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

The general microscopic appearance of yeast cells may be shortly stated as follows: they are round or oval cells, and by budding become daughter yeasts. Each consists of a membrane and clear homogeneous contents. As they perform their function of fermentation, vacuoles, fat-globules, and other granules make their appearance in the enclosed plasma. As in many vegetable cells a nucleus was detected by Schmitz by means of special methods of staining, Hansen has found the nucleus in old yeast cells from "films" without any special staining.

1. Alcoholic Fermentation.

It was Caignard-Latour who first demonstrated that yeast cells, by their growth and multiplication, set up a chemical change in sugar solutions which resulted in the transference of the oxygen from the hydrogen in the sugar compound to the carbon atoms, that is to say, in the evolution of carbonic acid gas and the production, as a result, of alcohol. If we were to express this in a chemical formula, it would read as follows:

C₆H₁₂O₆ (plus the yeast) = 2 C₂H₆O + 2 CO₂.

A natural sugar, like grape-sugar, present in the fruit of the vine, is thus fermented. The alcohol remains in the liquid; the carbonic acid escapes as bubbles of gas into the surrounding air. It is thus that brandy and wines are made. If we go a step further back, to cane-sugar (which possesses the same elements as grape-sugar, but in different proportions), dissolve it in water, and mix it with yeast, we get exactly the same result, except that the first stage of the fermentation would be the changing of the cane-sugar into grape-sugar, which is accomplished by a soluble ferment secreted by the yeast cells themselves. If now we go yet one step further back, to starch, the same sort of action occurs. When starch is boiled with a dilute acid it is changed into a gum-like substance named dextrin, and subsequently into a sugar named maltose, which latter, when mixed with these living yeast cells, is fermented, and results in the evolution of carbonic acid gas and the production of alcohol. In the manufacture of fermented drinks from cereal grains containing starch there is therefore a double chemical process: first the change of starch into sugar by means of conversion,32 and secondly the change of the sugar into alcohol and carbonic acid gas by the process of fermentation, an organic change brought about by the living yeast cells.

In all these three forms of alcoholic fermentation the principal features are the same, viz., the sugar disappears; the carbonic acid gas escapes into the air; the alcohol remains behind. Though it is true that the sugar disappears, it would be truer still to say that it reappears as alcohol. Sugar and alcohol are built up of precisely the same elements: carbon, hydrogen, and oxygen. They differ from each other in the proportion of these elements. It is obvious, therefore, that fermentation is really only a change of position, a breaking down of one compound into two simpler compounds. This redistribution of the molecules of the compound results in the production of some heat. Thus we must add heat to the results of the work of the yeasts.

When alcohol is pure and contains no water it is termed absolute alcohol. If, however, it is mixed with 16 per cent. of water, it is called rectified spirit, and when mixed with more than half its volume of water (56.8 per cent.) it is known as proof spirit.

We shall have to consider elsewhere a remarkable faculty which some bacteria possess of producing products inimical to their own growth. In some degree this is true of the yeasts, for when they have set up fermentation in a saccharine fluid there comes a time when the presence of the resulting alcohol is injurious to further action on their part. It has become indeed a poison, and, as we have already mentioned, a necessary condition for the action of a ferment is the absence of poisonous substances. This limit of fermentation is reached when the fermenting fluid contains 13 or 14 per cent. of alcohol.

Having discussed shortly the "medium" and the results, we may now turn to the bacteriology of the matter, and enumerate some of the chief forms of the yeast plant. Professor Crookshank33 gives more than a score of different members of this family of Saccharomycetes. Before dwelling upon some of the chief of these, it will be desirable to consider a number of properties common to the genus.

