ORIGINAL PAPERS ON BATING.

The reprints of these papers being no longer available, I have thought it best to print them in the present volume exactly as published, with the exception of the paper entitled “Fermentation in the Leather Industry” from which only so much is reproduced as relates to puering and bating.

In the next process, that of “bating,” the bacteria play a more important part, and may be put in our second category, viz. helpful to the tanner, though, from the very nature of the material used, it forms a nidus for various putrefactive ferments, and is therefore dangerous. The materials usually employed are bird dung and dog dung; the former of these is sharp and piercing in its action, while the latter has a more softening action on the skin. In both bates, however, a rapid solution of the gelatinous and albuminous hide substance (coriin) takes place. The hide fibres, however, are not attacked until all the nutrient material in the liquid has been consumed by the bacteria.

Eitner (Der Gerber, xv., 158) states that a sterile bate is without action on the skins, and appears in that article to attribute the entire working of the bate to the action of micro-organisms. He seems to have based his conclusions on experiments with an old bate sterilised with creolin, which he found had no action on the skins. If, however, a fresh bate be made and boiled for half an hour, then allowed to cool to 90°–95°F., it will be found to have a considerable action on the skins, though not so rapid a one as the unboiled bate. The boiling kills all organisms, and fresh ones have not time to develop from spores still remaining before the experiment is at an end.

Lately, the action of unorganised digestive ferments in the dung has been found to be considerable. Thus bating is an exceedingly complex process. The action appears to be threefold:—

(i)	A purely chemical action of the soluble salts present in the bate on the lime in the skin.113
(ii)	An action due to organised ferments.
(iii)	An action due to unorganised ferments.

The precise amount of influence each of these actions has on the skin is difficult to assign. The chemical action of the ammonia compounds dissolves the lime remaining in the skin, but the simple removal of the lime is not sufficient, as may be shown by removing it completely with dilute hydrochloric acid or other similar means, and washing perfectly free from acid in distilled water. When tanned, such a skin is hard and brittle.

The organised ferments or bacteria, of which there are many species in the bate, probably act on the skin by secreting soluble ferments, which have the power of dissolving hide fibre. I have isolated in plate cultivations several species which liquefy the gelatin.

If skins are allowed to lie in the bate, zoogloÆ of these bacteria collect in the folds and attack the fine “grain,” so that the leather is covered with lines and markings, or “flaked.” Thus the operation is a very critical one, requiring careful watching; under certain atmospheric conditions and at a temperature of 35°–40°C. the skin, if only left a short time too long, will completely melt away. The more skin substance is dissolved in the liquid, the more rapid and pronounced the bacterial action becomes.

So far as is at present known the unorganised ferments in the dung, besides those which are secreted by the bacteria, are mostly digestive ferments114 —pepsin, pancreatin, and trypsin—certain quantities of which pass out of the animal’s body in an unchanged condition. Of these, pepsin only acts in an acid solution, and, although fresh bate liquor is faintly acid to litmus, it quickly becomes neutralised by the lime in the skins, so that the action of this ferment can only be limited in extent. Pancreatin will act in a neutral solution, and has, therefore, a considerable effect on the skin.

Some Dung Analyses.

W.I. Macadam, J.S.C.I., 1888, 80. Viollet, Diction d’Analyses Chim.

The dog dung actually used in a tannery is from animals fed on a more vegetable diet, the one given having an extremely high percentage of lime, no doubt owing to the animal having eaten bones. A dung as brought from the kennels was found to contain:—

I find by experiments with the purified ferments that their action is very slow compared with the bate itself. Two portions of the same skin were taken: one of them was treated with a 1 per cent. solution of pepsin acidified with 0·2 per cent. of hydrochloric acid, the other in a bate liquor of dog’s dung; both at a temperature of 40°C. (104°F.). At the end of one hour, the skin in the pepsin solution was considerably “fallen,” but that in the manure solution was bated nearly away, i.e. the greater part of it was dissolved.

