  THE BACTERIOLOGY OF THE BATE.“Omne vivum ex ovo.”—Harvey (1578–1675). When a drop of liquid from a puer wheel in use is examined under the microscope73 with 112 o.i. objective, it is seen to be swarming with bacteria. The majority are short rods (bacilli), but other forms, cocci and spirilli, are seen in lesser numbers. Most of these bacteria move briskly in the liquid; as the temperature of the slide sinks, their movements become slower, and finally cease. The illustration, Fig.14, shows the various forms of bacteria observed by the author in puer liquors ×1000 diam. Fig. 14.—Various Forms of Bacteria in Puer Liquors. The living bacteria are best examined in a drop culture in the following manner. A clean cover-glass, of the proper thickness for the objective to be used, is laid upon a black glass plate. With a platinum loop, previously heated to redness in the flame, a drop of sterile physiological salt solution (0·6 to 0·75 per cent.) or sterile broth is placed in the centre of the cover-glass. If the ring of vaseline is continuous, and the cover well pressed down, the drop is preserved from evaporation, and the bacteria may be examined in their natural condition—best on the edge of the drop. For illuminating the drop culture, the concave mirror is used, and a small diaphragm without condenser; whereas, for stained preparations, the flat mirror is used in conjunction with the Abbe condenser. If the cover-glass be carefully removed, and dried under a bell glass, the culture may be preserved in a dry condition, or may be stained and mounted.74 If the dried preparation is on a cover-glass, it should be held in the fingers (prepared side upwards), and passed slowly three times through the flame of a Bunsen burner. By holding the preparation in this way, the exact temperature for proper fixation is obtained. A drop of fuchsin stain, or gentian violet, is allowed to remain on the preparation for five minutes; wash off the superfluous dye with water, and examine, either in the wet state or after drying and mounting in balsam. For a detailed account of the technique of staining and mounting, the following works may be consulted:— Methods and FormulÆ. P.W. Squire. (Churchill.) Taschenbuch fÜr den bakteriologischen Praktikanten. Dr. Rudolf Abel. (Stubers-Verlag, WÜrzburg.) Technique Microbiologique. Nicolle and Remlinger. (Octave Doin, Paris.) Practical Bacteriology. Kanthack and Drysdale. (Macmillan, London.) The recent researches of Tissier and Metchnikoff have shown that the flora of the intestines, both of men and animals, consist very largely of anaerobic bacteria. These have been overlooked in previous researches, owing to imperfect means of studying this class of organisms. Indeed, in one work on the microbes of the alimentary canal of the dog, no mention was made of them, whereas they are all very active. Most of these organisms, and the new methods by which they have been isolated, are fully described in a new work entitled “Les AnÆrobies,” by M. Jungano and A. Distaso, of the Pasteur Institute, Paris.75 The following bacteria have up to the present been isolated from dung (mostly dog dung), and studied in pure cultures:—
It will be surmised from the above list, to which additions are still being made, that the flora of the intestines is pretty extensive, and, consequently, the study of the part played by the various species of bacteria is a long and difficult one. The methods of isolating these bacteria, and the compositions of the media employed, would demand a treatise on bacteriology; but, for general purposes, a good liquid medium for the cultivation of puer bacteria is a gelatin peptone broth, made by digesting 10grm. gelatin with 6 12grm. 80 per cent. lactic acid in 100c.c. water under pressure for three hours, neutralizing with ammonia, adding 1grm. potassium phosphate, making up to 1000c.c., and filtering. A sterile infusion of fresh dung may be used, but it is troublesome to prepare and not easy to get uniform in strength or composition. The culture liquids are left slightly alkaline, an alkalinity equal to 0·0636 per cent. Na2CO3 or 12c.c. N/1 soda per litre. The amount of alkali may be increased to 0·15 per cent. Na2CO3 without affecting the growth of the bacteria. Of solid media, 10 per cent. nutrient gelatin, or in summer 15 per cent., is good if used at temperatures below 25°C. For higher temperatures, up to 39° and 40°, nutrient agar is required. The best nutrient gelatin for general work is made according to Klein’s formula.76 For media in general, a most useful compendium is Abel’s Taschenbuch. The number of bacteria in fresh fÆces varies greatly, Dr. A.C. Houston found in raw London sewage from 3,000,000 to 9,000,000 microbes per c.c., of which more than one-tenth were gelatin-liquefying organisms. There were only about 300 spores of aerobic bacteria, about 100,000 B. coli, 100 B. enteritidis sporogenes, and streptococci, in one gram of fÆces. With the object of ascertaining the effect of the various species of bacteria contained in the dung upon skins, a large number have been isolated, and the effect of pure cultures in different media has been tried upon skin.77 A number of the results have been published in the Journal of the Society of Chemical Industry. Professor H. Becker, who has done a great deal of this part of the work, is of opinion that the principal organisms concerned in the bating exist in the dog’s intestines, and belong to the group of coli bacteria. These are very widely distributed bacteria, and are found in the large intestines of mammals, and, as a consequence, in almost all soils, and in the mud of rivers and lakes. The principal variety is B. coli commune. Fig. 15.