The yeast cell is a round or oval body of the nature of a fungus, composed of granular protoplasm surrounded by a definite envelope, or capsule. It reproduces itself by budding, or, as it is sometimes termed, gemmation. At one end of the cell a slight swelling or protuberance appears, which slowly enlarges. Ultimately there is a constriction, and the bud becomes partly and at last completely separated from the parent cell. In many cases the capsules of the daughter cell and the parent cell adhere, thus forming a chain of budding cells. The character of the cell and its method of reproduction do not depend merely upon the particular species alone, but are also dependent upon external circumstances. There are differences in the behaviour of species towards different media at various temperatures, towards the carbohydrates (especially maltose), and in the chemical changes which they bring about in nutrient liquids. In connection with this Professor Hansen has pointed out that, whilst some species can be made use of in fermentation industries, others cannot, and some even produce diseases in beer.34

One of the most remarkable evidences of the adaptability of the yeasts to their surroundings and a specific characteristic occurs in what is sometimes called ascospore formation. If a yeast cell finds itself lacking nourishment or in an unfavourable medium, it reproduces itself not by budding, but by forming spores out of its own intrinsic substance, and within its own capsule. To obtain this kind of spore formation Hansen used some gypsum blocks as medium on which to grow his yeast cells. Well-baked plaster of Paris is mixed with distilled water, and made into a liquid paste. Small moulds are made by pouring this paste into cardboard dishes, where it hardens again. The mould is sterilised by heat, and a small portion of yeast is placed on its upper surface, and then the whole is floated in a small vessel of water and covered with a bell-jar. Under these conditions of limited pabulum the cell undergoes the following changes: it increases in size, loses much of its granularity, and becomes homogeneous, and about thirty hours after being sown on the gypsum there appear several refractile cells inside the parent cell. These are the ascospores. In addition to the gypsum, it is necessary to have a plentiful supply of oxygen, some moisture (gained from the vessel of water in which the gypsum floats), a certain temperature, and a young condition of the protoplasm of the parent yeast cells. Hansen found that the lowest temperature at which these ascospores were produced was .5–3° C., and at the other extreme up to 37° C., which is blood-heat. The rapidity of formation also varies with the temperature, the favourable degree of warmth being about 22–25° C.

Hansen pointed out that it was possible by means of sporulation to differentiate species of yeasts. For it happens that different species show slight differences in spore formation, e. g.:

(a) The spores of Saccharomyces cerevisiæ expand during the first stage of germination, and produce partition walls, making a compound cell with several chambers. Budding can occur at any point on the surface of the swollen spores. To this group belong S. pastorianus and S. ellipsoideus.

(b) The spores of Saccharomyces Ludwigii fuse in the first stage, and afterwards grow out into a promycelium, which produces yeast cells.

(c) The spores of Saccharomyces anomalus are different in shape from the others in that they possess a projecting rim round the base.

Another point in the cultivation of yeasts has been elucidated by a number of workers, chief among whom perhaps is Hansen, namely, methods of obtaining pure cultures. We know, generally speaking, what this term means, and there is no difference in its meaning here to what is understood as its meaning with regard to bacteria. There is, however, some difference in the mode of securing it. It is only by starting with one individual cell that we can hope to secure a pure culture of yeasts. For the study of the morphology of yeasts under the microscope the problem was not a difficult one. It was comparatively easy to keep out foreign germs from a cover-glass preparation enough to perceive germination of spores and growth of mycelium. But when we require pure cultures for various physiological purposes, then a different standard and method are necessary.

Pasteur and Cohn adopted a practice based upon the fact that when organisms find themselves in a favourable medium they multiply to the exclusion of others to which the medium is less favourable. Hence if an impure mixture be placed under such circumstances there comes a time when those organisms for which the circumstances are favourable multiply to such an extent that they form an almost pure culture. The method is open to fallacy, and will rarely result in a really pure culture; and even if that be secured, it is quite possible that it will be to the exclusion of the desired culture. Hansen has devised a much improved process for securing a pure culture of yeast which depends upon dilution. We believe Lister was one of the first who, in the seventies, introduced some such plan as this. Hansen employed dilution with water in the following manner:

