METHODS OF EXAMINING AIR FOR BACTERIA
The basis of the usual methods in practice is to pass air over or through some nutrient medium. By this means the contained organisms are waylaid, and finding themselves under favourable conditions of pabulum, temperature, and moisture, commence active growth, and thus reveal themselves in characteristic colonies. These are examined, as directed on page 43, by the microscope and sub-culture. Quantitative estimation is not generally made, as a fixed standard is even less a possibility than in milk and soil. Returns of the number of bacteria in the sample taken may be made for the sake of information, but little or no conclusion of value can be drawn from such data. The standard recognised in Europe is the cubic metre, and one may speak, for example, of the air of a room containing 500, 1000, or 3000 germs per cubic metre.
The following are the chief methods:
1. Pouchet's Aëroscope. This apparatus was in use some time ago in France, and by its means all the solid matter of a given quantity of air was drawn through an air-tight glass tube by aspiration and made to impinge upon a small plate of glycerine. The air escaped to the aspirator at the sides, leaving upon the glycerine plate only its particulate matter. This remnant could then be examined.
2. Koch adopted the simplest of all the culture methods, viz., exposing a plate of gelatine or agar for a longer or shorter time to the air of which examination is desired. By gravity the suspended bacteria fall on the plate and start growth. As a matter of quantitative exactitude, this method is not to be recommended, but it frequently proves an excellent method for qualitative estimation.
3. The Method of Miquel. Pasteur was the first to analyse air by the culture method, and he adopted a plan which in principle is washing the air in some fluid culture medium which will retain all the particulate matter, which may then be cultured directly or sub-cultured into any favourable medium.
Miquel has contrived a simple piece of apparatus for the carrying out of this principle. It consists of a flask with a
Miquel's Flask central tube through its own neck for the entrance of the air. On one side of the flask is a tube to be connected with the aspirator, on the other side of the flask a tube through which to pour off the contained fluid at the end of the process. In the flask are placed 30 cc. of sterilised water (or, indeed, if it be preferred, sterilised broth). The entrance tube is now unplugged, and the aspirator draws through a fair sample of the air in the room (say ten litres). This air perforce passes through the water and by the exit tube to the aspirator, and is thereby washed, leaving behind in the water all its bacteria. The aspiration is then stopped, and the entrance tube closed. The water (plus bacteria) is now poured out into test-tubes of media or plated out on Petri's dishes. Provided the apparatus has been absolutely sterilised, and that the water was also sterile, any colonies developing upon the Petri dish are composed of micro-organisms from the air examined.
4. The Method of Hesse. This method is somewhat akin to Pouchet's aëroscope, but is in addition a culture method. Hesse's tube is about 2 feet long and 1-1/2 inches bore throughout. At one end is an india-rubber stopper bored for a glass tube to the aspirator. The other end is open. Before using, the tube is sterilised, and 40 or 50 cc. of sterilised gelatine replaced in it. The tube is now rapidly rotated in a groove on a block of ice or under a cold-water tap, and by this simple means the gelatine becomes fixed and forms a layer inside the tube throughout. We have therefore, so to speak, a tube of glass with a tube of gelatine inside it. The apparatus is now ready for use. It is fixed on the tripod, and fifteen litres of air are drawn through, and the tube is properly plugged and incubated at room temperature. In a day or two days the colonies appear upon the gelatine. They are most numerous generally in the first part of the tube, and might be roughly estimated as follows:
15 litres of air, 6 colonies.
⁂ 6/15 × 10,000 = 4000 aërobic bacteria in the cubic metre.
The disadvantages of this process are that dried gelatine does not catch germs like the broth cultures of Pasteur or Miquel, and that many organisms are able to go straight through the tube, and failing to be deposited, pass out at the aspirator exit, and thus are neither caught nor counted. The Hesse tube is generally used in practice with a pump consisting of two flasks and a double-way india-rubber tube. The flasks have a capacity for one litre of water. By a simple adaptation it is possible to secure siphon action, and hence measure with considerable exactitude the amount of air passing through the tube.
