136 



Messrs. E. C. Grey and E. G. Young. 



Technique. 



Growth of the Organisms anaerohically prior to Ferrnentation. 



Winchester quarts containing agar were inoculated with B. coli communis 

 which had been grown anaerohically on broth in an atmosphere of hydrogen. 

 The procedure was to sterilise the whole apparatus, consisting of Winchester 

 quart containing agar and side tube containing broth. The apparatus was 

 cooled in nitrogen, and when cold the . side tubes containing broth were 

 inoculated. The bottles were placed side by side in a large incubator and 

 connected in series ; hydrogen was then passed through the whole series, and 

 in this way the broth in the side tube of each bottle was anaerobic during the 

 growth of the bacteria. Strict anaerobiosis was maintained by exhausting at 

 the pump from time to time and allowing fresh hydrogen to enter. After 

 seventy-eight hours the broth of the side tubes was allowed to run into the 

 bottles and the growth on agar continued for forty-eight hours. To remove 

 the bacterial growth, a vacuum was created in the bottles and saline solution, 

 such as has been used in all previous experiments of this series, was allowed 

 to enter. The saline emulsion of bacteria was removed to the fermentation 

 flask by previously exhausting the latter of air and allowing the hydrogen to 

 enter the Winchester quarts. The hydrogen was subsequently pumped out 

 of the fermentation flasks by boiling the contained fluids in vacuo, and 

 nitrogen was admitted. The fermentation was allowed to proceed anaerohi- 

 cally, so that the whole of the operation of growth of the bacteria and of 

 fermentation by them of the sugar was carried out with rigorous exclusion of 

 air. The operations were found exceedingly difficult. 



As regards the fermentations themselves, the technique was that described 

 in Part III of this series, with the addition of an arrangement for regulating 

 the admission of oxygen or air at a measured rate where aerobic fermentations 

 were desired. 



Experiment I. — The bacteria were grown anaerohically on agar and 

 approximately equal portions of bacterial emulsion were added to each of 

 three flasks containing sugar, water, atd some chalk. The fermentation was 

 allowed to continue forty-eight hours at 37° C. Experiment N was com- 

 pletely anaerobic, A received two litres of air, two litres of oxygen during 

 the fermentation. 



The analysis of the fermentation products gave the following results, 

 expressed as percentages upon the sugar employed : — 



