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 250 c.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 showed[132] 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.