5. Methods of Filtration. To-day most of the above methods have been discarded, with the exception, perhaps, of Miquel's and modifications thereof.
Sedgwick's Sugar-Tube
Frankland, Petri, Pasteur, Sedgwick, and others have suggested the adoption of methods of filtration. These depend upon catching the organisms contained in the air by filtering them through sterilised sand or sugar, and then examining these media in the ordinary way. Many different kinds of apparatus have been invented. Petri aspirates through a glass tube containing sterilised sand, which after use is distributed in Petri dishes and covered with gelatine. The principal objection to this method is the presence of the opaque particles of sand in and under the gelatine. Probably it was this which suggested the use of soluble filters like sugar. Pasteur introduced the principle, and Frankland and others have followed it out. The apparatus most largely used is that known as Sedgwick's Tube. This consists of a comparatively small glass tube, about a foot long. Half of it has a bore of 2.5 cm., and the other half a bore of .5 cm. It is sterilised at 150° C., after which the dry, finely granulated cane-sugar is inserted in such a way as to occupy an inch or more of the narrow part of the tube next the wide part. Next to it is placed a wool plug, and the whole is again sterilised at 130° C. for two hours, care being taken that the sugar does not melt. After sterilisation an india-rubber tube is fixed to the end of the narrow portion, and thus it is attached to the aspirator. The measured quantity (5–20 litres) of air is drawn through, and any particulate matter is caught in the sugar. Warm, nutrient gelatine (10–15 cc.) is now poured into the broad end of the tube, and by means of a sterilised stilette the sugar is pushed down into the gelatine, where it quickly dissolves. We have now in the gelatine all the micro-organisms in the air which has been drawn through the tube. After plugging with wool at both ends, the tube is rolled on ice or under a cold-water tap in order to fix the gelatine all round the inner wall of the tube, which is incubated at room temperature. In a day or two the colonies appear, and may be examined.
Sedgwick's Tube
Fixed upon Tripod for Air Examination
Micro-organisms in the Air. Schwann was one of the first to point out that when a decoction of meat is effectually screened from the air, or supplied solely with calcined air, putrefaction does not set in. Helmholtz and Pasteur confirmed this, but it may be said with some truth that Schwann originated the germ theory, and Lister applied it in the treatment of wounds. Lister believed that if he could surround wounds with filtered air the results would be as good as if they were shut off from the air altogether.
It was Tyndall[21] who first laid down the general principles upon which our knowledge of organisms in the air is based. That the dust in the air was mainly organic matter, living or dead, was a comparatively new truth; that epidemic disease was not due to "bad air" and "foul drains," but to germs conveyed in the air, was a prophecy as daring as it was correct. From these and other like investigations it came to be recognised that putrefaction begins as soon as bacteria gain an entrance to the putrefiable substance, that it progresses in direct proportion to the multiplication of bacteria, and that it is retarded when they diminish or lose vitality.
Tyndall made it clear that both as regards quantity and quality of micro-organisms in the air there neither is nor can be any uniformity. They may be conducted on particles of dust—"the raft theory"—but being themselves endowed with a power of flotation commensurate with their extreme smallness and the specific lightness of their composition, dust as a vehicle is not really requisite. Nevertheless the estimation of the amount of dust present in a sample of air is a very good index of danger. It is to Dr. Aitken that we are indebted for devising a method by which we can measure dust particles in the air, even though they be invisible. His ingenious experiments, reported in the Transactions of the Royal Society of Edinburgh (vol. xxxv.), have demonstrated that by supersaturation of air the invisible dust particles may become visible. As is now well known, Dr. Aitken has been able to prove that fogs, mists, and the like do not occur in dust-free air, and are due to condensation of moisture upon dust particles. But it should be remembered that, though dust forms a vehicle for bacteria, dusty air is often comparatively free from bacteria. Hence, after all, the necessary conditions for dissemination of bacteria in air are two, namely, some degree of air-current and dry surfaces.