Yeast is diluted with a certain amount of sterilised water. A drop is carefully examined under the microscope, a single cell of yeast is taken, and a cultivation made upon wort. When it has grown abundantly a quantity of sterilised water is added. From this, again, a single drop is taken and added, to, say 20 cc. of sterilised water in a fresh flask. This flask will contain we will suppose ten cells. It is now vigorously shaken, and the contents are divided into twenty portions of 1 cc. each, and added to twenty tubes of sterilised water. It is highly probable that half of those tubes have received one cell each. In the course of a few days it can be seen how far a culture is pure. If only one colony is present, the culture is a pure one, and as this grows we obtain an absolutely pure culture in necessary quantity. Even when the gelatine-plate method is used it is desirable to start with a single cell (Hansen). The advantage of Hansen's yeast method over Koch's bacterial-plate method is that it has a certain definite starting-point. This is obviously impossible when dealing with such microscopic particles as the bacteria proper.

A third matter in the differentiation of yeast species is the question of films. Hansen set to work, after having obtained pure cultures and ascospores, to examine films appearing on the surface of liquids undergoing fermentation. The object of this was to ascertain whether all yeasts produced the same mycelial growth on the surface of the fermenting fluid. To produce these films the process is as follows: Drop on to the surface of sterilised wort in a flask a very small quantity of a pure culture of yeast; secure the flask from movement, and protect it, not from air, which is necessary, but from falling particles in the air. In a short time small colonies appear, which coalesce and form patches, then a film or membrane which covers the liquid and attaches itself to the sides of the flask. By the differences in the films and the temperatures at which they form it is possible to obtain something of a basis for classification. The further advances in a yeast culture and in our knowledge of the agencies of fermentation have, however, tended to show that no strict dividing lines can be drawn. Hansen's researches have, notwithstanding, been of the greatest moment to the whole industry of fermentation. What has been found true in bacteriology has also been demonstrated in fermentation, namely, that though many yeasts differ but little in structure and behaviour, they may produce very different products and possess very different properties. Industrial cultivation of these finer differences in fermentative action has to a large extent revolutionised the brewing industry.

The formation of films is not a peculiarity of certain species, but must be regarded as a phenomenon occurring somewhat commonly amongst yeasts. The requisites are a suitable medium, a yeast cell, a free, still surface, direct access of air, and a favourable temperature. The wort loses colour, and becomes pale yellow. Microscopic differences soon appear between the sedimentary yeast and the film yeast of the same species, the latter growing out into long mycelial forms, the character of which depends in part upon the temperature. This often varies from 3° to 38° C.

A fourth point helpful in diagnosis is the temperature which proves to be the thermal death-point. Saccharomyces cerevisiæ is killed by an exposure to 54° C. for five minutes, and 62° C. kills the spores. As a rule, yeasts can resist a considerably higher temperature when in a dry state than in the presence of moisture.

Lastly, yeasts may be cultivated on solid media. Hansen employed wort-gelatine (5 per cent. gelatine), and found that at 25° C. in a fortnight the growths which develop show such microscopic differences as to aid materially in diagnosis. Saccharomyces ellipsoideus I. exhibits a characteristic network which readily distinguishes it.

There is one other point to which reference must be made. The process of fermentation may be set up by a "high" or a "low" yeast. These terms apply to the temperature at which the process commences. "High" yeasts rise to the surface as the action proceeds, accomplish their work rapidly, and at a comparatively high temperature, say about 16° C.; "low" yeasts, on the contrary, sink in the fermenting fluid, act slowly, and only at the low temperature of 4° or 5° C. This is maintainable by floating ice in the fluid. Formerly all beer was made by the "high" mode, but on the continent of Europe "low" yeast is mostly used, while the "high" is in vogue in England. This latter method is more conducive to the development of extraneous organisms, and therefore risky in all but well-ordered brewing establishments. Whether high and low yeasts consist of one or several species is not known.