—B. Coli Commune. Stained to show Flagellae. Lortet found it, along with other organisms, in the mud of the Lake of Geneva, at a spot where the water was chemically very pure. Dr. A.C. Houston, the bacteriologist of the Metropolitan Water Board, enumerates sixteen varieties of this organism, 80 per cent. of which produced acid and gas in lactose-peptone cultures, indol in peptone-water cultures, and when grown in milk produced acid and clot. The bacterium (Fig.15) resembles that of typhoid fever, and has frequently been mistaken for it. It is, however, much more resistent to destructive influences. It is a short bacillus, possessing flagellae, by which it moves more or less rapidly. B. coli forms short rods 0·8µ wide, 1 to 3µ long. It moves somewhat slowly by means of flagellÆ, which may be demonstrated by staining with Loffler’s method.78 It grows equally well in absence or presence of air, that is, it is a facultative anaerobe. Although it will grow at room temperature, the optimum growth is at 37°C. In plate cultures the appearance of the colonies below the surface of the gelatin is quite different from that of the surface colonies. The former are small B. coli does not liquefy the gelatin. When grown in nutrient solutions containing sugars, it produces much acid, and at the same time gases are given off, consisting of CO2 and hydrogen. If the growth in this solution be allowed to continue a secondary fermentation ensues, and the culture eventually becomes alkaline. Indol is produced by B. coli, and may be demonstrated by adding to 10c.c. of the culture, 1c.c. of a 150 per cent. solution of pure potassium nitrite; then adding a few drops of concentrated sulphuric acid, when, if indol be present, a red coloration (nitroso-indol) is produced. This bacterium reduces nitrates to nitrites. Cultivated in a 1 per cent. solution of peptone, to which 110000 per cent. of potassium nitrate has been added, after four hours at 37°C., the presence of nitrite may be shown; after the growth has continued for seventeen hours, the nitrite is further reduced to ammonia. Among other products of B. coli, Harden found lactic, formic, acetic, and succinic acids, ethyl-alcohol, CO2 and hydrogen. In Germany, W. Lembke and H. Becker have specially investigated the bacterial flora of the dog’s intestines. Lembke, in 189679 cultivated the bacteria from the fÆces of the dog, fed in various ways—bread, meat, and fat diet—and found B. coli constantly present, although the form of the individuals, as well as the colonies, and the intensity of the indol and gas formation, showed great variations. The other species of bacteria present varied with the kind of food; this has a great influence on the flora of the intestines, which was found to be very different when the dogs were fed on bread to what it was when they were fed on meat. Lembke describes two other species of bacteria closely resembling B. coli, one of which he calls B. coli anindolicum, which, as the name implies, gives no indol reaction; the other, B. coli anaerogenes, is non-motile, possesses no flagellÆ, and differs from B. coli by the absence of gas production in the fermentation of sugars. Besides B. coli, there are several species of bacteria which liquefy gelatin, and a number of facultative organisms, whose presence is more or less accidental. By changing the food, and introducing with it quantities of foreign organisms, the composition of the intestinal flora may be changed. By introducing for a considerable period B. coli anindolicum, Lembke succeeded in entirely suppressing B. coli commune. On returning to normal feeding, the foreign organisms in some cases entirely disappeared. The researches of Dr. H. Becker80 were applied more directly to the use of bacterial cultures for the bating of skins, and to the elucidation of the bacterial action of dog-dung infusions. He isolated 54 varieties of bacteria from dog-dung, and tried the action of pure cultures of many of them on a skin. A list of the various bacteria isolated by Becker is given in tabular form on pp.98–101. Fig. 16.—B. Erodiens (Becker). Fig. 17.—Plate Culture from Fresh Puer. Professor Becker’s Bacterium No. 12, which he has B. erodiens does not secrete any tryptic enzymes, hence its action on the skin is to be attributed either to an intracellular enzyme, or to its chemical products, which, being secreted in situ, have a more favourable and powerful action than if merely added to the bating liquid. It was for this reason that I proposed to use a mixed culture of bacteria, especially bacteria from the sweating process (see p.105), which secrete a mild form of proteolytic ferment, capable of dissolving the more easily soluble portion of the skin fibres (or certain constituents), but not capable of attacking the hyaline layer.