This latter condition is one of essential importance. Bacteria cannot leave a moist surface either under evaporation or by means of air-currents.[22] Only when there is considerable molecular disturbance, such as splashing, can there possibly be microbes transmitted to the surrounding air. This fact, coupled with the influence of gravitation, is the reason why sewer gas and all air contained within moist perimeters is almost germ-free; whereas from dry surfaces the least air-current is able to raise countless numbers of organisms. Quite recently this principle has been admirably illustrated in two series of investigations made upon expired and inspired air. In a report to the Smithsonian Institution of Washington (1895) upon the composition of expired air, it is concluded that "in ordinary quiet respiration no bacteria, epithelial scabs, or particles of dead tissue are contained in the expired air. In the act of coughing or sneezing such organisms or particles may probably be thrown out." The interior of the cavity of the mouth and external respiratory tract is a moist perimeter, from the walls of which no organisms can rise except under molecular disturbance. The position is precisely analogous to the germ-free sewer air as established by Messrs. Laws and Andrewes for the London County Council. The popular idea that infection can be "given off by the breath" is contrary to the laws of organismal pollution of air. The required conditions are not fulfilled, and such breath infection must be of extremely rare occurrence. The air can only be infective when filled with organisms arising from dried surfaces.
The other series of investigations were conducted by Drs. Hewlett and St. Clair Thompson, and dealt with the fate of micro-organisms in inspired air and micro-organisms in the healthy nose. They estimated that from 1500 to 14,000 bacteria were inspired every hour. Yet, as we have pointed out, expired air contains practically none at all. It is clear, then, that the inspired bacteria are detained somewhere. Lister has pointed out, from observation on a pneumo-thorax caused by a wound of the lung by a fractured rib, that bacteria are arrested before they reach the air-cells of the lung; hence it is at some intermediate stage that they are detained. Hewlett and Thomson examined the mucus from the wall of the trachea, and found it germ-free. It was only when they reached the mucous membrane and moist vestibules and vibrissæ of the nose that they found bacteria. Here they were present in abundance. The ciliated epithelium, the moist mucus, and the bactericidal influence of the wandering or "phagocyte" cells probably all contribute to their final removal.[23]
There can be no doubt that the large number of bacteria present in the moist surfaces of the mouth is the cause of a variety of ailments, and under certain conditions of ill-health organisms may through this channel infect the whole body. Dental caries will occur to everyone's mind as a disease possibly due to bacteria. As a matter of fact, probably acids (due to acid secretion and acid fermentation) and micro-organisms are two of the chief causes of decay of teeth. Defects in the enamel, inherent or due to injury, retention of débris on and around the teeth, and certain pathological conditions of the secretion of the mouth are predisposing causes, which afford a suitable nidus for putrefactive bacteria. The large quantity of bacteria which a decayed tooth contains is easily demonstrated.
From the two series of experiments which we have now considered we may gather the following facts:
(a) That air may contain great numbers of bacteria which may be readily inspired.
(b) That in health those inspired do not pass beyond the moist surface of the nasal and buccal cavities.
(c) That here there are various influences of a bactericidal nature at work in defence of the individual.
(d) That expired air contains, as a rule, no bacteria whatever.
The practical application of these things is a simple one. To keep air free from bacteria, the surroundings must be moist. Strong acids and disinfectants are not required. Moisture alone will be effectual. Two or three examples at once occur to the mind.
Anthrax spores are conveyed from time to time from dried infected hides and skins to the hands or bodies of workers in warehouses in Bradford and other places. If the surroundings were moist, and the hides moist, anthrax spores and all other bacteria would not remain free in the air.