A 1 per cent. solution of pancreatin (Merck’s) was found to act far more rapidly than pepsin. At 40°C. in a neutral solution the skin fell rapidly, and the action continued even in the cold. In this experiment it was found that in 15 hours the liquid was swarming with minute bacteria. In order to guard against the influence of bacteria a similar experiment, at the suggestion of Mr. H.R. Procter, of the Yorkshire College, Leeds, was tried with the addition to the pancreatic solution of 1·5 per cent. of chloroform; this prevents the development of bacteria, while it does not interfere with the action of the pancreatin. The skin was reduced as before, but in neither case had it the peculiar touch of a “puered” skin, nor were the characteristics of the leather the same.

In a paper read before the Society of Chemical Industry (J.S.C.I., 1894, 219), the author gave an outline of the bating process as far as he had then investigated it. Further researches have shown that, although the views then put forward must be somewhat modified, in the main they are correct.

In 1895–96 a series of experiments was undertaken with a view of ascertaining how much of the reducing action of the bate was due to: (1) the chemical constituents; (2) the bacteria; (3) the digestive ferments and enzymes.

The puer used was dog dung obtained freshly every week from hunting kennels. It contained on an average: water, 85 per cent.; organic matter, 10 per cent.; mineral matter, 5 per cent.; about 3 per cent. of the organic is soluble and about 1 per cent. of the mineral matter.

According to Gamgee (Phys. Chem. 2), a dog fed on flesh diet excretes from 27 to 40grm. of fÆces in 24 hours, of which 12·9grm. are solids. On a bread diet the amount of fÆces is much more.

An ultimate analysis gave the following results:—

These figures alone are sufficient to show what variable quantity is the puer employed in a tannery.

The mineral matter is composed as follows:—

The ammonia compounds combine with the lime remaining in the skin, but the constitution of these compounds was unknown.

The puer was examined for ammonia by SchlÖsing’s method, 50c.c. being mixed with 50c.c. milk of lime placed under an air-tight bell-glass, together with an open vessel containing a measured quantity of standard acid, for 36 hours. No NH₃ was given off.115

The mixture of dung and Ca(OH)₂ was filtered and a perfectly clear filtrate of a golden-yellow colour obtained. This contained free amines, calcium salts of volatile and non-volatile acids.

It was distilled until the distillate was no longer alkaline, and made up to 500c.c. 50c.c. required for neutralisation 5·7c.c. of (N/100) H₂SO₄, equivalent to 0·1938grm. of NH₃ per litre of original puer, being the amines set free by Ca(OH)₂ in cold solution.

A further 50c.c. was distilled with NaOH, the amines given off neutralised 8c.c. of (N/1) HCl, equivalent to 2·72grm. of NH₃ per litre. The neutralised liquid was evaporated to dryness; the residue, consisting of amine hydrochlorates, amounted 0·27grm. A qualitative test showed both primary and secondary amines to be present.

The action of these amine hydrochlorides was tried upon skin, pieces of limed sheep grain previously washed in water being used. The solution was maintained at 35°C. In the first experiment 0·27grm. was dissolved in 100c.c. of water. In two hours the skin was considerably reduced, but had not the touch of a “puered” skin. It tanned well and was a good colour, showing that the lime had been removed, but the leather was not sufficiently soft.

With a strength of 1grm. per 100c.c. the action was hastened, the same result being obtained in 1 1/4 hours, but the coriin of the skin was not dissolved. A comparison piece of the same skin in dung was bated too much.

With a view of comparing the action of analogous bodies, skin was treated in a solution of anilin hydrochloride, 1grm. per 100c.c. for 1 1/4 hours at 35°C. The solution was acid, lime was removed, and the skin felt very similar to that in the previous experiment. Coriin was not dissolved, and the skin did not “come down.”

Of the mineral constituents mentioned above, only the chlorides116 have a reducing action on skin, the other compounds being inert, e.g. silicates, or merely supply food for the bacteria.

