Sir George Newman

Surface soils and those rich in organic matter supply a varied field for the bacteriologist. Indeed, it may be said that the introduction of the plate method of culture and the improved facilities for growing anaËrobic micro-organisms have opened up possibilities of research into soil microbiology unknown to previous generations of workers.

From the nature of bacteria it will be readily understood that their presence is affected by geological and physical conditions of the soil, and in all soils only within a few feet of the surface. As we go down below two feet, bacteria become less, and below a depth of five or six feet we find only a few anaËrobes. At a depth of ten feet, and in the "ground water region," bacteria are scarce or absent. This is held to be due to the porosity of the soil acting as a filtering medium. Regarding the numbers of micro-organisms present in soil, no very accurate standard can be obtained. Ordinary earth may yield anything from 10,000 to 5,000,000 per gram, whilst from polluted soil even 100,000,000 per gram have been estimated. These figures are obviously only approximate, nor is an exact standard of any great value. Nevertheless, FrÄnkel, Beumer, Miquel, and Maggiora have, as the result of experiments, arrived at a number of conclusions respecting bacteria in soil which are of much more practical use. From these results it appears that, in addition to the "ground water region" being free, or nearly so, virgin soils contain much fewer than cultivated lands, and these latter, again, fewer than made soils and inhabited localities. In cultivated lands the number of organisms augments with the activity of cultivation and the strength of the fertilisers used. In all soils the maximum occurs in July and August.

But the condition which more than all others controls the quantity and quality of the contained bacteria is the degree and quality of the organic matter in the soil. The quantity of organic matter present in soil having a direct effect upon bacteria will be materially increased by placing in soil the bodies of men and animals after death. Dr. Buchanan Young two or three years ago performed some experiments to discover to what degree the soil bacteria were affected by these means. "The number of micro-organisms present in soil which has been used for burial purposes," he concludes, "exceeds that present in undisturbed soil at similar level, and this excess, though apparent at all depths, is most marked in the lower reaches of the soil."35 The numbers were as follows:—

Virgin soil, 4 ft. 6 in. = 53,436 m.o. per gram of soil.
Burial soil (8 years), 4 ft. 6 in. = 363,411 m.o. per gram of soil.
Burial"lsoil(3"), 6 ft. 6 in. = 722,751m.o. pe"per gra"

Methods of Examination of Soil. Two simple methods are generally adopted. The first is to obtain a qualitative estimation of the organisms contained in the soil. It consists simply in adding to test-tubes of liquefied gelatine or broth a small quantity of the sample, finely broken up with a sterile rod. The test-tubes are now incubated at 37°C. and 22°C., and the growth of the contained bacteria observed in the test-tube, or after a plate culture has been made. The second plan is adopted in order to secure more accurate quantitative results. One gram or half-gram of the sample is weighed on the balance, and then added to 1000 cc. of distilled sterilised water in a sterilised flask, in which it is thoroughly mixed and washed. From either of these two different sources it is now possible to make sub-cultures and plate cultures. The procedure is, of course, that described under the examination of water (p. 41 et seq.), and Petri's dishes, Koch's plates, or Esmarch's roll cultures are used. Many of the commoner bacteria in soil will thus be detected and cultivated. But it is obvious that this by no means covers the required ground. It will be necessary for us here to consider the methods generally adopted for growing anaËrobic bacteria, that is to say those species which will not grow in the presence of oxygen. This anaËrobic difficulty may be overcome in a variety of ways.

1. The air contained in the culture tube may be removed by ebullition and rapid cooling. And whilst this may accurately produce a vacuum, it is far from easy to introduce the virus without also reintroducing oxygen.