(continuation of table to right)
The practical difficulty is to keep such cultures uniform during propagation, and so far this has prevented their introduction in practice. Similar difficulties have influenced the use of pure cultures of yeast in the brewing of English beers, although the use of a single species of yeast is common in the low fermentation breweries on the Continent. I found in studying the bacteria of dog dung, that the species existing in the fresh dung, which developed in ordinary plate cultures, appeared to belong to four or five species only, mostly bacilli. At the end of two or three weeks, the original species had given place to others, mostly cocci, in a very similar way to the change which takes place in putrefaction. In fact, many of the organisms are identical with those which cause putrefaction. It will be seen, therefore, that no single species produces the complex chemical and physiological changes which take place, or the bodies necessary for the bating of skin, as some observers have supposed; but the various species succeed one another as the medium changes its reaction and composition, until finally the organic portion is resolved into the simplest bodies such as carbon dioxide, ammonia, and hydrogen. There is thus a moment when the dung is at its best so far as the bating action is concerned, and this moment is due to the vital activity of bacteria, and consequently varies according to the temperature and some other influences (electrical condition of the atmosphere, etc.). One may say it is at its best at about fifteen days in summer, and one month or more in winter. Puer which has been dried, is not so powerful in its Fig. 18.—Plate Culture from Puer Wheel. Fig. 19.—B. Putrificus. Pigeon-Dung Bate as used for Hides.—The bacteria contained in the intestines of birds and in bird dung have not been studied to the same extent as those of mammals, so that it is not possible to give anything but a meagre account of them. A microscopical examination of fresh pigeon dung, collected on a sterile Petri dish, showed debris of food, cellulose, etc., among the debris, a large number of dumb-bell bacteria (b) (Fig.20), and a few motile pairs (c); no bacilli were seen. Cells of a saccharomyces (a) were also observed. From this pigeon dung attenuations were made by a modification of Soyka’s method,82 and from the fourth attenuation a plate culture was made in ordinary nutrient gelatin. The colonies from this plate Fig. 20.—Organisms in Pigeon Dung. ×1000. A microscopical examination of a bating pit used for kips, showed an extraordinary mixture of bacteria, bacilli, vibrios, and monads; some comparatively large It would be unsafe to say from these two experiments that the bacterial effect of the pigeon-dung bate is negligible, but we may assume that it is different and not so great as with the dog-dung bate or puer. A complete research as to the various species of bacteria developing in the bird-dung bate is necessary before this question can be answered. General Considerations on the Growth of Bacteria in Various Media.—Since the publication of Further notes on the action of the dung bate (Chapter VI.), I have found that the bating organisms grow better in the special medium, when it is neutralized with ammonia, than when it is neutralized with sodium carbonate, i.e. the presence of organic ammonium salts is more favourable to the growth of the bacteria than the corresponding sodium salts. I also found that bacteria obtained from other sources than dung, viz. from the roots of wool just beginning to “slip” in a sweating stove, were equally effective in causing the skin to fall. Now these bacteria produce ammonia, and it seems clear that they are Fig. 21.—Bacillus d. Some of the fermentations taking place in the dung come under the heading of putrefactive processes (see p.116). Tyrosin is formed in considerable quantities during putrefactive fermentation, but is soon further decomposed, according to Nencki, with formation of indol, CO2 and hydrogen. Leucin gives valerianic acid, ammonia, CO2 and hydrogen; nitrogenous bodies of the aromatic series are also produced. Fig. 22.—Bacillus e. Bacillus ureaÆ, B. prodigiosus, and B. fluorescens putridus, evolve trimethylamine (Herfeldt), and, as the writer has shown, this amine has an important action in the puering process. In combination with organic acids, it removes lime from the skin, and in addition it favours the growth of bacteria, such as bacillus d and e (Figs.21 and 22) and B. coli. The albumens and peptones of the dung are pretty well decomposed and absorbed before evacuation; the bacteria subsequently split up the amido acids into fatty acids and ammonia. The fatty acids are then decomposed generally in the form of the calcium salts, in the manner shown in the table (p.108), for which I am indebted to Dr. E. Herfeldt, of Bonn. We have already treated of the action of these various products in Chapter II., but it will be seen from what has been said in the present chapter that the chemistry and bacteriology of the puer overlap, and that it is difficult, if not impossible, to separate them entirely. The bacteria are continually manufacturing chemical compounds, and decomposing others. In this respect it is interesting and instructive to note that Nencki, in his classical work “The Chemical Mechanism of Putrefaction,”85 considers the processes by which the putrefaction of proteids is brought about by bacteria, to be analogous to those taking place by melting the bodies with potash, and he holds the view that in the hydration processes brought about by bacteria, the water plays the same part as the potash.