For a general outline of the decompositions taking place in dung, see Dr. Herfeld’s paper (J.S.C.I., May 1895). We need only consider the action of the chief organic compounds, as the amount of chlorine is too small (in one case 0·053 per cent. on the dry dung) to be of importance. The principal compounds are:—

Organic Acids.—Formic, acetic, butyric, valerianic, lactic, malic, tartaric, citric, and glyceric.

Amido Compounds.—Leucin, tyrosin, glutamic acid, glutamine, asparagin, glycocol.

Bases, consisting of amines, skatol, and indol, and varying amounts of ammonia, according to the age of the dung.

The organic acids exist partly as salts of the alkaline earths, partly combined with amines. A distillation of “puer,” after acidifying with H₂SO₄, gave 2·2grm. per litre of volatile acids reckoned as acetic. No HCl came over. On decomposing the sodium salts of the acids with H₂SO₄, the smell of butyric acid predominated. I have also shown the presence of lactic acid in the dung, but have been unable to determine the amount.

Action of Amido Compounds.—A mixture of glycocol and leucin117 was prepared by boiling 8grm. of gelatin in 400c.c. of water, acidified with 1c.c. of HCl for two hours with inverted condenser. The acid was then neutralised with ammonia. The solution had a very considerable reducing action on skin at 35°C. in 90 minutes, but not sufficient for practical purposes. It appears to dissolve a little coriin in a similar way to dilute acids. It was found on further investigation that the action was largely due to the NH₄Cl present.

Action of Dilute Acids.—Although the bate in practice is almost always alkaline, yet a fresh bate is acid, and it may be as well, before proceeding, to consider briefly the action of weak and dilute acids on skin.

The fibres of the skin have only a limited capacity for holding acids, and soon begin to swell abnormally and partially dissolve. Reimer118 has found that the material may be reprecipitated by lime water. It forms a fibrous mass, which has not the sticky feel of gelatin, but is at once converted into that body by boiling.

The author has examined the action of dilute H₂SO₄ on sheep skins, and this may be considered typical of the action of acids on skin generally.

When puered and drenched sheep skin is treated for 20 minutes with a very dilute solution of H₂SO₄ ( 1/280), it swells up considerably and becomes soft and semi-transparent, The fibres of the skin, which under normal conditions have a diameter of 4µ–6µ become 20µ or more; on staining with picro-carmin there are seen to be certain fibres which are unaffected or only slightly affected by the acid. These are the elastic fibres119 and the capillary blood vessels.

On filtering a solution of the above strength in which skin in excess has been treated for 20 minutes, and evaporating 100c.c. to dryness in a platinum dish, a residue of 0·4992grm. was obtained. A second experiment gave 0·496grm. of which 61 per cent. was organic matter.

By Kjeldahling another portion of the residue, the N in 0·12grm. amounted to 0·0122grm. equivalent to 0·073grm. of skin substance; i.e. 60 per cent. of the soluble matter is skin. It is evident that the matter in solution is a compound of skin substance and H₂SO₄ together with a little soluble mineral matter contained in the skin. The amount of substance dissolved depends upon the condition and previous treatment of the skin.

In the presence of NaCl, which prevents the swelling of the fibres, puered sheep grains take up 7·0c.c. of (N/1) H₂SO₄ from a solution containing 17c.c. of (N/1) H₂SO₄ (0·833grm. H₂SO₄) and 7·8grm. of NaCl per 100c.c. About 300grm. wet skin per litre were used. The skin substance dissolved by the acid in the time named does not appear to be more than that dissolved by the weak acids of a drench, although an equivalent weight of H₂SO₄ has a much more powerful swelling action on skin than these acids.

A piece of puered and drenched sheep skin was carefully washed in distilled water and then dried in vacuo until it ceased to lose weight; the absolute dry weight was 4·2670grm. The skin was soaked down in distilled water and again drenched for 30 hours in a clean bran drench, developing approximately 0·8grm. lactic acid, and 0·5grm. acetic acid per litre (see this Journal, May 1893). It was again washed and dried in vacuo. It weighed 4·12grm., a loss of 0·14grm. = 3·44 per cent.