The bacilli or spores of tubercle present in sputum in great abundance cannot, by any chance whatever, infect the air until, and unless, the sputum dries. So long as the expectorated matter remains on the pavement or handkerchief wet, the surrounding air will contain no bacilli of tubercle. But when in the course of time the sputum dries, then the least current of air will at once infect itself with the dried spores and bacilli.
Typhoid Fever, too, occupies the same position. Only when the excrement dries can the contained bacteria infect the air. It is of course well known that the common channel of infection in typhoid fever is not the air, whereas the reverse holds true of tuberculosis. The writer recently obtained some virulent typhoid excrement, and placed it in a shallow glass vessel under a bell-jar, with similar vessels of sterilised milk and of water, all at blood-heat. So long as the excrement remained moist, even though it soon lost its more or less fluid consistence, the milk and water remained uninfected. But when the excrement was completely dried it required but a few hours to reveal typhoid bacilli in the more absorptive fluid, milk, and at a later stage the water also showed clear signs of pollution. This evidence points in the same direction as that which has gone before. If the excrement of patients suffering from typhoid dries, the air will become infected; if, on the other hand, it passes in a moist state into the sewer, even though untreated with disinfectants, all will be well as regards the surrounding air.
Before passing on to consider other matters concerning organisms in the air, we may draw attention to some interesting observations recorded by Mr. S. G. Shattock[24] on the negative action of sewer air in raising the toxicity of lowly virulent bacilli of diphtheria. Some direct relationship, it has been surmised, exists between breathing sewer air and "catching" diphtheria. Clearly it cannot be that the sewer air contains the bacillus. But some have supposed that the sewer air has had a detrimental effect by increasing the virulent properties of bacilli already in the human tissues. Two cultivations of lowly virulent bacilli were therefore grown by Mr. Shattock in flasks upon a favourable medium over which was drawn sewer air. This was continued for two weeks or five weeks respectively. Yet no increased virulence was secured. Such experiments require ample confirmation, but even from this it will be seen that sewer air does not necessarily have a favouring influence upon the virulence of the bacilli of diphtheria.
It should be noted that the bacilli of diphtheria are capable of lengthened survival outside the body, and are readily disseminated by very feeble air-currents. The condition necessary for their existence outside the body for any period above two or three days is moisture. Dried diphtheria bacilli soon lose their vitality. It is probably owing to this fact that the disease is not as commonly conveyed by air as, for example, tubercle.[25]
The influence of gravity upon bacteria in the air may be observed in various ways, in addition to its action within a limited area like a sewer or a room. Miquel found in some investigations in Paris that, whereas on the Rue de Rivoli 750 germs were present in a cubic metre, yet at the summit of the Pantheon only 28 were found in the same quantity of air. At the tops of mountains air is germ-free, and bacteria increase in proportion to descent. As Tyndall has pointed out, even ultra-microscopic cells obey the law of gravitation. This is equally true in the limited areas of a laboratory or warehouse and in the open air.
The conditions which affect the number of bacteria in the air are various. After a fall of rain or snow they are very markedly diminished; during a dry wind they are increased. In open fields, free from habitations, they are fewer, as would be expected, than in the vicinity of manufactories, houses, or towns. A dry, sandy soil or a dry surface of any kind will obviously favour the presence of organisms in the air. Frankland found that fewer germs were present in the air in winter than in summer, and that when the earth was covered with snow the number was greatly reduced. Miquel and Freudenreich have declared that the number of atmospheric bacteria is greater in the morning and evening between the hours of six and eight than during the rest of the day. But we venture to express the hope that such coincidental facts may not be exalted into principles.
