Bacterial fermentation and its products are however the main factor in the action of puers and bates, and on this subject we owe most of our knowledge to the work of J. T. Wood, since, though Popp and Becker have worked over much of the same ground, they have not nearly so freely published their results.
Wood showed that a fresh puer liquor, even when boiled for half an hour and so freed from living organisms and albuminoid ferments, has still considerable action on a limed skin, though much less than the unboiled puer. He found that this action was principally due to amines and their compounds with organic acids, which removed lime, but did not remove the interfibrillary substance or give the proper feel of puered skin. A very similar result was obtained with aniline (phenyl-amine) hydrochloride in 1 per cent. solution.
A considerable variety of bacteria from dung and other sources were cultivated in various media and their puering power tested, but though greater than that of the unorganised chemical compounds such as amine salts and organic acids, it was in no case equal to that of an ordinary puer, or sufficient for practical use. When, however, a small quantity of the amine salts obtained from the puer were added to a mixed bacterial culture the effect on the skin was almost as rapid and considerable as with an actual puer.
In order to determine whether the puering effect was due to the direct action of the bacteria or to their enzyme-products, the latter were separated from a filtered puer solution by adding it to a large volume of 98 per cent. alcohol in which the enzymes are insoluble. When redissolved in water, they had a decided puering effect, and a solution of 0·5 grm. of the mixed enzymes and 0·5 grm. of the mixed amine hydrochlorides in 100 c.c. of water at 350° C. brought down a piece of limed sheep-skin in thirty minutes exactly like a puer. The action is therefore dependent on the mutual action of the enzymes and amine salts, but as the separation of these would be too costly for practical use, and the puering proved more effectual when they were formed in contact with the skin by active bacteria, Wood adopted the method of preparing a suitable sterilised nutritive liquid, which was inoculated before use with a mixed culture of suitable bacteria. For laboratory purposes a suitable culture-medium was obtained by digesting 10 grm. of gelatine with 5 grm. of lactic acid (reckoned water-free) and 100 c.c. of water for three hours in a closed vessel on the water-bath. The resultant solution was neutralised with sodium carbonate and diluted to 1 litre with addition of a small quantity of potassium phosphate.
The bacteria of fresh dog-dung were not found to possess a satisfactory puering effect, but those from dung which had been fermented a month (as in practice) gave a result nearly equal to actual puer. A still better result was obtained by a mixed culture from the roots of wool loosened by sweating. The bacteria were principally of two species, of which neither separately was capable of satisfactory puering; but which together acted more rapidly than an actual puer. These bacteria do not liquefy gelatine.
During the course of his experiments, Wood found that filtered puer solutions were less active than turbid ones and that their activity was increased even by the addition of inert substances, such as kaolin.
Wood attributes the differences in action between dog-dung and bird-dung not only to different bacteria, but to the fact that in the latter case the urinary products, and especially uric acid are contained in the dung.
From the results of these and similar researches, Wood in England, and Popp and Becker in Germany succeeded in producing a practical artificial puer, which they now manufacture in conjunction under the name of “Erodin.”
“Erodin” consists of a solid nutrient medium and a liquid “pure culture” of the bacteria necessary to effect the required bating or puering.
The following are the directions for working with erodin bate, as supplied by the manufacturers:—