The Puering, Bating & Drenching of Skins

THE BACTERIOLOGY OF THE BATE.

When a drop of liquid from a puer wheel in use is examined under the microscope73 with  1/12 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.

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. With a platinum needle a minute quantity of the puer liquor is stirred into the drop. A slide with a depression in the centre is taken, the edge of the depression painted round with vaseline, and pressed over the cover-glass, so that the drop is exactly central. If the whole be now turned smartly over, the drop will hang central in the hollow space.

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:—

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 10 grm. gelatin with 6 1/2 grm. 80 per cent. lactic acid in 100 c.c. water under pressure for three hours, neutralizing with ammonia, adding 1 grm. potassium phosphate, making up to 1000 c.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 12 c.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, but is of the order of 10,000,000 per grm. of dry matter, capable of developing in nutrient gelatin. Of this number, about 100,000 are spore-bearing organisms. This estimate applies to healthy animals; in a diseased condition, the numbers vary enormously.

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.

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 0·8 µ wide, 1 to 3 µ 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 round colonies, about the size of a pin head; the latter spread into a whitish iridescent film, with irregular edges.

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 10 c.c. of the culture, 1 c.c. of a  1/50 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  1/10000 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.

Professor Becker’s Bacterium No. 12, which he has named Bacillus erodiens (Fig. 16), is undoubtedly a variety of B. coli, but has a more rapid motion, and does not coagulate milk, although it renders it somewhat thick. Cultivated in broth it gives off much gas, consisting of 12 per cent. carbon dioxide, 85 per cent. hydrogen, 3 per cent. oxygen. If glucose be added, the quantity of carbon dioxide rises to 40 per cent., and acid is produced. The most rapid growth is at 37° C., and at this temperature a broth culture has a distinct reducing action on skin. According to the medium in which it is grown, it produces acid or alkali, and thus comes under the heading of mixed bacteria. In sugar81 solutions acid is produced, and in proteid solutions ammonia compounds, indol, and evil-smelling gases are given off. Thus, by varying the medium, the effect produced may also be varied.

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 action as that which is immediately made into a paste with water. It appears to lose its “nature,” partly owing to irreversible dehydration processes, and partly because some of the bacteria are killed. Plate cultures on agar from fresh puer (Fig. 17), and from a puer wheel in use (Fig. 18), show the number of bacteria in the puer wheel to be much greater than in the fresh puer. Whence it is evident, that the bacteria continue to develop in the puer and to produce their various products, enzymes, etc. We have already considered the action of the chemical products, and in Chapter V. we propose to discuss the action of enzymes.

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 were principally of two varieties (both non-liquefying organisms), corresponding to the bacteria observed in the original dung. Large cultures were made in a Carlsberg vessel, as described in Chapter VI., and the effect of these cultures tried upon skin. No particular reducing effect was obtained.

A microscopical examination of a bating pit used for kips, showed an extraordinary mixture of bacteria, bacilli, vibrios, and monads; some comparatively large dumb-bell shaped bacteria, very motile, were present. The difference between the bating liquor and the fresh dung was very marked, especially in the variety of species present. Cultures made from several colonies isolated from the above bate, in a nutrient liquid, consisting of 10 litres water, and 20 grm. gelatin, peptonized by heating under pressure with 10 c.c. sulphuric acid, afterwards neutralizing with ammonia, and adding the soluble matter from 200 grm. bone-meal, had no action on skin.

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 essential to the chemical part of the process. They also produce proteoclastic enzymes, which act upon the skin fibre (see chapter on Enzyme Action). The products of the bacteria depend very much upon the composition of the nutrient medium. Many organisms grown in media containing sugar or other carbohydrates produce acids, but, grown in proteids free from sugars, they produce alkaline compounds. Villon (“The Leather Industry,” 1901, p. 408) describes a bacterium which he considers to be the special micro-organism concerned in the depilation of skins, which resembles Bacillus d (Wood) (Fig. 21), but he does not describe the appearance of the cultures; he states, however (p. 410), that this is the only bacterium which can develop in the limes,83 and that it is the cause of the unhairing in this case also. Since the production of ammonia in limes is known to be due to bacterial action, it is very probable that this bacterium, which is ubiquitous, is also of use in the bate, and a research in this direction would be interesting.84