There is no numerical standard for bacteria in the air as there is in water. The open air possibly averages about 250 per cubic metre. On the seacoast this number would fall to less than half; in houses and towns it would rise according to circumstances, and frequently in dry weather reach thousands per cubic metre. When it is remembered that air possesses no pabulum for bacteria as do water and milk, it will be understood that bacteria do not live in the air. They are only driven by air-currents from one dry surface to another. Hence the quality and quantity of air organisms depend entirely upon environment and physical conditions. In some researches which the writer made into the air of workshops in Soho in 1896, it was instructive to observe that fewer bacteria were isolated by Sedgwick's sugar-tube in premises which appeared to the naked eye polluted in a large degree than in other premises apparently less contaminated. In the workroom of a certain skin-curer the air was densely impregnated with particles from the skin, yet scarcely a single bacterium was isolated. In the polishing-room of a well-known hat firm, in which the air appeared to the naked eye to be pure, and in which there was ample ventilation, there were found four or five species of saprophytic bacteria. Quite recently Mr. S. R. Trotman, public analyst for the city of Nottingham, estimated the bacterial quality of the air of the streets of that town during "the goose fair" held in the autumn. He used a modification of Hesse's apparatus in which the gelatine is replaced by glycerine. The air was slowly drawn through and measured in the usual way. Sterilised water was then added to bring the glycerine to a known volume, the liquid thoroughly mixed, and a series of gelatine and agar plates made with quantities varying from 0.1 to 2 cc. By this method a large number of bacteria were detected in this particular investigation, including Staphylococcus pyogenes aureus et albus, the common Bacillus subtilis, and B. coli communis.[26]
During a six years' investigation the air of the Montsouris Park yielded, according to Miquel, an average of 455 bacteria per cubic metre. In the middle of Paris the average per cubic metre was nearly 4000. Flügge accepts 100 bacteria per cubic metre as a fair average. From this fact he estimates that "a man during a lifetime of seventy years inspires about 25,000,000 bacteria, the same number contained in a quarter of a litre of fresh milk."[27] Many authorities would place the average much below 100 per cubic metre, but even if we accept that figure it is at once clear how relatively small it is. This is due, as we have mentioned, to sunlight, rain, desiccation, dilution of air, moist surfaces, etc. So essentially does the bacterial content of air depend upon the facility with which certain bacteria withstand drying that Dr. Eduardo Germano[28] has addressed himself first to drying various pathogenic species and then to mixing the dried residue with sterilised dust and observing to what degree the air becomes infected. Typhoid appears to withstand comparatively little dessication, without losing its virulence. Nevertheless, it is able to retain vitality in a semi-dried condition, and it is owing to this circumstance in all probability that it possesses such power of infection. Diphtheria, on the other hand, is, as we have pointed out, capable of lengthened survival outside the body, particularly when surrounded with dust. The question of their power of resisting long drying is an unsettled point. The power of surviving a drying process is, according to Germano, possessed by the streptococcus. This is not the case with cholera or plague. Dr. Germano classifies bacteria, as a result of his researches, into three groups: first, those like plague, typhoid, and cholera, which cannot survive drying for more than a few hours; second, those like the bacilli of diphtheria, and streptococci, which can withstand it for a longer period; thirdly, those like tubercle, which can very readily resist drying for months and yet retain their virulence. It will be obvious that from these data it is inferred that Groups 1 and 2 are rarely conveyed by the air, whereas Group 3 is frequently so conveyed. Miquel has recently demonstrated that soil bacteria or their spores can remain alive in hermetically sealed tubes for as long a time as sixteen years. Even at the end of that period the soil inoculated into a guinea-pig produced tetanus.[29]
The presence of pathogenic bacteria in the air is, of course, a much rarer contamination than the ordinary saprophytes. Tubercle has been not infrequently isolated from dry dust in consumption hospitals, and in exit ventilating shafts at Brompton the bacillus has been found. From dried sputum it has, of course, been many times isolated, even after months of desiccation. M. Lalesque failed to isolate it from the dry soil surrounding some garden seats in a locality frequented by phthisical patients. The writer also failed to isolate it from the same soil. But a very large mass of experimental evidence attests the fact that the air in proximity to dried tubercular sputum or discharges may contain the specific bacillus of the disease. Diphtheria in the same way, but in a lesser degree, may be isolated from the air, and from the nasal mucous membrane of nurses, attendants, and patients in a ward set apart for the treatment of the disease. Delalivesse, examining the air of wards at Lille, found that the contained bacteria varied more or less directly with the amount of floating matter, and depended also upon the vibration set up by persons passing through the ward and the heavy traffic in granite-paved streets adjoining. Bacillus coli, staphylococci, and streptococci, as well as B. tuberculosis, were isolated by this observer.
