nodules existence of two or more species of bacteria in one medium is not, of course, symbiosis. Most frequently such a condition would result in injury and the subsequent death of the weaker partner, an effect precisely opposite to that defined by this term.

The example of bacteriological symbiosis with which we are concerned here is that partnership between bacteria and some of the higher plants (Leguminosæ) for the purpose of fixing nitrogen in the plant and in the surrounding soil.

The Nitrogen-fixing Bacteria, the third group of micro-organisms connected with the soil, exist in groups and colonies situated inside the nodules appearing, under certain circumstances, on the rootlets of the pea, bean, and other Leguminosæ. It was Hellriegel and Wilfarth who first pointed out that, although the higher chlorophyllous plants could not directly obtain or utilise free nitrogen, some of them at any rate could acquire nitrogen brought into combination under the influence of bacteria. Hellriegel found that the gramineous, polygonaceous, cruciferous, and other orders depended upon combined nitrogen supplied within the soil, but that the Leguminosæ did not depend entirely upon such supplies.

It was observed that in a series of pots of peas to which no nitrogen was added most of the plants were apparently limited in their growth by the amount of nitrogen locked up in the seed. Here and there, however, a plant, under apparently the same circumstances, grew luxuriantly and possessed on its rootlets abundant nodules. The experiments of Sir John Lawes and Sir Henry Gilbert at Rothamsted[46] demonstrated further that under the influence of suitable microbe-seeding of the soil in which Leguminosæ were planted there is nodule formation on the roots, and coincidentally increased growth and gain of nitrogen beyond that supplied either in the soil or in the seed as combined nitrogen. Presumably this is due to the fixation, in some way, of free nitrogen. Nobbe proved the gain of nitrogen by non-leguminous plants (Elœagnus, etc.) when these grow root nodules containing bacteria, but to all appearances, bacteria differing morphologically from the Bacillus radicicola of the leguminous plants.

	—Cellular sheath of Rootlet forming capsule of nodule. —Colonies of bacteria in situ.
Nitrogen Fixing Bacteria in Situ in Nodule on Rootlet of Pea × 400

Nitrogen Fixing Bacteria in Situ in Root-Nodule of Pea (Section of Nodule) × 500	Nitrogen Fixing Bacteria in Situ in Root-Nodule of Pea (Section of Nodule) × 600

These facts being established, the question naturally arises, How is the fixation of nitrogen to be explained, and by what species of bacteria is it performed? In the first place, these matters are simplified by the fact that there is very little fixation indeed by bacteria in the soil apart from symbiosis with higher plants. Hence we have to deal mainly with the work of bacteria in the higher plant. Sir Henry Gilbert concludes[47] that the alternative explanations of the fixation of free nitrogen in the growth of Leguminosæ seem to be:

"1. That under the conditions of symbiosis the plant is enabled to fix the free nitrogen of the atmosphere by its leaves;

"2. That the nodule organisms become distributed within the soil and there fix free nitrogen, the resulting nitrogenous compounds becoming available as a source of nitrogen to the roots of the higher plant;

"3. That free nitrogen is fixed in the course of the development of the organisms within the nodules, and that the resulting nitrogenous compounds are absorbed and utilized by the host." "Certainly," he adds, "the balance of evidence at present at command is much in favour of the third mode of explanation."

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.

Bacillus of Tetanus

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.

B. of Symptomatic Anthrax	B. of Malignant Œdema

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.

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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. Cautley[53] 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:

"A cow that had been pastured in a meadow was taken for the experiment, and the milking done out of doors, to eliminate as much as possible the influence of germs in the barn air. Without any special precaution being taken the cow was partially milked, and during the operation a covered glass dish, containing a thin layer of sterile gelatine, was exposed for sixty seconds underneath the belly of the cow in close proximity to the milk-pail. The udder, flank, and legs of the cow were then thoroughly cleaned with water, and all of the precautions referred to before were carried out, and the milking then resumed. A second plate was then exposed in the same place for an equal length of time, a control also being exposed at the same time at a distance of ten feet from the animal and six feet from the ground to ascertain the germ contents of the surrounding air. From this experiment the following instructive data were gathered. Where the animal was milked without any special precautions being taken there were 3250 bacterial germs per minute deposited on an area equal to the exposed top of a ten-inch milk-pail. Where the cow received the precautionary treatment as suggested above, there were only 115 germs per minute deposited on the same area. In the plate that was exposed to the surrounding air at some distance from the cow there were 65 bacteria. This indicates that a large number of organisms from the dry coat of the animal can be kept out of milk if such simple precautions as these are carried out."[54]

