8o ANAEROBIC ORGANISMS : ISOLATION AND CULTURE 



sufficient strength to allow (in the case of " C ") of a vacuum being 

 set up in the interior. The former is useful when hydrogen or 

 other inert gas is alone employed, the latter when it is desired to 

 study the phenomena of fermentation " in vacuo." 



Flask " B " is fitted with a two-holed india-rubber stopper, 

 through one hole of which a long length of glass tubing is led to 

 the bottom of the flask, and through the other a shorter length 

 extending into the flask to a point just below the under surface of 

 the stopper. Both tubes are constricted at the point at which they 

 enter the cork, are plugged lightly with cotton-wool, and fitted at 

 their exterior extremities as shown — the one with a short length of 

 pressure tubing fitted with a screw pinchcock — the other with a 

 small india-rubber stopper fitting tightly into the tube ending, 

 which should have been previously " coned " in the flame. The 

 flask is filled to about one-third its capacity with the medium 

 required, a piece of wrapping paper is tied loosely 

 over the tube terminals and neck of the flask, and 

 the whole is then sterilised in the autoclave, care 

 being taken that the screw pinchcock is tightly 

 closed and the small stopper removed during the 

 process. Inoculation is made in the usual manner 

 by withdrawing the central stopper sufficiently to 

 Fig. i6.— Best allow the insertion of a finely drawn pipette, but 

 pinchcock. without the removal of the loose paper covering 



mentioned above. The stopper is then replaced, 

 the pinchcock opened, and a current of hydrogen passed through 

 the liquid for ten or fifteen minutes. The small stopper is then 

 fitted tightly into place, the screw pinchcock closed, and the flask 

 incubated at any desired temperature. As soon as interior 

 pressure is set up by the evolution of gas, the small stopper is 

 slightly loosened in its place in order to allow the escape of any 

 great excess. 



Flask " C " is treated in a similar manner, the only difference ■ 

 being that instead of a current of hydrogen being passed through i 

 the liquid, a vacuum is formed in the flask. 



Cultivation in hydrog-en— 



Although a supply of hydrogen is not absolutely essential in j 

 anaerobic work, yet a convenient apparatus for generating and I 

 holding in reserve a sufficient supply of this gas will be found of i 

 the greatest possible value, and will considerably extend the range 

 of methods at the disposal of the worker. 