The action, therefore, of the acids is a reducing one, inasmuch as skin is dissolved, but as the fibres hold a certain amount of acid, the skin appears to be plumped. Where the acid is a mineral one the skin tans plump, but with a brittle and inelastic fibre, weak organic acids make a plump, soft leather with a moderately elastic fibre, giving to the skin a somewhat india-rubber feel. We thus see that weak acids alone do not produce a similar result to a dung bate, which causes the skin to fall and produces a flat soft leather with a stretch in it, which will not spring back.

I have also tried the action of the following Na and NH₃ salts upon skin:—

Sodium lactate, C₃H₅O₃Na, prepared by neutralising 2grm. lactic acid with sodium carbonate in 1000c.c. water. The solution was used under the same conditions as the other bating experiments, viz. at 37°C. the reducing action was nil, but on prolonging the digestion, the medium was found to be very suitable for the development of putrefactive organisms, and, as a consequence, the skin was attacked and partly peptonised.

Ammonium lactate, prepared in a similar way, had an almost identical action on the skin, but was more effective in removing the last traces of lime.

Ammonium butyrate, prepared as above, removed lime, but instead of a reducing action it appeared to plump the skin slightly.

Action of Bile.

Amongst other compounds in the dung which might have some action on skin were the bile salts and bile colouring matters. (For a full description of these, see Gamgee, Phys. Chem. ii.)

Experiments were made with ox bile, 25c.c. being diluted with 250c.c. of water. Pieces of sheep grain previously washed in water were digested in this solution at 37°C. for 1–4 hours. The bile had no reducing action on the skin and in fact hardened it, at the same time staining it a dirty yellowish brown, the colour differing from that of the colouring matter of dung (hydrobilirubin). Even after continued standing there was little or no development of bacteria, the bile appearing to have an antiseptic action.

Action of Bacteria.

Having ascertained the action of the chief chemical constituents of the dung, that of the bacteria was next examined. In the first experiment, eliminating all but bacterial action, a tube of nutrient gelatin was inoculated from an active bate. In two days the gelatin had liquefied along the needle track.

The culture was now transferred to the following solution:—

The growth in this was fairly rapid and the smell very slight at the end of a week. A piece of washed sheep grain worked in this culture at 37°C. for four hours was considerably reduced, the grain was not attacked, the action differing in this respect from a prolonged puering. This mixed culture was grown in various other nutrient media, gelatin, gelatin and mineral salts, broth made from sheep and calf fleshings, etc., with practically the same results. In no case did the bating action equal that of dung, and was insufficient for practical purposes.

A cursory bacteriological examination of dung will show that the number of species of bacteria it contains is very large, but on making a plate cultivation from a sufficiently dilute infusion of fresh dog dung, most of the colonies developing appear to belong to four or five species. The following is a list of all the organisms which, so far as my knowledge goes, have been isolated from different kinds of dung:—

List of Known Bacteria in Dung.

There are besides many unnamed species of bacteria in dung, among which are the two following isolated by me from puer—

(a) Plate Cultures.—Small yellowish colonies, slightly fluorescent, liquefy the gelatin slowly. Rods resembling B. subtilis when at rest, but moving with a rapid undulatory motion. Cultures in nutrient gelatin have a considerable reducing action on skin.

(b) Plate Cultures.—Bluish colonies liquefying gelatin rapidly, micro-bacteria in pairs. Not so marked an action on skin as (a). The organism resembles Proteus vulgaris, but swarming islets not observed.

Of the species mentioned above, a pure culture of B. subtilis in meat broth, maintained for one week at 35°C., had no reducing action on skin. B. fluorescens liquefaciens had a moderate reducing action.

The species developing on the plates vary according to the age of the dung. In horse manure, according to Severin,121 bacilli predominate in the early stages. At the end of two to three weeks micro-bacteria, cocci and diplococci make their appearance, while the bacillar form becomes scarce. In three months cocci predominate, forming zoogloÆ; strepto- and staphylo-cocci and spirilli were seldom found. Yeasts and sarcina were never observed. From observations extending over several years it is evident that a similar cycle occurs in dog dung. When quite fresh it contains comparatively few bacteria; then certain groups of species take possession, causing decompositions in the dung; these in their turn give place to other species, which decompose the products formed by the first, so that no one species produces or can produce the complex chemical and physiological changes which take place, or the bodies necessary for the bating of skin, as has been supposed by some observers.