2. The oxygen may be displaced by some other gas, and though coal-gas, nitrogen, and carbon dioxide may all be used for this purpose, it has become the almost universal practice to grow anaËrobes in hydrogen. The production of the hydrogen is readily obtained by Kipp's or some other suitable apparatus for the generation of hydrogen from zinc and sulphuric acid. The free gas is passed through various wash-bottles to purify it of any contaminations. Lead acetate (1–10 per cent. water) removes any traces of sulphuretted hydrogen, silver nitrate (1–10) doing the same for arseniated hydrogen; whilst a flask of pyrogallic acid will remove any oxygen. It is not always necessary to have these three purifiers if the zinc used in the Kipp's apparatus is pure. Occasionally a fourth flask is added of distilled water, and this or a dry cotton wool pledget in the exit tube will ensure germ-free gas. From the further end of the exit tube of the Kipp's apparatus an india-rubber tube will carry the hydrogen to its desired destination. With some it is the custom to place anaËrobic cultures in test-tubes, and the test-tubes in a large flask having a two-way tube for entrance and exit of the hydrogen; others prefer to pass the hydrogen immediately into a large test-tube containing the culture (FrÄnkel's method). Either method ends practically the same, and the growth of the culture in hydrogen is readily observed. Yet another plan is to use a yeast flask, and after having passed the hydrogen through for about half an hour, the lateral exit tube is dipped into a small flask containing mercury. The entrance tube is now sealed, and the whole apparatus placed in the incubator. The interior containing the culture is filled with an atmosphere of hydrogen. No oxygen can obtain entrance through the sealed entrance tube, or through the exit tube immersed in mercury. Yet through this latter channel any gases produced by the culture could escape if able to produce sufficient pressure.

Kipp's Apparatus for the production of Hydrogen

Kipp's Apparatus
For the Production of Hydrogen

3. The Absorption Method. Instead of adding hydrogen to the tube or flask containing the anaËrobic culture, it is FrÄnkel's Tube FrÄnkel's Tube
For Cultivation of AnaËrobes feasible to add to the medium some substances, like glucose Buchner's Tube Buchner's Tube
For Cultivation of AnaËrobes or pyrogallic acid, which will absorb the oxygen which is present, and thus enable the anaËrobic requirement to be fulfilled. To various media—gelatine, agar, or broth (the latter used for obtaining the toxins of anaËrobes)—2 per cent. of glucose may be added. Pyrogallic acid, or pyrogallic acid one part and 20 per cent. caustic potash one part, is also readily used for absorptive purposes. A large glass tube of 25 cc. height, named a Buchner's cylinder, having a constriction near the bottom, is taken; and about two drachms of the pyrogallic solution are placed in the bottom of it. A test-tube containing the culture is now lodged in the upper part above the constriction. The apparatus is now placed in the incubator at the desired temperature, and the contained culture grows under anaËrobic conditions. As the pyrogallic solution absorbs the oxygen it assumes a darker tint.

4. Mechanical Methods. These include various ingenious tricks for preventing an admittance of oxygen to the culture. An old-fashioned one was to plate out the culture and protect it from the air by covering it with a plate of mica. A more serviceable mode is to inoculate, say, a tube of agar with the anaËrobic organism, and then pour over the culture a small quantity of melted agar, which will readily set, and so protect the culture itself from the air. Oil may be used instead of melted agar. Another mechanical method is to make a deep inoculation and then melt the top of the medium over a bunsen burner, and thus close the entrance puncture and seal it from the air.

5. Absorption of Oxygen by an AËrobic Culture. This method takes advantage of the power of absorption of certain aËrobic bacteria, which are planted over the culture of the anaËrobic species. It is not practically satisfactory, though occasionally good results have been obtained.

6. Lastly, there is the Air-pump Method. By this means it is obviously intended to extract air from the culture and seal of it in vacuo. The culture tubes are connected with the air-pump, and exhausted as much as possible.

Of these various methods it is on the whole best to choose either the hydrogen method, the vacuum, or the plan of absorption by grape-sugar or pyrogallic. In anaËrobic plate cultures grape-sugar agar plus 0.5 per cent. of formate of soda may be used. The poured inoculated plate should be placed over pyrogallic solution under a sealed bell-glass and incubated at 37°C. Pasteur, Roux, Joubert, Chamberland, Esmarch, Kitasato, and others have introduced special apparatus to facilitate anaËrobic cultivation, but the principles adopted are those which have been mentioned.

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 oedema, 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

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 denitrifying37 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.

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 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:

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:

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:

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.

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 recently42 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 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 Rothamsted46 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 (Eloeagnus, etc.) when these grow root nodules containing bacteria, but to all appearances, bacteria differing morphologically from the Bacillus radicicola of the leguminous plants.

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 concludes47 that the alternative explanations of the fixation of free nitrogen in the growth of LeguminosÆ seem to be:

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