Before proceeding to mention shortly some of the commoner forms of yeast we must again emphasise Hansen's method of analysis in separating a species. The shape, size, and appearance of cells are not sufficient for differentiation, because it is found that the same species when exposed to different external conditions can occur in very different forms. Hence Hansen established the analytical method of observing (1) the microscopic appearance, (2) the formation of ascospores, and (3) the formation of films. In addition, the temperature limits, cultivation on solid media, and behaviour towards carbohydrates, are characters which aid in the separation of yeasts. By basing differentiation of species upon these features, the following can be distinguished:

Saccharomyces Cerevisiæ. Oval or ellipsoidal cells; reproduction by budding; ascospores, rapidly at 30° C., slowly at 12° C., not formed at all at lower temperatures; film formation, seven to ten days at 22° C.; an active alcoholic ferment, producing in a fortnight in beer wort from 4 to 6 per cent. by volume of alcohol (Jörgensen). This species is a typical English "high" yeast, possessing the power of "inverting" cane-sugar previous to producing alcohol and carbonic acid. It is said to have no action on milk-sugar.

Saccharomyces Ellipsoideus I. Round, oval, or sausage-shaped cells, single or in chains; ascospores in twenty-four hours at 25° C. (not above 30° C., not below 4° C.). Grown on the surface of wort-gelatine, a network is produced by which they can be recognised (in eight to twelve days at 33° C.). At 13–15° C. a characteristic branching mass is produced. It is an alcoholic ferment as active as S. cerevisiæ. S. Ellipsoideus II. Round and oval, rarely elongated, a widely distributed yeast, causing "muddiness" in beer and a bitter taste. It is essentially a "low" yeast.

Saccharomyces Conglomeratus is a round cell, often united in clusters, and occurring in rotting grapes, and at the commencement of fermentation.

Saccharomyces Pastorianus I. Oval or club-shaped cells, occurring in after-fermentation of wine, etc., and producing a bitter taste, unpleasant odour, and turbidity. The spores frequently occur in the air of breweries.

S. Pastor. II. Elongated cells, possessing an invertose ferment. They do not, like S. pastor I., produce disease in beer.

S. Pastor. III. Oval or elongated cells, producing turbidity in beer. Grown on yeast-water gelatine, the colonies show after sixteen days crenated hairy edges.

Saccharomyces Apiculatus. Lemon-shaped cells. They give rise to a feeble alcoholic fermentation, and produce two kinds of spores—round and oval; they appear at the onset of vinous fermentation, but give way later on to S. cerevisiæ.

Saccharomyces Mycoderma. Oval or elliptical cells, often in branching chains. They form the so-called "mould" on fermented liquids, and develop on the surface without exciting fermentation. When forced to grow submerged they produce a little alcohol.

Saccharomyces Exiguus. Conical cells, appearing in the after-fermentation of beer.

Saccharomyces Pyriformis. Oval cells, converting sugary solutions containing ginger into ginger-beer.

Saccharomyces Illicis, Hansenii, et Aquifolii produce a small percentage of alcohol.

2. Acetous Fermentation.

If alcohol be diluted with water, and the specific ferment mixed with it and exposed to the air at 22° C., it is rapidly converted into vinegar. The change is accompanied by the absorption of oxygen, one atom of which combines with two of hydrogen to form water, and a substance remains called aldehyde, further oxidation of which produces the acetic acid. We may express it chemically thus:

The aldehyde becomes further oxidised:

C₂H₄O + O = C₂H₄O₂ (acetic acid).

Now this method of simply oxidising alcohol to obtain acetic acid may be carried out chemically without any ferment. If slightly diluted alcohol be dropped upon platinum black, the oxygen condensed in that substance acts with energy upon the spirit, and union readily occurring, acetic acid results. Here the whole business of the platinum sponge is to persuade the oxygen of the air and the hydrogen of the alcohol to unite. In the ordinary manufacture this is accomplished by the vegetable cells of Mycoderma aceti.