Some new light has been thrown upon the subject of pathogenic organisms in air by Neisser in his investigations concerning the amount and rate of air-currents necessary to convey certain species through the atmosphere. He states that the bacteria causing diphtheria, typhoid fever, plague, cholera, and pneumonia, and possibly the common Streptococcus pyogenes, are incapable of being carried by the molecules of atmospheric dust which the ordinary insensible currents of air can support, whilst Bacillus anthracis, B. pyocyaneus, and the bacillus of tubercle are capable of being aërially conveyed. This work will require further confirmation, but if its truth be established, it proves that attempted aërial disinfection of the first group of diseases is useless.
CHAPTER IV
BACTERIA AND FERMENTATION
It was Pasteur who in 1857 first propounded the true cause and process of fermentation. The breaking down of sugar into alcohol and carbonic acid gas had been known, of course, for a long period. Since the time of Spallanzani (1776) the putrefactive changes in liquids and organic matter had been prevented by boiling and subsequently sealing the flask or vessel containing the fluid. Moreover, this successful preventive practice had been in some measure correctly interpreted as due to the exclusion of the atmosphere, but wrongly credited to the exclusion of the oxygen of the air. It was not until the beginning of the present century that authorities modified their view and declared in favour of yeast cells as the agents in the production of fermentation. That this process was due to oxygen per se was disproved by Schwann, who showed that so long as the oxygen admitted to the flask of fermentative fluid was sterilised no fermentation occurred. It was thus obvious that it was not the atmosphere or the oxygen of the atmosphere, but some fermenting agent borne into the flask by the admission of unsterilised air. It was but a step to further establish this hypothesis by adding unsterilised air plus some antiseptic substance which would destroy the fermenting agent. Arsenic was found by Schwann to have this germicidal faculty. Hence Schwann supported Latour's theory that fermentation was due to something borne in by the air, and that this something was yeast. Passing over a number of counter-experiments of Helmholtz and others, we come to the work of Liebig. He viewed the transformation of sugar into alcohol and carbonic acid gas simply and solely as a non-vital chemical process, depending upon the dead yeast communicating its own decomposition to surrounding elements in contact with it.
Liebig insisted that all albuminoid bodies were unstable, and if left to themselves would fall to pieces—i. e., ferment—without the aid of living organisms, or any initiative force greater than dead yeast cells. It was at this juncture that Pasteur intervened to dispel the obscurities and contradictory theories which had been propounded.
As in all the conclusions arrived at by Pasteur, so in those relating to fermentation, there were a number of different experiments which were performed by him to elucidate the same point. We will choose one of many in relation to fermentation. If a sugary solution of carbonate of lime is left to itself, after a time it begins to effervesce, carbonic acid is evolved, and lactic acid is formed; and this latter decomposes the carbonate of lime to form lactate of lime. This lactic acid is formed, so to speak, at the expense of the sugar, which little by little disappears. Pasteur demonstrated the cause of this transformation of sugar into lactic acid to be a thin layer of organic matter consisting of extremely small moving organisms. If these be withheld or destroyed in the fermenting fluid, fermentation will cease. If a trace of this grey material be introduced into sterile milk or sterile solution of sugar, the same process is set up, and lactic acid fermentation occurs.