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:

	3 hours.	6 hours.	9 hours.	24 hours.
59° F.	1 +	2.5	5	163
77° F.	2	18.5	107	62,100
95° F.	4	1,290	3,800	5,370

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:

				77° F.	95° F.
2	hours	after	milking	(liquefied the plate of gelatine)	1,275,000
7	"	"	"	14,620,000	45,900,000
9	"	"	"	36,550,000	57,800,000
24	"	"	"	13,702,000,000	13,812,500,000
				[Bacteria per cub. inch.]

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:

Milk drawn at 59° C.			=	153,000	m.o.	per	cub.	in.
After	1	hour	=	616,000	"	"	"	"
"	2	hours	=	539,000	"	"	"	"
"	4	"	=	680,000	"	"	"	"
"	7	"	=	1,020,000	"	"	"	"
"	9	"	=	2,040,000	"	"	"	"
"	24	"	=	85,000,000	"	"	"	"
(Conn.)

Freudenreich gives another example, as follows:

Milk drawn at 15.5° C.			=	27,000	m.o.	per	cc.
After	4	hours	=	34,000	"	"	"
"	9	"	=	100,000	"	"	"
"	24	"	=	4,000,000	"	"	"

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:

Ordinary fresh milk = 100 per cent.		(1) Water	87.5	per	cent.
		(2) Milk-sugar	4.9	"	"
		(3) Fat	3.6	"	"
		(4) Proteids (casein, etc.)	3.3	"	"
		(5) Mineral matter	0.7	"	"
			—— 100.0

Another mode of expressing average milk constitution would be thus:

Fat	4.1	per	cent.
Solids not fat	8.8	"	"
	——
	12.9	"	"

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.

Town Shippons.			Country Shippons.			Total.
Total. 228	Infected. 12	Per cent. 5.2	Total. 67	Infected. 9	Per cent. 13.4	295

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.

Professor W. J. Simpson, lately the Medical Officer of Health for Calcutta, has placed on record an interesting series of cholera cases on board the Ardenclutha, in the port of Calcutta, which arose from drinking milk which had been polluted with one quarter of its volume of cholera-infected water. This water came from a tank into which some cholera dejecta had passed. Of the ten men who drank the milk four died, five were severely ill, and one, who drank but very little of the milk, was only slightly ill. There was no illness whatever amongst those who did not drink the milk.

Diphtheria. Recent observations on the infectivity of diphtheria in milk by Schottelius have established the fact that milk is a good medium for the bacillus of diphtheria, but that it rarely acts as a vehicle for transmitting the disease. Klein has emphasised the possibility of this means of infection. In the first place, it is obvious that the milk may become infected from a human source—from pollution with diphtheritic discharges or dried "fomites." Secondly, from a variety of different quarters evidence has been forthcoming to throw some suspicion upon the cow itself as the agent. Klein states that "a new eruptive disease on the teats and udder of the cow," consisting of papules, vesicles, and induration, may be set up by the subcutaneous inoculation of a pure culture of the Bacillus diphtheriæ. In these eruptions a bacillus similar to the B. diphtheriæ was demonstrated. On a priori grounds this evidence substantiates a belief that diphtheria, in some form or other, may be a disease of cows. Other observers have not been able to confirm these observations, and the whole matter of cow diphtheria must remain for the present sub judice.

As long ago as 1879 W. H. Power traced an epidemic of diphtheria in North London to the milk supply. In 1887 the same authority studied another outbreak, and other observers have produced further evidence in favour of the conveyance of this disease by milk. Air infection of milk by the Bacillus diphtheriæ probably occurs only very rarely, on account of the fact that the organism is readily killed by desiccation, and yet such is necessary before it can be airborne. The most frequent mode of infection of milk with this disease is from the throats, hands, bodies, or clothing of dairy workers suffering from a mild or acute form of the disease.

The specific and proved cases in which milk has acted as the vehicle of diphtheria are, it is true, comparatively few. Yet, nevertheless, the possibility of milk infection in this disease is not one which we can afford to neglect.