There are two chief methods adopted in the commercial manufacture of vinegar, both of which depend upon the presence of the Mycoderma. The method in vogue at Orleans when Pasteur (about 1862) commenced his studies of the vinegar organism was to fill vats nearly to the brim with a weak mixture of vinegar and wine. Where the process is proceeding the surface is covered with a fragile pellicle, "the mother of vinegar," which is produced by and consists of certain micro-organisms whose function is to convey the oxygen of the air to the liquor in the vats, thus oxidising the alcohol into vinegar. This oxidation may be carried on even beyond the stage of acetic acid (when no more alcohol remains to be oxidised), resulting in carbonic acid gas, which escapes into the air. But as in the alcoholic, so in the acetic, fermentation, there comes a time when the presence of an excess of the acid inhibits the further growth of the organism. This point is approximately when the acetic acid has reached a percentage as high as 14. But if the acid be removed, and fresh alcohol added, the process recommences.

The second method, sometimes called by the Germans the "quick vinegar process," is to pour the weakened alcohol through a tall cylinder filled with wood-shavings, having first added some warm vinegar to the shavings. After a number of hours the resulting fluid is charged with acetic acid. What has occurred? Liebig maintained that a chemical and mechanical change had brought about the change from the alcohol put into the cylinder and the vinegar drawn off at the exit tube. It was reserved for Pasteur to demonstrate by experiment that the addition of the warm vinegar to the shavings was in reality an addition of a living micro-organism, which, forming a film upon the shavings, became "the mother of vinegar," and oxidised the alcohol which passed over it, inducing it to become aldehyde and then acetic acid.

Mycoderma Aceti (described by Persoon 1822, Kützing 1837, and Pasteur 1864). It must be understood that this term is the name rather of a family than an individual. Pasteur believed it to be a specific individual, but Hansen pointed out that it was composed of two distinctly different species (Bacterium aceti and B. pasteurianum), and subsequently other investigators have added members to the acetic fermentation group of which M. aceti is the type.

This bacterium is made up of small, slightly elongated cells, with a transverse diameter of 2 or 3 µ, sometimes united in short chains of curved rods. They frequently show a central constriction, are motile, and produce in old cultures involution forms. The way in which the cells act and are made to perform their function is as follows: A small quantity, taken from a previous pellicle, is sown on the surface of an aqueous liquid, containing 2 per cent. of alcohol, 1 per cent. of vinegar, and traces of alkaline phosphates. Very rapidly indeed the little isolated colonies spread, and, becoming confluent, form a membrane or pellicle over the whole area of fluid. When the surface is covered the alcohol acidifies to vinegar. After this it is necessary to add each day small quantities of alcohol. When the oxidation is completed the vinegar is drawn off, and the membrane is collected and washed, and is then again ready for use. It ought not to remain long out of fermenting liquid, nor ought it to be allowed to over-perform its function, for thus having oxidised all the alcohol it will commence oxidation of the vinegar.

In wort-gelatine Bacterium pasteurianum develops round colonies with a smooth or wavy border, whilst B. aceti has a tendency towards stellate arrangement. Spores have not been observed, and from a morphological point of view the two species behave alike. Neither produces any turbidity in the liquid containing them. In order to flourish, B. aceti requires a temperature of about 33° C. and a plentiful supply of oxygen. In a cool store or cellar there is, therefore, nothing to fear from B. aceti. Frankland has isolated a Bacillus ethaceticus, which is a fermentative organism producing ethyl-alcohol and acetic acid. By oxidation the ethyl-alcohol may be converted into acetic acid.

3. Lactic Acid Fermentation.

The process set up by the lactic ferment is simply a decomposition, an exact division of one molecule of sugar into two molecules of lactic acid, there being neither oxidation nor hydration. The conditions under which the ferment acts are very similar to those we have already considered. There is frequently carbonic acid gas formed; there is a cessation of fermentation when the medium becomes too acid; there is the same method of starting the process by inoculation of sour milk or cheese or any substance containing the specific bacillus. It is probable that such inoculated matter will contain a mixture of micro-organisms, but if the lactic bacillus is present, it will grow so vigorously and abundantly that the fermentation will be readily set up.