Pasteur examined the elements of this organic layer by aid of the microscope, and found it to consist of small short rods of protoplasm quite distinct from the yeast cells which previous investigators had detected in alcoholic fermentation. One series of experiments was accomplished with yeast cells and these bacteria, a second series with living yeast cells only, a third series with bacteria only, and the conclusions which Pasteur arrived at as the result of these labours were as follows:
"As for the interpretation of the group of new facts which I have met with in the course of these researches, I am confident that whoever shall judge them with impartiality will recognise that the alcoholic fermentation is an act correlated to the life and to the organisation of these corpuscles, and not to their death or their putrefaction, any more than it will appear as a case of contact action in which the transformation of the sugar is accomplished in the presence of the ferment without the latter giving or taking anything from it."
Pasteur occupied six years (1857–1863) with further elucidation of his wonderful discovery of the potency of these hitherto unrecognised agents, and the establishment of the fact that "organic liquids do not alter until a living germ is introduced into them, and living germs exist everywhere."
It must not be supposed that to Pasteur is due the whole credit of the knowledge acquired respecting the cause of fermentation. He did not first discover these living organisms; he did not first study them and describe them; he was not even the first to suggest that they were the cause of the processes of fermentation or disease. But, nevertheless, it was Pasteur who "first placed the subject upon a firm foundation by proving with rigid experiment some of the suggestions made by others." Thus it has ever been in the times of new learning and discovery: many contributors have added their quota to the mass of knowledge, even though one man appearing at the right moment has drawn the conclusions and proved the theory to be fact.
In order that no confusion may arise in the mind of the reader, we may here say that, although fermentation is always due to a living agent, as proved by Pasteur, the process is conveniently divided into two kinds.[30] (1) When the action is direct, and the chemical changes involved in the process occur only in the presence of the cell, the latter is spoken of as an organised ferment; (2) when the action is indirect, and the changes are the result of the presence of a soluble material secreted by the cell, acting apart from the cell, this soluble substance is termed an unorganised soluble ferment, or enzyme. The organised ferments are bacteria or vegetable cells allied to the bacteria; the unorganised ferments, or enzymes, are ferments found in the secretions of specialised cells of the higher plants and animals. With the former this book deals in an elementary fashion; with the latter we have little concern. It will be sufficient to illustrate the enzymes by a few of the more familiar examples. They form, for example, the digestive agents in human assimilation. This function is performed, in some cases, by the enzyme combining with the substance on which it is acting and then by decomposition yielding the new "digested" substance and regenerating the enzyme; in other cases, the enzyme, by its molecular movement, sets up molecular movement in the substance it is digesting, and thus changes its condition. These digestive enzymes are as follows: in the saliva, ptyalin, which changes starch into sugar; in the gastric juice of the stomach, pepsin, which digests the proteids of the food and changes them into absorptive peptones; the pancreatic ferments, amylopsin, trypsin, and steapsin, capable of attacking all three classes of food stuffs; and the intestinal ferments, which have not yet been separated in purer condition. In addition to these, there are ferments in bitter almonds, mustard, etc. Concerning these unorganised ferments we have nothing further to say. Perhaps the commonest of them all is diastase, which occurs in malt, and to which some reference will be made later.
Its function is to convert the starch which occurs in barley into sugar. These unorganised ferments act most rapidly at about 75° C. (167° F.).[31]
We may now return to the work of Pasteur and the question of organised ferments. Let us preface further remark with an axiom with which Professor Frankland sums up the vitalistic theory of fermentation, which was supported by the researches of Pasteur: "No fermentation without organisms, in every fermentation a particular organism." From these words we gather that there is no one particular organism or vegetable cell to be designated the micro-organism of fermentation, but that there are a number of fermentations each started by some specific form of agent. It is true that the chemical changes induced by organised ferments depend on the life processes of micro-organisms which feed upon the sugar or other substance in solution, and excrete the product of the fermentation. Fermentation nearly always consists of a process of breaking down of complex bodies, like sugar, into simpler ones, like alcohol and carbonic acid. Of such fermentation we may mention at least five: the alcoholic, by which alcohol is produced; the acetous, by which wine absorbs oxygen from the air and becomes vinegar; the lactic, which sours milk; the butyric, which out of various sugars and organic acids produces butyric acid; and ammoniacal, which is the putrefactive breaking down of compounds of nitrogen into ammonia. We have already referred at some length to this process when considering denitrifying organisms in the soil.