Nencki explains, for example, the metamorphosis of leucin by putrefaction in this way: The bacteria decompose the water into hydrogen and hydroxyl, which act upon the leucin as follows:—
The resulting oxycaproic acid is then split up by the second water molecule into methylenglycol and valerianic acid:—
The methylenglycol, which changes into formaldehyde and water, is now split up into CO2 and hydrogen, as it would be by melting with caustic alkali.
As we shall see in the chapter on the action of enzymes, the phenomena are of a catalytic nature. Any urea present is decomposed, by the direct action of micrococcus ureÆ, into ammonium carbonate and ammonium carbamate, so that it does not play any part If, however, dung containing the urinary products be used in a fresh condition, the urea has indirectly a very important influence on the bating, as it favours the permeability of the skin fibre. (See p.72.) The fermentation of the cellulose in the dung has not been studied from the bating standpoint, but it is well known that it is fermented by various species of bacteria, which have been grouped together under the generic name of Amylobacter. Deherain and Gayon first showed that the solution and fermentation of cellulose in the form of dead vegetable matter, which had previously been observed, also took place in dung. Van Tieghem, in 1879, showed that the solution of cellulose is caused by bacteria, whose properties correspond with those described by him as Amylobacter. Tappeiner was able to ferment cellulose by mixed cultures of bacteria from the intestines of oxen—in neutral solution, CO2, methane, H2S, aldehyde, butyric acid, and acetic acid, were all recognized. In alkaline solutions, the principal products were CO2 and hydrogen, together with the same by-products as before. From the researches of Van Sennis, in 1890, it seems pretty certain that the fermentation of cellulose is due to the symbiotic action of at least two different organisms The decomposition of the cellulose may be explained by considering that first a sugar-like carbohydrate is Another group of organisms which have some influence in the bating process, are the class called by Beijerinck, Granulobacter. They produce butyric acid, and this acid, combining with the ammonia compounds of the dung, forms salts which undoubtedly exert an effect on the lime in the skins, though its action on the fibre is, perhaps, not so great as the compounds of lactic and propionic acids. The most common butyric ferment is the old Clostridium butyricum, now known as B. butyricus, (Prazmowsky), which is anaerobic. It forms spindle-shaped spores, hence the name Clostridium (from ???st??, a spindle). Another species (Fig.23), found in milk by Hueppe (1884), is aerobic, and ferments lactic acid and its salts to butyric acid, CO2, and hydrogen; it appears to correspond with Granulobacter polymyxa of Beijerinck. Oxalic acid is known to be produced by some bacteria and the moulds Penicillium and Sclerotinia, and in the white rot of the turnip it is produced by Pseudomonas; it is also produced by some saccharomycetes, such as B. Hansenii.87 There is reason to believe that its pro There are, of course, a large number of putrefactive bacteria in the puer, among these B. putrificus (Fig.19), isolated by Bienstock; it is a spore-bearing anaerobic bacillus, and is interesting as specially attacking fibrin. Now fibrin is extremely resistant to the action of most putrefactive bacteria, and it is very probable that specific organisms ferment the different albuminous compounds, in the same way that the different carbohydrates are each decomposed by specific ferments. Very interesting are the various forms of spirilla met with in dung; Figs.24 and 25 show Spirillum volutans in the unstained condition, and also stained to show the flagellÆ. It will be noted that the appearance is so different that, to an inexperienced observer, they might be taken for different species. The rÔle played by these organisms still requires investigation. Fig. 23.—B. Butyricus. (Hueppe.) Fig. 24.—Spirillum Volutans. (Kutscher.) Stained to show Flagellae. Fig. 25.