ACID FERMENTATION 153 



to seal the apparatus and prevent diffusion, whilst at the same 

 time it readily allowed gases to pass out of the flask. After 

 inoculation, which was effected by removing the stopper 

 and introducing a loop fuU of the culture, the air of the flask 

 was displaced by nitrogen, prepared by the action of ammonia 

 solution on copper. 



After passing nitrogen for about one to two hours, the 

 flask was removed and the long vertical tube sealed off at a 

 constriction previously made near the top. It was then placed 

 in an incubator (Fig. 24), the side of which was pierced by a 

 brass tube, with which the tube A is connected by indiarubber 

 tubing. The apparatus for collecting and measuring the gas 

 was connected with the other end of the brass tube. It 

 consisted of a Winchester quart bottle (B), fitted as an aspirat- 

 ing bottle, and provided with a long piece of indiarubber 

 tubing passing to the bottom of a second bottle, graduated in 

 volumes of 100 c.c. on a piece of paper pasted to the glass. On 

 the tube between the flask and the collecting bottle was 

 placed a three-way tap (D), by means of which samples of 

 gas can be withdrawn either directly from the flask or from 

 the collecting bottle. The collecting bottle was fiUed with 

 saturated brine, on the surface of which a little oil was poured, 

 to prevent absorption of carbonic acid gas. Direct experiment 

 showed that a mixture of carbonic acid gas and air could be 

 preserved over this liquid for a considerable time without 

 undergoing any perceptible alteration in composition. 



About 100 c.c. of brine were placed in C, and the connecting 

 rubber tube was also filled with brine so that the volume of gas 

 evolved could be measured by that of the liquid displaced. 



During the period of incubation the flask was agitated at 

 frequent intervals in order to secure the neutralisation of the 

 acid produced, and the volume of the liquid displaced was 

 read off, the measuring bottle being raised or lowered until 

 the surface of the liquid in it was at the same level as that in the 

 collecting bottle. 



