ISOLATION OF ANAEROBIC ORGANISMS 79 



shown ; the other curved in a downward direction on the exterior, 

 drawn in the form of a pipette, and prolonged downwards to 

 within about i inch of the bottom level, the end being sealed off in 

 the flame. Through the centre stopper a short length of similar 

 tubing is inserted, the lower end (the orifice of which should be 

 sHghtly constricted in the flame) projecting not more than half an 

 inch beyond the lower level of the cork, and the upper to about 

 I inch above it, this latter being constricted at the point of entry 

 into the cork, lightly plugged with cotton-wool, and fitted with a 

 tightly fitting india-rubber cap of the form shown, which must be 

 removed when sterilising liquid in the flask or when passing a 

 current of hydrogen through it. 



The flask, after sterilisation in the autoclave, is filled to one- 

 third its capacity with fresh milk, or (if it is desired to study the 

 action of some particular organism) is sterilised with the milk or 

 bouillon in it,^ and then inoculated in the usual manner by slightly 

 withdrawing the centre stopper. In this latter case the central 

 stopper with its fitting should be covered with a loosely fitting 

 paper cap lightly tied on during process of sterilisation and until 

 the inoculation is made. The short length of india-rubber tubing 

 is then connected up with the hydrogen apparatus (as explained 

 on p. 82) and a current of hydrogen (escaping by the central 

 orifice) is passed through the medium for ten or fifteen minutes. 

 The cap is then placed on the outlet tube, and the inlet closed with 

 the pinchcock. If thought desirable successive currents of hydrogen 

 can be passed through the medium in a similar manner. The 

 flask is then incubated at 37° or other temperature. 



As soon as pressure of gas induced by fermentation is set up in 

 the flask — the first indication of which will be increased tension of 

 the india-rubber cap, and the rising of the liquid in the inner portion 

 of the outlet tube — the extreme point of the bent tube is snipped 

 off, and the contents allowed to fall drop by drop into a receptacle 

 placed below. If it is desired to obtain a freer flow of the culture 

 it is only necessary to connect up the inlet with the hydrogen 

 apparatus, and allow a current of gas under pressure to enter the 

 flask. 



Flasks " B " and " C " are convenient when it is desired only to 

 study the final results of the fermentative processes. They consist 

 of flasks of bottle shape, of sufficient temper to withstand exposure 

 to a high temperature during course of sterilisation, and yet of 



' Care must be taken in this case to remove the india-rubber cap and close 

 down tightly the pinchcock of the inlet tube before placing in the autoclave. 