—Spirillum Volutans. (Kutscher.) Unstained Preparation. I have pointed out previously the importance of the nutrient medium, or substratum, in which the bacteria grow, on the species surviving. In it one can see on a small scale the Darwinian process of natural selection. There is a great struggle for existence between the various species, and the circumstances determining the survival of this or that organism are extremely complicated, and we are yet very much in the dark as to the action of the various chemical compounds contained in the puer, so that it is unsafe to If we inoculate a nutrient material with a pure culture of bacteria, and the medium is not exactly adjusted to the needs of the particular organism, it will not thrive, and will speedily be overgrown by some other species obtaining access from the air. This fact very much discounts the use of pure cultures of bacteria which have been proposed for bating, although in the case of erodin, where the medium has been adjusted to suit the organism, considerable success has been attained. The whole of the enzymes and chemical compounds essential for a perfect bate, are not present in the dung when it leaves the animal’s body, but these compounds are produced by the continued action of the intestinal bacteria and other organisms which obtain access from the air. The production of the enzymes depends, too, upon the composition of the nutrient medium, since this exerts a selective influence on the species of bacteria obtaining access to it. Just as in the spontaneous souring of milk numerous bacteria have free access to it, yet the lactic ferment is generally so pure that it may be, and is, used as a pure culture on a large scale in the manufacture of lactic acid. Coming to the action of the bacteria on the skin fibres, from the work of Abt and Stiasny,88 we may conclude that the substance of the conjunctive fibres is less profoundly decomposed by bacterial fermentation than by the action of lime. The latter dissolves about 2 per cent. of skin substance from a fresh skin, whereas a puer acting normally dissolves about 1 per cent. The nuclein of the skin fibres appears to be all removed by the puer, since Abt confirms the fact that no nuclei can be seen under the microscope in a puered skin. The actual solution of the skin substance is brought about by enzymes of a tryptic character. (See Chapter V.) While the main lines of the bacteriology of the dung bate are now pretty well known and understood, it will be seen that much work still remains to be done as to details, and this principally with the anaerobic bacteria of the dung, which have been studied by few investigators.89 I have suggested90 that such a research might well be undertaken by the bacteriological laboratories of our Leather Industries Schools in Leeds and London. Moulds and Putrefaction.—In view of the fact that moulds are of frequent occurrence on dog dung, a brief mention of them is necessary. So far as our present knowledge goes the researches of Van Tieghem, De Bary, Rankin, Marshall Ward, V.H. Blackman and The following species have been noted and classified as growing on dog dung, though probably not all of them are specific. 1. Pilaira dimidiata (Grove). Certain myxobacteria are found on dung, among these Chondromyces, described as long ago as 1857 by Berkeley, and at that time included among the Hyphomycetes. It was rediscovered in 1892 by Thaxton, and owing to his researches the whole class of myxomycetes is now generally considered as a division of Bacteria. Another myxomycete, Polyangium primigenum (Quehl), The following abstract gives some account of putrefaction, and may be of use in conjunction with the account of the bacteriology of the bate which has been given. Since it was written Dr. G. Abt (see Bibliography 51) has also given a very full description of putrefactive processes as affecting leather manufacture. The subject is still occupying the attention of a large number of bacteriologists, and we may expect more light to be thrown on the whole question during the next few years. Abstract of Paper on Recent Advances in the Bacteriology of Putrefaction. Read before the Nottingham section of the Society of Chemical Industry, January 24, 1906.91 To those who have to do with the manufacture of leather, the changes which take place in the skin from the time it leaves the animal are of the utmost interest. The most important of these changes is the natural process of decomposition known as putrefaction. Putrefaction may be defined as the decomposition of nitrogenous organic matter by living organisms, Dr. Sims Woodhead (59) gives a concise account of the earliest researches on the organisms causing putrefaction by Leeuwenhoek (1692), Plenciz of Vienna, MÜller of Copenhagen (1786), Needham (1749), Spallanzani (1769), Schwann (1837), Schroeder and Van Dusch (1854), Tyndall (1870), Lister (1878). These names show that the history of putrefaction proceeds parallel with the evolution of the microscope and the development of the comparatively recent science of bacteriology. I propose to-night briefly to carry it up to the present day. I need scarcely say that putrefaction is not a specific fermentation like alcoholic or acetic fermentation, but that it is extremely complex. In any putrefying matter, such as gelatin or albumin, a large number of different species of bacteria may be observed as well as monads and infusoria, and in some cases moulds, all of which take part in the process. The first stage is a process of oxidation in the presence of