Chapter II., but the organisms producing it and their mode of action

still remain to be investigated.

[87] Reynolds Green, Fermentation, p. 350.

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.

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 neglect even those which are present in only small amounts. Very minute quantities of certain bodies, almost too small for detection by chemical means, are sufficient to cause large differences in the growth of certain organisms. For instance, Raulin found that the addition of a trace of zinc to his nutrient liquids increased the crop of the mould Aspergillus niger more than four times the weight of a crop grown in the same liquid free from zinc.

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.

[88] Sensibilité de la peau verte, et de la peau après l’échauffe, les pelains, et les confits, à l’égard de la chaux du sel, et de l’acide acétique. Georges Abt et Edmund Stiasny, Collegium 1910, p. 1 9.

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 suggested[90] that such a research might well be undertaken by the bacteriological laboratories of our Leather Industries Schools in Leeds and London.

[89] See Les Anaerobies, Jungano and Distaso (Masson et Cie Paris, 1910).

[90] The Bacteriology of the Leather Industry, J.S.C.I., 1910, p. 666.

*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 others indicate that their action on the essential bating constituents of the dung is a destructive one. They grow usually on acid media, and in so doing break down the acids present into simple inorganic bodies, such as CO_{2} and water, utilizing the carbon and nitrogen for their own growth. Although these fungi secrete almost all varieties of enzymes (Bourquelot), yet we have no evidence that any of the enzymes contained in dog dung are from this source. In the usual case of dung preserved in pits or casks, the upper surface only becomes mouldy, since moulds require a free supply of oxygen. The mycelium penetrates but a very little way into the body of the dung, and cannot therefore effect any decomposition, except of the surface layer. The dung exposed to the action of the mould is generally a bad colour, and is rejected as unsuitable for puering.

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).

2. Mucor caninus (a variety of Mucor mucedo).

3. Circinella simplex (Van Tieghem).

4. Pilobolus crystallinus (also on cow-dung).

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), forming a red fructification on dog dung, is figured in the Encyclopædia Britannica, XI. edition, vol. 3, p. 163.

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]

[91] Reprinted from the Journal of the Society of Chemical Industry, February 15, 1906, No. 3, vol. xxv. The numbers in brackets refer to the Bibliography, Chapter XI.

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, accompanied by the evolution of malodorous gases. The study of it may be divided into two parts--(1) the biological, (2) the chemical. The first concerns the organisms which break down the proteid molecule either directly or by means of enzymes; the second that of the different products of the action of these organisms. It is extremely difficult to separate these two studies.

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 is a process of reduction. It has been shown that there are no bacteria in healthy tissues, and if a muscle or any organ is taken from an animal under antiseptic conditions it may be preserved indefinitely in a sterile vessel to which filtered air has free access. Solid matter is usually liquefied by organisms like B. liquefaciens magnus, which are invariably present in the air, and which prepare the way for more specifically putrefactive bacteria, such as Proteus vulgaris and B. putrificus, but if one observes a number of putrefactions of the same kind of matter under natural conditions, scarcely any two follow the same course. The modern study of putrefaction dates from Hauser (58), who, in 1885, isolated from putrefying flesh the three organisms--Proteus vulgaris, P. mirabilis, and P. zenkeri. He studied the action of these in pure cultures, and came to the following conclusions:--

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 produce a powerful poison, of which small quantities injected into animals produce septicæmia.

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, and B. putidus gracilis, and the peptolytic bacteria, Diplococcus magnus anaerobius and Proteus zenkeri, which can only decompose peptones.

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 Bacillus putrificus, Bacillus putidus gracilis, and Diplococcus griseus non liquefaciens.

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 2–4 µ 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 amœboid 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 Paramœcium and a colourless transparent piece of protoplasm, shaped like a dumb-bell, with a slow rotating motion. On the fifth day the number of vibrios and spirilli had greatly increased, some with a swifter motion than others. There were also many large infusoria present; one of a peculiar double form, which appeared to be a development of the dumb-bell shaped piece of protoplasm seen on the third day. On the seventh day the most striking feature was the great increase in the number of vibrios; the field of the microscope was crowded; masses of the bacilli could be seen clustered round small particles of the disintegrating skin as if feeding upon it; there were more infusoria, many of them short, boat-shaped monads, with a trembling motion, refracting light strongly; these evidently accompany the putrefaction bacteria, and assist in the final disintegration. On the ninth day the piece of skin was entirely dissolved.

[92] J.S.C.I., 1894, 218.

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 shown that the fermentation of cellulose is an anærobic process, caused by two species of bacteria belonging to the class of butyric ferments. Morphologically the organisms closely resemble one another, but one of them decomposes the cellulose with evolution of hydrogen, the other with evolution of methane; in both cases considerable amounts of acetic acid and normal butyric acid are produced.

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) by reduction of the sulphates in the liquid by an anærobic organism Spirillum desulfuricans; (2) by bacteria capable of growing in the presence of oxygen such as B. coli commune and B. lactis ærogenes, which ferment glucoses with formation of lævorotatory lactic acid and evolution of CO_{2} and hydrogen, and if at the same time the material contains albumin or sulphur, H_{2}S is given off; these organisms are incapable of reducing sulphates. Beijerinck (64) has investigated this process, and found a variety of different forms intermediate between the two above-mentioned, but all possessing the same characteristics so far as their chemical action is concerned, so that they may be classed as one order, which he calls Aerobacter.

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 resembles butyric fermentation. An analysis of the gases given off during the later stages of the process gave the following result: nitrogen, 58·88; hydrogen, 37·43; methane, 3·15. In the residue from the putrefaction he found acetic, butyric and valerianic acids, but not caproic or caprilic acid. In the normal steeping process employed for barley the gases given off consisted almost entirely of carbon dioxide and nitrogen. This observation is interesting to compare with the evolution of nitrogen in the fermentation of bran shown by Wood and Willcox.[93]

[93] J.S.C.I., 1893, 442.

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 CO_{2} according to the following formula:--

COH(CHOH)_{4}COOH = CO_{2} + COH[CHOH]_{3}CH_{2}OH.

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). 5. Bacillus Chauvæi = B. sarcophyematos bovis. 6. B. Liquefaciens magnus. 7. B. spinosus. 8. B. putrificus (Bienstock). 9. B. pseudo œdematicus (Liborius). 10. B. enteritidis sporogenes (Klein). 11. B. tetani. 12. Clostridium fœtidum. 13. B. cadaveris sporogenes (Klein). 14. Spirillum desulfuricans (Beijerinck). 15. B. coli commune. 16. B. lactis ærogenes. 17. B. fermentationis cellulosæ. 18. Micrococcus flavus liquefaciens (Flügge). 19. Diplococcus griseus non liquefaciens (n. sp.). 20. Streptococcus pyogenes. 21. Staphylococcus pyogenes albus. 22. Bacillus filiformis ærobius (n. sp.). 23. Diplococcus magnus anærobius (n. sp.). 24. Bacillus putidus gracilis (n. sp.). 25. B. perfringens (Frankel). 26. B. bifermentans sporogenes (n. sp.).

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. Cephalothecium roseum.