There are four chief conditions common to all these five kinds of organised fermentation. They are as follows:—
1. The presence of the special living agent or organism of the particular fermentation under consideration. This, as Pasteur pointed out, differs in each case.
2. A sufficiency of pabulum (nutriment) and moisture to favour the growth of the micro-organism.
3. A temperature at or about blood-heat (35–38° C., 98.5° F.).
4. The absence from the solution or substance of any obnoxious or inimical substances which would destroy or retard the action of the living organism and agent. Many of the products of fermentation are themselves antiseptics, as in the case of alcohol; hence alcoholic fermentation always arrests itself at a certain point.
We are now in a position to consider particular fermentations and their causal micro-organisms. These latter are of various kinds, belonging, according to botanical classification, to various different subdivisions of the non-flowering portion of the vegetable kingdom. A large part of fermentation is based upon the growth of a class of microscopic plants termed yeasts. These differ from the bacteria in but few particulars, mainly in their method of reproduction by budding (instead of dividing or sporulating, like the bacteria). Their chemical action is closely allied to that of the bacteria. Secondly, there are special fermentations and modifications of yeast fermentation due to bacteria. Thirdly, a group of somewhat more highly specialised vegetable cells, known as moulds, make a perceptible contribution in this direction. According to Hansen, these latter, so far as they are really alcoholic ferments, induce fermentation, not only in solutions of dextrose and invert sugar, but also in solutions of maltose. Mucor racemosus is the only member that is capable of inverting a cane-sugar solution; M. erectus is the most active fermenter, yielding eight per cent. by volume of alcohol in ordinary beer wort. Each of these will be referred to as they occur in considering the five important fermentations already mentioned.
Saccharomyces Cerevisiæ
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.
Cause, yeast; medium, sugar solutions; result, alcohol and carbonic acid.
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 Crookshank[33] 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]
Ascospore Formation
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.
Gypsum Block
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.
| Yeast (Saccharomyces Cerevisiæ) × 1000 | Ascospore Formation in Yeast (The capsule of the parent cell around the spores is invisible) × 1000 |
| Nitrogen Fixing Bacteria from Rootlet-Nodules (Subculture) × 1000 | Bacillus of Tetanus (From broth culture, showing spore formation) × 1000 By permission of the Scientific Press, Limited |
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.
| S. Ellipsoideus | S. Pastorianus |
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.
Cause, Mycoderma aceti; medium, wine and other alcoholic liquids; result, the formation of vinegar.
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:
| Alcohol. | Aldehyde. Water. |
| C2H6O (+ oxygen and the ferment) = C2H4O + H2O. | |
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.
Cause, Bacillus acidi lactici; medium, milk-sugar, cane-sugar, glucose, dextrose, etc.; result, lactic acid.
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.
B. Acidi Lactici
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.
Cause, Bacillus butyricus and B. amylobacter; medium, milk, butter, sugar and starch solutions, glycerine; result, butyric acid.
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:—
| C6H12O6(by simple decomposition) = 2 C3H6O3 | |
| Glucose, | Lactic acid. |
which is followed by the fermentation of the lactic acid:—
| 2 C3H6O3 = C4H8O2 + 2 CO2 + 2 H2 | |||
| Lactic acid. | Butyric acid. | Carbonic acid gas. | Free hydrogen. |
B. Butyricus
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:—
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
3. The Absorption Method. Instead of adding hydrogen to the tube or flask containing the anaërobic culture, it is
Fränkel's Tube
For Cultivation of Anaërobes feasible to add to the medium some substances, like glucose
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.