None of the experiments, either with mixed or pure cultures of bacteria, had a perfect reducing action on skin, although the action was considerably greater than that of the chemical solutions. It was found, however, that by adding a small quantity of the amines above mentioned to the bacterial cultures, the reducing action became nearly as rapid and effective as with dung itself; and it was, therefore, clear that the principal bating effect was due to a combination of two things, viz. an active growth of bacteria in the presence of amine compounds.

It was now necessary to ascertain the mode of action of the bacteria on the skins.

I have previously shown122 the action of digestive ferments or enzymes to be notable in the case of pancreatin. A careful study of the behaviour of the digestive enzymes in the animal body shows, however, pretty conclusively, that they are all destroyed before the fÆcal matter is discharged,123 and, therefore, the enzymes in the dung are not from this source.

An attempt was made to filter the dung diluted with water through a Berkefeld filter, and thus obtain a clear filtrate containing all the enzymes and free from bacteria; it was found impossible to filter the liquid in this way.

The method of Claudio Fermi124 was also tried, but a clear filtrate could not be obtained.

The author has succeeded in obtaining the enzymes best in the following manner:—About 150c.c. of the above-mentioned “puer” was well mixed with an equal quantity of glycerin and allowed to stand for seven days; it was then capable of being filtered on a filter pump, although very slowly, and yielded a clear filtrate of a deep golden brown colour; the filtrate was poured in a thin stream into a tall vessel containing about 1500c.c. of 98 per cent. alcohol. A copious flocculent precipitate of the albuminous matters and enzymes was thrown down, the alcohol was filtered off and the precipitate washed on the filter with absolute alcohol, and dried over sulphuric acid. The resulting amorphous body was of light brown colour, and became darker when exposed to the air. The amount obtained from 150c.c. of dung was 0·55grm. (3·66grm. per litre). The body consists of a mixture125 of all the enzymes existing in the dung along with other albuminous bodies. It has a very slight diastatic action on starch. 0·5grm. in 100c.c. of water at 35°C. had a very considerable reducing action on skin. A further experiment with 0·5grm. of amine hydrochlorides, 0·5grm. of enzymes and 100c.c. of water, at 35°C. brought down a piece of limed sheep grain in 30 minutes exactly like puer; the reaction of the solution at the commencement of the experiment was faintly alkaline; at the end of the experiment it was considerably alkaline.

It was noted that the fÆcal odour of the glycerin solution disappeared on standing for two or three weeks, and the solution smelt strongly of ethyl butyrate, the enzymes being still present.

These enzymes were prepared from dung, and it now remained to prepare them in the laboratory by the action of bacteria alone. For this purpose 200c.c. of a mixed culture of dung bacteria in the solution above described, seven days old, was mixed with 200c.c. of dilute alcohol (alcohol 65, water 100) and well shaken. Gelatin and albuminoid bodies are by this means precipitated. The liquid was filtered and poured into eight times its volume of 98 per cent. alcohol. The precipitate which came down was washed with absolute alcohol and dried in the usual way. The enzymes thus obtained were redissolved in water and the former experiment with skin repeated with this solution. The same effect was produced, showing conclusively that it is the enzymes produced by the bacteria, acting in conjunction with the amines, which bate the skin. It would seem that the special action of the enzymes is aided by the presence of amine compounds, in addition to the chemical action which these latter have upon the skin. The action is shown to be interdependent, i.e., bacterial action alone is insufficient, and chemical action alone is insufficient, the true bating action being a combination of the two.

In conclusion the author has pleasure in acknowledging the valuable assistance of Mr. H.S. Shrewsbury in carrying out some of the experiments.

The present notes are a continuation of those published in 1898. It has been impossible for me to follow up the subject except in a desultory manner, but I think it desirable to put further results, however meagre, before you with the object of throwing more light on the complex process of bating hides and skins.