air, in which Ærobic bacteria use up the oxygen present and only simple inorganic compounds are formed, carbon dioxide, nitrates and sulphates; this part of the process is generally without odour. The second stage, or true putrefaction, takes place in the absence of oxygen by anÆrobic bacteria, and That Bacterium termo (Ehr.) is not a single definite species; various forms and stages of other organisms have been described under this name. The various species of Proteus go through a wide range of forms during their development in which cocci, short and long rods, thread forms, vibrios, spirilli, and spirochÆtÆ occur. Under special nutritive conditions Proteus goes through a swarm stage, in which condition it is capable of moving over the surface and in the solid gelatin. The Proteus bacteria are facultatively anÆrobic, they all cause putrefaction; P. vulgaris and P. mirabilis are the commonest and most active of all putrefactive bacteria. They do not secrete an unorganised ferment, but decompose albuminous bodies by direct action. They also Tito Carbone (60) found amongst the products of P. vulgaris, choline, ethylenediamine, gadinine, and trimethylamine. MacÉ (61), criticising Hauser’s work, considers the cocci form of Proteus to be spores. Bienstock (62) believes the rÔle of the Proteus group somewhat doubtful. He discovered (1884) another widely distributed putrefactive organism, which he called Bacillus putrificus; it is a spore bearing, drumstick shaped bacillus found in fÆces; it is anÆrobic and specially attacks fibrin. Now fibrin is extremely resistent to the action of most putrefactive bacteria, and it is very probable that specific organisms ferment the different albuminous compounds in the same way that the different carbohydrates are each decomposed by specific ferments. A certain number of species of bacteria are able to decompose both carbohydrates and proteids. Tissier and Martelly (70) call these mixed ferments, and divide them further into two groups (1), mixed proteolytic ferments, including B. perfringens, B. bifermentans sporogenes, Staphylococcus albus, Micrococcus flavus liquefaciens, Proteus vulgaris, this group decompose albumin by means of tryptic enzymes. (2) Mixed peptolytic ferments are only able to attack the albumin when it has undergone a preliminary decomposition. This group comprises B. coli, B. filiformis, Streptococcus pyogenes, Diplococcus griseus non liquefaciens. The second class of bacteria are those which are without action on carbohydrates, and only attack proteids; these consist of the true proteolytic bacteria B. putrificus, These authors state that B. putrificus is always present in putrefying albumin, but always accompanied by facultative Ærobes which favour the growth and development of the special putrefactive bacteria. In the putrefaction of meat the reaction is first acid owing to the action of the mixed ferments on the sugars present. In the next stage ammonia is formed by the tryptic enzymes secreted by the Ærobic bacteria, and so the anÆrobic organisms are enabled to develop. We can thus understand how it is that putrefaction proceeds more rapidly the more mixed ferments there are present, although these were formerly supposed to hinder putrefaction from taking place. When meat is exposed to air it is first attacked by the mixed ferments, Micrococcus flavus liquefaciens, Staphylococcus, Bacillus coli, Bacillus filiformis, Streptococcus and Diplococcus, and becomes acid; at the same time, the presence of decomposition products of albumin may be detected, proteoses, amidoacids, amines and ammonia; the latter quickly neutralise the acids, and in three to four days the meat is alkaline, and has a faint putrid smell. Bacillus perfringens and Bacillus bifermentans sporogenes now make their appearance; the latter of these organisms produces amines, amido-acids and ammonia. In this stage the simple anÆrobic ferments are able to begin their work, and real putrefaction sets in; as this proceeds, the mixed ferments gradually disappear, and finally the only organisms remaining are Another organism, which appears to play an important part in the decomposition of animal bodies, is described by Klein (63); he found that in bodies, which had been buried from three to six weeks, bacteria such as B. coli and B. proteus had almost disappeared, and an anÆrobic bacterium, which he calls B. cadaveris sporogenes, was very active. It is a motile bacillus 2–4µ long, with flagellÆ all over its surface. Spores are formed at the rounded ends, giving it a drumstick form. It coagulates milk, the clot gradually dissolving. It grows on all the usual nutritive media, but only under strictly anÆrobic conditions. In a paper, entitled “Fermentation in the Leather Industry,”92 I gave a short account of the progress of putrefaction as it takes place in the