The septic tank is a large underground vault of cemented brick, having a capacity of thousands of gallons, according to the population. That at Exeter has a capacity of 53,800 gallons, and takes the average sewage of 1500 inhabitants in twenty-four hours. Near the entrance is a submerged wall, seven feet from the entrance and twelve inches below the surface of the liquid in the full tank. Within this are caught, by gravity, gravel and such-like deposits. The remaining solid matter of the sewage becomes deposited in the tank itself. Both in the sediment at the bottom of the tank and in the thick scum on the surface the organic compounds are broken down and made soluble. In the former position this is accomplished by anaërobic bacteria, in the latter on the surface by aërobic bacteria. It need hardly be added that these are denitrifying and putrefactive bacteria, and that those at the bottom of the tank perform greater service than those at the top. When the liquid sewage passes out of the tank it differs from the crude sewage which enters the tank in the following particulars: (a) The gravel and particulate débris have been removed; (b) the organic solids in suspension are so greatly diminished that they are almost absent; (c) there is an increase of organic matter in solution; (d) the sewage is darker in colour and more opalescent; (e) compounds like albuminoid ammonia, urea, etc., have been more or less completely broken down, and reappear in elementary conditions, like ammonia, methane, carbon dioxide, and sulphuretted hydrogen. These latter bodies may be in solution or may have escaped as gas.

The cultivation beds are four or five filters, to which the sewage from the tank flows in such a manner as to produce a weir. By an automatic arrangement the fluid is distributed to each filter in turn. When the second filter is full the first is discharged, and remains empty during the time that the third and fourth are being filled. Each filter is thus full, say, about six hours, and has from ten to twelve hours' rest. These filter-beds (at Exeter) have an area of eighty square yards and a depth of five feet; collecting drains are laid on the bottom of the filters, joining main collectors, the latter terminating in discharging wells. The filtrant is broken furnace clinker or broken coke.

The changes occurring in these filters are of the nature of oxidation, with the result that the proportion of the oxidised nitrogen increases (as nitrites and nitrates), the ammonia becomes less, and the total solids and organic nitrogen almost disappear. It will thus be seen that the work of these filters is not merely a straining action. It is true that particulate matter in the effluent from the tank is caught on the surface by the film (resulting from previous effluents), but the real work of the bed is nitrification, an oxidation of ammonia into nitrites and nitrates. This change obviously begins when the tank effluent flows over the "weir" on to the filter-beds, and the oxygen thus obtained by the effluent is carried down in solution into the coke-breeze. Upon the surface of the filtrant are oxidising bacteria. When the effluent is on the bed they oxidise its contained products; when the bed is empty and "resting" they oxidise carbon. An advantage arising from the periodical emptying and filling of the filter is that the products of decomposition which would eventually inhibit the action of the aërobic bacteria are washed away, and pass into the nearest stream, where they become absolutely innocuous.

The "filter" is more correctly termed a cultivation bed, for its purpose is to furnish a very large surface upon which the nitrifying organisms present, as we have seen, in all soils, may flourish, and thus feeding upon the organic matter of the sewage, may perform their function of oxidation.

It is not possible to lay down exact limits as to where denitrification ends and oxidation begins. To a certain extent, and in varying degree, they overlap each other. But roughly we may say that in the tank there is a breaking down (denitrification and decomposition) and in the filter-beds a building up (nitrification). The case is precisely parallel to similar changes occurring in soil, and which we have dealt with elsewhere. The advantage indeed of this biological treatment of sewage is that it exactly follows the processes of nature, in contradistinction to the mechanical and chemical methods hitherto adopted.

At Sutton and some other places the same principles are applied,—that is to say, bacterial filtration,—but there is no tank. A metal screen in some measure takes its place, and holds back solid matter from being carried on to the beds. The filtrant is burnt clay, and it is forked over occasionally to let in oxygen. The crude sewage is run over the top of the burnt ballast, where it is left for two or three hours. It is then slowly run off on to a finer filter, where it also stays two hours. Thence the effluent is run into the stream.

It must be admitted that the bacterial treatment of sewage, though exhibiting such excellent results where it has been given a fair trial, is still in a probationary stage. It appears to stand on reason. The sludge of previous methods is avoided. The sewage is entirely broken down, and the effluent is a comparatively pure one, yet taking back nitrogen, as nitrate, to the soil. The whole change, indeed, in the opinion of Dr. Dupré, is more effective and radical than in chemical treatment. Further, it has been tested as regards its action upon the pathogenic bacilli—those of tubercle and typhoid—with the result that these infective bacteria have been completely destroyed. It appears that such destruction of infective germs occurs in the tank, and depends in degree upon the rapidity with which sewage is passed through the tank. The cultivation beds also have an inimical effect upon infective bacteria. Hence the final effluent is practically germ-free as regards pathogenic organisms.

CHAPTER III

BACTERIA IN THE AIR

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 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.

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.

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 Tyndall21 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. Shattock24 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 Germano28 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:

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.