Since the previous notes were written, Dr. Th. KÖrner127 has called attention to the physics of the process, a matter which I had not previously mentioned. He explains part of the action by differences of osmotic pressure. The ammonium salts and salts of organic bases contained in the dung, possess a smaller osmotic pressure than the solution of calcium hydrate contained in the skins. The ammonium ion endeavours to unite with the hydroxyl ion of the calcium hydrate to form undissociated ammonium hydroxide. In consequence of the osmotic pressure and the electrostatic attraction of the oppositely charged ions, the calcium hydrate solution is withdrawn from the skin and the latter falls.

Experiments with Bacteria on a Large Scale.

I have shown128 that simple cultures of bacteria in certain nutrient media, in the absence of amines and other bodies, have an imperfect bating action on skin. This was the case, whether the cultures were pure or mixed, but the mixed cultures have a better action than the pure cultures of any of the organisms I have hitherto used. Bacteriological processes in nature are usually carried on by a mixture of species of bacteria; still there are fermentations like the spontaneous souring of milk, the formation of nitrites from nitrates, and the ammoniacal fermentation of urea, which may be considered as natural pure cultures. These are examples of what I may call the selective influence of the nutrient medium. The nutrient medium used in my former experiments was a solution of gelatin and mineral salts, such as is used in general bacteriological work, modified in various ways. On careful consideration, this did not appear to be a medium at all corresponding to the natural bate. The albuminoids in the process of digestion are peptonised, and dung contains only those bodies which the animal is incapable of assimilating. It is from these bodies that the necessary enzymes are produced by bacteria. Of the numerous trials with pure cultures of different organisms from dung, I found that most of those having the property of secreting bating enzymes, were non-liquefying bacteria. Popp and Becker have also pointed out that peptonising bacteria do not exert a favourable reducing action on skin. It is impracticable to grow these non-peptonising organisms on a large scale in a solid medium, and it occurred to me that gelatin, previously peptonised by chemical means, would be a favourable medium. Experiments with hydrochloric and sulphuric acids gave hopeful results. The acid was neutralised with ammonia after the chemical action was complete, and the bacteria cultivated in this solution.

A better result was obtained by digesting 10grm. gelatin, 5grm. lactic acid (anhydrous by calculation), 100c.c. of water, in a closed vessel on the water bath for three hours. A slight black precipitate of melanoidic acid (humic acid) is formed. The liquid is of a clear brown colour, and contains a large number of nitrogenous bodies resulting from the breaking down of the gelatin molecule. It has been impossible for me to ascertain its exact composition, but I have partially examined it with the following results:—

1. A small quantity of the liquid poured into absolute alcohol, gives a white precipitate which re-dissolves on the addition of about 30 per cent. water. This shows absence of gelatin.

2. On saturating with ammonium sulphate, a brownish precipitate is thrown down which is perfectly soluble in cold water. It consists of gelatose lactates which are evidently formed in an analogous manner, to the gelatose hydrochlorides prepared by C. Paal.129 The gelatoses correspond to the protoses formed during the digestion of albumin. In one sample 1·3grm. gelatose lactates per 100c.c. was found.

3. The filtrate from (2) was dialysed against running water for 15 hours to get rid of the ammonium sulphate. The resulting gelatin-peptones, or gelatones, amounted to 9·1grm. per 100c.c. In other words, the heating under pressure had transformed 87 per cent. of the original gelatin into true peptones.

It may be presumed that the other and simpler nitrogenous bodies formed, are the same as those produced by the treatment of gelatin solutions by dilute mineral acids, and that the bases combine with the free lactic acid. An excellent list of these and a very interesting account of the protamines and hexones which go to form the complex albumin molecule is given in a paper by Dr. A. Kossel.130

The solution of gelatone lactates and free lactic acid prepared as above, was neutralised with sodium carbonate and diluted to 1000c.c.; it was found to be a very good medium for the growth of the bating organisms after the addition of a small quantity of potassium phosphate.