The Bacillus Acidi Lactici. Rods about 2 µ long and 4 µ wide, occurring singly or in chains and threads. It is non-motile. Spore formation is present, the spores appearing irregularly or at one end of the rod.

On the surface of gelatine a delicate growth appears along the track of the needle, with round colonies appearing at the edges of the growth. It does not liquefy gelatine. It grows best at blood-heat; but much above that it fails to produce its fermentation, and it ceases to grow under 10° C. It inverts milk-sugar and changes it to dextrose, from which it then produces lactic acid. Sugars do, however, differ considerably in the degrees to which they respond to the influence of the lactic ferment, and some which are readily changed by the alcoholic ferment are untouched by the Bacillus acidi lactici. It will be necessary to refer again to this micro-organism when we come to speak of milk and other dairy products.

Van Laer has described a saccharobacillus which produces lactic acid amongst other products, and brings about a characteristic disease in beer, named tourne. The liquid gradually loses its brightness and assumes a bad odour and disagreeable taste. The bacillus is a facultative anaërobe. A number of workers have separated organisms, having a lactic acid effect, which diverge considerably from the orthodox type of lactic acid bacillus. This is but further evidence of a fact to which reference has been made: that nomenclature restricted to one individual has now become adapted to a family.

4. Butyric Acid Fermentation.

When sugars are broken down by the Bacillus acidi lactici the lactic acid resulting may, under the influence of the butyric ferment, become converted into butyric acid, carbonic acid, and hydrogen. Neither butyric acid nor lactic acid is as commonly used as alcohol or vinegar. Both, like vinegar, can be manufactured chemically, but this is rarely practised. Butyric acid is a common ingredient in old milk and butter, and its production by bacteria is historically one of the first bacterial fermentations understood. Moreover, in its investigation Pasteur first brought to light the fact that certain organisms acted only in the absence of oxygen. In studying a drop of butyric fermenting fluid, it was observed that the organisms at the edge of the drop were motionless and apparently dead, whilst in the central portion of the drop the bacilli were executing those active movements which are characteristic of their vitality. To Pasteur's mind this at once suggested what he was able later to demonstrate, namely, that these bacilli were paralysed by contact with oxygen. When he passed a stream of air through a flask containing a liquid in butyric fermentation, he observed the process slacken and eventually cease. So were discovered the anaërobic micro-organisms. The aërobic ferments give rise to oxidation of certain products of decomposition; the anaërobic organisms, on the other hand, only commence to grow when the aërobic have used up all the available oxygen. Thus in such fermentations certain bodies (carbohydrates, fatty acids, etc.) undergo decomposition, and by oxidation become carbonic acid gas, and the remainder is left as a "reduced" product of the whole process. Hence sometimes this is termed fermentation by reduction. The chemical formula of this butyric reaction may be expressed thus:—

which is followed by the fermentation of the lactic acid:—

Bacillus Butyricus. Long and short rods, generally straight, with rounded ends, single or in chains, reproducing themselves both by fission and spores, and sometimes growing out into long threads, actively motile, anaërobic, and liquefying. The spores are widely distributed in nature, and grow readily on fleshy roots, old cheese, etc. The favourable temperature is blood-heat, and on liquid media they produce a pellicle. The resistant spores are irregularly placed in the rod, and may cause considerable variations in morphology. The culture gives off a strong butyric acid odour. It grows most readily at a temperature of about 40° C.

Although, according to Pasteur's researches, the butyric acid ferment performs its functions anaërobically, many butyric organisms can act in the presence of oxygen, and yield somewhat different products.

