Since the previous notes were written, Dr. Th. Körner[127] 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the osmotic pressure and the electrostatic attraction of the oppositely charged ions, the calcium hydrate solution is withdrawn from the skin and the latter falls.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 shown[128] 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 10 grm. gelatin, 5 grm. lactic acid (anhydrous by calculation), 100 c.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 1·3 grm. gelatose lactates per 100 c.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 9·1 grm. per 100 c.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 1000 c.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.