In looking round for suitable means of testing the action of both pure and mixed cultures of bacteria on a practical scale, it occurred to me that the Carlsberg vessels, as used by Hansen for the pure cultivation of yeast,131 would answer for the cultivation of bacteria also. I was unable to obtain any information on the subject, and was surprised to find that the apparatus is very little used in England. I procured two of these vessels from Jensen of Copenhagen, and found they answered very well. The bacteria were transferred from the test tube in which the original inoculation had been made to a Pasteur flask of 250c.c. capacity containing the nutrient solution; when the growth in this flask was sufficiently vigorous the Carlsberg vessel was inoculated from it in the manner described by Hansen, and with all the usual precautions.

After three days at a temperature of 37°C. the whole of the 10 litres was used for inoculating 100 litres of the nutrient medium above mentioned contained in a clean barrel standing in a room, the temperature of which was maintained at 37°C. By using this comparatively large volume of pure culture for pitching, if I may be allowed to use a familiar brewing term, the large culture was kept practically pure, although it could not be supplied with germ-free air, as in the case of the Carlsberg vessel.

Using a nutrient medium containing 1 per cent. original gelatin, the maximum bating effect was obtained with the majority of organisms in three days.

In my previous notes I showed132 that a mixture of digestive enzymes and amine hydrochlorates was effective in bating skin. The liquid in this case was free from bacteria. It was found, however, that the action of the bate was hastened when an active growth of bacteria was going on at the same time in the liquid, even though the quantity of enzyme present was smaller. This is what one might infer from a careful study of the process. It appears better for small quantities of enzymes to be produced in the liquid as required, than to use larger quantities of stale enzymes.

Mr. Loxley Meggitt was kind enough to concentrate for me about 170 litres of a culture of bating organisms. The concentration was conducted in vacuo at a temperature below 50°C., at which the enzymes were certainly not injured, and it was found that when this concentrated culture was diluted to the same strength as the original, the action was considerably diminished. This diminution of activity was due in part to a loss of volatile products in the evaporation, but more especially to the absence of an active fermentation going on in the liquid.

The nutrient medium above described, besides acting in a selective manner on a mixture of bacteria, and providing them with food stuff, contains amido compounds and other bodies, which are eminently favourable to the proteolytic action of the enzymes, and therefore act in the same way as the amine hydrochlorates which I used in former experiments.

The action of the following bacteria grown in this medium has been tried on skin. The pure cultures are lettered in continuation of previous descriptions (see previous paper, p.162).

1. Bacillus c, isolated from pigeon dung bate. Small, round, bluish-white colonies, standing up slightly above the surface of the gelatin, but not spreading out on the surface, non-liquefying pairs of micro-bacteria. This was the principal organism found in a pigeon dung bate as used for the bating of E.I. kips. Grown in nutrient broth or in the special medium it had no reducing effect on skin.

2. Bacillus pyocyaneus from blue skin, i.e. skin in an early stage of putrefaction. No action.

3. Mixed culture from fresh pigeon dung, collected in a sterile vessel. This contained very few bacteria capable of developing in the special medium. The action on skin was no more than that due to the chemical compounds present.

4. Mixed culture from fresh dog dung. This contained remarkably few species of bacteria which came to a good development in the special medium. The principal organisms were a jointed bacillus with extremely rapid vibrionic movement, and a small bacterium in pairs; the action on skin was perceptible but slight.

5. Mixed culture from dog dung one month old, as used in the bate. The action of this in a certain medium has already been described (see p.160). In the special medium it was distinctly better and almost equal to that of the dung bate.

6. Mixed culture from fresh fÆces gave a similar result to (3). A microscopic examination showed pairs of small bacteria and micrococci, but no bacillar forms.

