The cultivation used in the first experiments for this purpose was one isolated in 1889, and used in the cellulose and starch experiments described in the above-mentioned paper.
This cultivation had not been obtained from a single colony from gelatin, and in order to make quite sure that the cultures used were pure, it was decided to make another attempt to isolate the bacillus by plate cultivation. Previous attempts to do this had failed, bate organisms and gelatin liquefying bacilli, developing in such numbers that the plates were spoiled before the organism, which caused the fermentation, had time to develop; beside which the organisms, as obtained direct from the drenches, grew with difficulty in the ordinary nutrient gelatin. A special gelatin was therefore prepared of the following composition:—
| Gelatin | 100 grm. | |
| Glucose | 30 " | |
| [174] | Salt solution | 200 c.c. |
| Water | 800 " |
Plates of this gelatin in Petri dishes were prepared from the previously used supposed pure cultures which had been preserved in sealed tubes. These were found to be dead. A modification of the method previously described by one of us[175] was adopted.
Fig. 32.—Cultures of α in Glucose Gelatin, showing Bubbles of Gas.
A solution of nutrient glucose was inoculated from a working drench, and as soon as the liquid was observed to become cloudy, a tube of the solid glucose gelatin was inoculated from it by plunging in a platinum needle. In two days the bacteria developed along the needle track. Fig. 32 shows the appearance of the tube four days after inoculation, a bubble of gas being formed in the solid gelatin. On the following day, the tube was broken, and from the portion where gas was given off most vigorously other tubes of solid and liquid media were inoculated. Acid was quickly formed in the nutrient glucose solutions. In the gelatin tubes, the bacteria developed well in the depth. The now purified culture was passed through three more glucose gelatin tubes, each time also a glucose tube being inoculated. From the last of these tubes a very minute quantity was taken 12 hours after inoculation on the point of a platinum needle, and a streak culture made on glucose gelatin. In 24 hours a growth could be seen on the surface of the gelatin in the form of minute dots perfectly separated one from another.