Scarlet Fever. Here again the evidence is not complete, chiefly owing to the fact that no specific organism of scarlet fever has yet been discovered. Many cases have, however, illustrated the undeniable conveyance of the disease by milk. Even before 1881 a number of milk epidemics of scarlet fever had been traced out. In 1882 these were further added to by Mr. W. H. Power's report concerning a series of cases in Central London. That report was remarkable for the introduction of a new feature, viz., the evidence produced in favour of the infection of milk from some disease of the cow. The Medical Department of the Local Government Board from that time took up a position of suspended judgment concerning the belief hitherto credited that milk could only be infected by human scarlet fever. In 1886 there was a remarkable epidemic in Marylebone, and the theory was suggested by Dr. Klein and Mr. Power that the cow from which the milk was derived suffered from scarlet fever.

Into the extensive controversy which raged round "the Hendon disease," as it was called, affecting the cows supplying the Marylebone milk, we cannot here enter. It will be sufficient to say that a long discussion took place as to whether or not this Hendon disease was or was not scarlet fever. The difficulty of course largely arose from the fact before mentioned that we do not at present know the specific micro-organism of scarlet fever. The Agricultural Department supported the view of Professor Crookshank that the cow disease at Hendon was cowpox, and Professor Axe further pointed out that there was evidence of the Hendon milk having been contaminated with human scarlet fever. Whichever conclusion was adopted, all were agreed upon one point, viz., that the disease had been conveyed from Hendon to persons in Marylebone by means of the milk.

Mr. Ernest Hart in 1897 published a very large number of records of scarlatinal milk infection from all parts of the country, and though the cause of the disease is obscure, there is now no doubt that it may be and is conveyed by means of milk.

Other Diseases Conveyed by Milk. In addition to the above, there are other diseases spread by means of polluted milk. From time to time exceptional cases have occurred in which a disease like anthrax has been spread by this means. But it is not to such rare cases that we refer. There are two very common diseases in which milk has been proved to play a not inconsiderable part, viz., thrush and diarrhœa.

The mould which gives rise to the curd-like patches in the throats of children, and which is known as Oidium albicans, frequently occurs in milk. Soft white specks are seen on the tongue and mucous membrane of the cheeks and lips, looking not unlike particles of milk curd. If a scraping be placed upon a glass slide with a drop of glycerine and examined by means of the microscope, the spores and mycelial threads of this mould will be seen. The spores are oval, and possess a definite capsule. The threads are branched and jointed at somewhat long intervals. Milk affords an excellent medium for the growth of this parasite. Thus undoubtedly we must hold milk partly responsible for spreading this complaint. Penicillium, Aspergillus, and Mucor are also frequent moulds in milk.

Professor MacFadyen[58] has given a full account of the ways in which milk becomes pathogenic, and his views have received further support from Professor Sheridan Delépine, who has examined more than one hundred samples of milk from Liverpool and Manchester. The result of this investigation has been that milk must be held to be one of the most potent causes of the summer diarrhœa of children. Indeed, a bacillus has been isolated identical with one which was apparently the cause of this complaint, which carries off such a large number of infants every summer. It resembles closely the Bacillus coli communis, which is an almost constant inhabitant of the alimentary canal, and is held by many bacteriologists to play, especially in conjunction with yeasts and other saprophytic organisms, an active rôle in the intestine of man.

In a recent official report[59] Dr. Hope, of Liverpool, states that "the method of feeding plays a most important part in the causation of diarrhœa; when artificial feeding becomes necessary, the most scrupulous attention should be paid to feeding-bottles." Careless feeding, in conjunction with a warm, dry summer, invariably results in a high death-rate from this cause. These two causes interact upon each other. A warm temperature is a favourable temperature for the growth of the poisonous micro-organism; a dry season affords ample opportunity for its conveyance through the air. Unclean feeding-bottles are obviously an admirable nidus for these injurious bacteria, for in such a resting-place the three main conditions necessary for bacterial life are well fulfilled, viz., heat, moisture, and pabulum. The heat is supplied by the warm temperature, the moisture and food by the dregs of milk left in the bottle; and the dry air assists in transit.

Before passing on to other matters, reference must be made to poisonous products other than bacteria which occur in milk and set up ill-health. Vaughan, of Michigan, pointed out at the London Congress of Hygiene in 1891 that he had separated a poisonous alkaloid, which he called tyrotoxicon. This, as its name denotes, was a toxic or poisonous substance, probably produced by some form of microbe. It may be taken as a type of the organic chemical substances frequently occurring in milk.