7. Mixed culture from fresh horse dung had a moderate but distinct action on skin, about equal to (4).

8. Bacillus d, isolated from wool infusion. Very slight action.

9. Bacillus e, isolated from wool infusion. Similar result to (8).

10. Mixed culture of bacilli d and e only. Very powerful bating action, skin bated more rapidly than with dung.

These experiments tend to confirm the conclusion I previously arrived at, viz. that no single species of bacteria produces the complex chemical and physiological changes which take place in the dung, and which result in the production of the bodies necessary for the proper bating of skin. It is a well-known fact that dog dung requires keeping for at least a month before it gives the best result. During this time it undergoes a kind of fermentation and continues to improve up to two months, after which it deteriorates. The rate of fermentation depends upon the season.

It is evident from these facts that the bacteria present in the dung when it leaves the animal’s body do not produce the required enzymes and chemical compounds, and that these are produced by bacteria which obtain access from the air. It is also evident that the production of these enzymes depends upon the composition of the nutrient medium, since this exerts a selective influence on the species of bacteria obtaining access to it. In the spontaneous souring of milk numerous bacteria in the air have free access to the milk, 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. Dog dung is a favourable medium for the bacteria secreting the bating enzymes; but if dextrose or other carbohydrate be added to fresh dung an acid fermentation is set up which effectually prevents the development of the bating organisms.

Reasoning along these lines I was led to try some cultures of air bacteria in my special medium, and I found that a good source of such organisms likely to have a useful effect was a sweating stove as used for the depilation of skins. The hair roots are loosened by bacterial action; the wool, when it slips, brings away with it the epidermis. The root portions of the wool were cut off and digested in water at 35°C., the liquid strained off and used for making plate cultures in the usual way. In making the attenuations for the plate cultures, the fourth was found to be practically a pure culture of the organism I have called bacillus d, or the sweating bacillus. It forms large whitish colonies, spreading on the surface, with irregular contour. The bacilli are very small, mostly in pairs, but sometimes joining together in thread-like forms. (Fig.21.)

Grown in the special medium it had little or no action on skin. At the same time I found that a culture made from the original liquid, i.e., a mixed culture of the sweating organisms, had an exceedingly powerful bating action; indeed, the skin was bated more rapidly than with dung.

All the experiments carried out so far tend to prove that mixed cultures of suitable bacteria possessed the required action, whereas pure cultures do not. A further examination of the infusion of wool roots showed that the bacteria contained in it, consisted practically of two species only. The first of these I have already described, the second one, bacillus e (Fig.22), forms small brownish-yellow boat-shaped colonies on gelatin plates, very similar to one of the dung bacteria. They consist of plump cells, two or three times the size of bacillus d, united in pairs and short chains and surrounded by a capsule; the cells appear to vary considerably in size. Cultivated in the same way as the others it has very little action on skin. It is evident from these facts that the growth of the bacteria is a symbiotic one; separately they exert little or no action, whereas used together the action is most remarkable.

The author wishes to express his indebtedness to Mr. J. Golding, F.I.C., of University College, Nottingham, who made the first pure cultures of these “wool” bacteria.

The Influence of Solid Matter in the Bate.

In making comparative tests of my artificial bates and dog dung, I usually took the latter from the paddles in which it was diluted ready for use, and maintained both solutions at exactly the same temperature. Parts of the same skin were then digested in each solution, and the results noted after 1 1/2 hours. In order to make them more strictly comparable I filtered the dung bate, so as to employ the matter actually in solution when the bate was made up. It was found that the filtered bate had far less action than when it was used in an unfiltered condition. That this is not wholly due to the passing into solution of some of the solid matter during the bating was shown by adding an inert solid, viz., kaolin, to the filtered bate, when the action was greatly hastened. On adding kaolin to the artificial bate and keeping the liquid agitated, the same result was obtained. It seems that the finely-divided solid matter acts as a carrier for the enzymes, perhaps by a kind of mass action, each particle offering a surface vastly greater than the molecules of dissolved solids. In the case of “puer,” the organic insoluble matter is gradually brought into solution by bacterial action as the bating proceeds, though I do not believe this occurs to any great extent.

The following quantities of soluble and insoluble matter were found in puer wheels in actual practice per 100c.c.:—

1. Wheel in constant use for a week.
2. Wheel freshly made up.
3. The same wheel after one lot of skins.
4. From pigeon dung bate pit.