All of them, however, ferment most actively at a temperature at or about blood-heat, and the spores are able to withstand boiling for from three to twenty minutes (Fitz). It will be observed that as in lactic acid fermentation so in butyric, the results are not due to one species only.

5. Ammoniacal Fermentation (see under Soil).

Diseases in Beer. We have seen how a knowledge of fermentation has been compiled by a large number of workers. Spallanzani, Schwann, Pasteur, and Hansen all made epoch-making contributions. In the same way the investigations of diseases in beers and wines were carried out by many observers, and were closely connected with those relating to spontaneous generation and mixed cultures of bacteria in fermentation. These so-called "diseases" are analogous to the taints occurring in milk and due to fermentations. Turning (tourne), turbidity, ropiness, bitterness, acidity, mouldiness, are all terms used to describe these diseases. They are chiefly brought about by four agencies:—

1. Bacteria.
2. Mixed yeasts.
3. "Wild" yeasts.
4. Moulds.

To each species of wild yeast there belongs some taint-producing power in the fermentations for which it is responsible. Saccharomyces ellipsoideus II. and S. pastorianus I., III., are such yeasts; they only produce their diseases when introduced at the commencement of the fermentation.

Saccharomyces pastorianus I. is a low fermentative yeast in elongated cells, producing a bitter taste to beer and an unpleasant odour. It can also produce turbidity. S. pastorianus III. produces turbidity, and S. ellipsoideus II. has a similar effect.

In 1883 Hansen demonstrated that the much-dreaded turbidity and disagreeable tastes and smells in beer may be due to mixture of two yeasts, each of which by itself gives a faultless product.

Industrial Application of Bacterial Ferments. From what has been said we trust it has been made evident that bacteriology has a place of ever-increasing importance in regard to fermentative processes. Not only have the causal agents of various fermentations been isolated and studied, but from their study practical results follow. The question of pure cultures alone is one of practical importance; the recognition of the causes of "diseases" of beer is another.

We cannot enter into a full discussion of the rôle of bacteria in industrial processes, but several of the chief directions may be pointed out. Without exception, bacteria have a part in them on account of their powers of fermentation. In securing their food, bacteria break down material, and bring about chemical and physical change. The power which organisms have of chemically destroying compounds is in itself of little importance, but the products which arise as a result are of an importance in the world which has not hitherto been recognised. We have used bacteria abundantly in the past, but we have not perceived that we were doing so. The maceration industries may be mentioned as illustrative of this use without acknowledgment. The flax stem is made up of cellular substance, flax fibres, and wood fibres; the later are of no service in the making of linen, but the whole is bound together by a gummy, resinous substance. Now this connective element is got rid of in the process of retting. There is dew-retting and water-retting. The former is practised in Russia, and consists in spreading the flax on the grass and exposing it to the influence of dew, rain, air, and light. The result is a soft and silky fibre. Water-retting is accomplished by means of steeping the flax in bundles, roots downwards, in tanks or ponds. In ten to fourteen days, according to the weather, fermentation sets in, and breaks the "shore" or "shive" from the fibre, and the process is complete. This is always done by the aid of bacteria, which, under the favourable circumstances, multiply rapidly, and cause decomposition of the pectin resinous matter. The same operation occurs in jute and hemp. Sponges, too, are cleared in this manner by the rotting of the organic matter in their interstices. The preparation of indigo from the indigo plant is brought about by a special bacterium found on the leaves. If the leaves are sterilised, no fermentation occurs, and no indigo is formed. Tobacco-curing is also in part due to decomposition bacteria, and several bacteriologists have experimented independently in fermenting tobacco leaves by the action of pure cultures obtained from tobacco of the finest quality.

In all these applications we have advanced only the first stage of the journey. Nevertheless, here, as in nature on a big scale in the formation of fertile soils and coal-measures, we find bacteria silently at work, achieving great ends by co-operating in countless hordes.

CHAPTER V

BACTERIA IN THE SOIL

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:—

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.