animal skin, and also described some of the organisms I had observed in putrefying skin. A small piece of skin was placed in water and allowed to stand at room temperature. During the first two days there was little change, but on the third day a number of swiftly moving darting monads made their appearance. Some of these were propelled by flagellÆ, but a few had assumed amoeboid forms. A slowly moving bacillus consisting of a long straight rod, apparently broken up into cells exactly like the Vibrio subtilis, illustrated in the “Micrographic Dictionary,” was observed, accompanied by some species of spirillum. Higher organisms present were a Paramoecium and a colourless transparent piece of protoplasm, shaped like a dumb-bell, with a slow rotating Procter calls attention to the relative putrescibility of the different constituents of skin, and especially to the rapid putrescence of the lymph and serum. So far as I know, this part of the subject has not been studied at all thoroughly, and there is a considerable field open to workers in our research laboratories. Pure fat is not decomposed by bacteria, but if albuminous matter is present, the fat is split up by several species of bacteria and moulds. Schreiber (73) has shown that the presence of oxygen is necessary. As this subject scarcely comes within the category of putrefaction, I refer you to Schreiber’s paper, and also to an important paper by Otto Rahn (74) recently published. In the putrefaction of vegetable matter the cellulose is attacked by specific organisms, which have been thoroughly investigated by Omeliansky (75). He has I have previously stated that monads and infusoria take part in the process of putrefaction, but I do not know that their action has been studied in the same way as that of bacteria. The life history and morphology of some of these monads was studied in 1871 to 1875 by Dallinger and Drysdale (76). These authors, in their researches into the life history of the monads found in a putrefying infusion of cod’s head, came to the conclusion that “bacteria are not the only or even (in the end) the chief organic agents of putrefaction, for most certainly in the later stages of a disintegration of dead organic matter the most active agents are a large variety of flagellate monads.” Dallinger cultivated some of the monads in Cohn’s fluid, and found that they lived and multiplied in it. Their spores were killed at a temperature of 250°F. There is a big field of research open in this direction. The consideration of the chemical aspect of putrefaction is a vast subject, and would demand a special treatise. I shall only call your attention to one or two points of interest. Taking the simpler bodies first, sulphuretted hydrogen is formed in putrefying liquids in two ways: (1) Stich (65) found phosphorus pentoxide in the residue from the putrefaction of casein, nuclein, lecithin, and protagon; and in the putrefaction of certain organs of animals and plants, gases containing phosphorus are evolved. The nucleic acid of yeast yielded phosphoric acid along with hypoxanthin and xanthin. Vitali (66) found in the putrefaction of muscle, which had been freed from sugars and fat, that some alcohol was produced. He considers that a hexose is split off from the albumin in a similar manner to the splitting off of a fermentable sugar from the glucoproteids (compounds of simple proteins with carbohydrates). The formation of alcohol in the putrefaction of muscle occurs in the alkaline stage. Thus alcoholic fermentation is caused not alone by saccharomyces, but also by certain putrefactive bacteria. Lermer (77) finds that the putrefaction of barley The action of putrefactive bacteria has been found capable of transforming hexoses into pentoses. Salkowski and Neuberg (78) inoculated a solution of d-glukuronic acid with putrefying meat, and showed that it was changed into l-xylose with evolution of CO2 according to the following formula:— COH(CHOH)4COOH = CO2 + COH[CHOH]3CH2OH. This is an interesting fact, especially as, according to Neuberg, the pentose contained in animal nucleo-proteids is l-xylose. I wish to express my indebtedness to Dr. Alfred Koch’s “Jahresbericht Über GÄrungs-organismen” for some of the abstracts. The following is a list of putrefactive bacteria which have been studied in pure cultures:— 1. Proteus Vulgaris (Hauser). 2. Proteus mirabilis. 3. Proteus Zenckeri. 4. Bacillus Oedematis maligni (Kerry, Nencki, Bovet). Moulds taking part in putrefaction, principally of fruit and vegetable matter:— 1. Penicillium glaucum. 2. Mucor mucedo. 3. Mucor piriformis (Fischer, possibly identical with 2). 4. Mucor stolonifer (Ehrenberg). 5. Botrytis cinerea (Pers). 6. Mucor racemosus (Fres). 7. Monilia fructigena (Pers). 8. Fusarium putrefaciens (Osterwalder). 9. 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