602 T. J. MURRAY 



Some further experiments with pure cultures were carried out. 

 A pure culture was isolated from an ammonified urea solution 

 and grown on urea agar under anaerobic conditions. A very- 

 simple method was devised for anaerobic plate work. It con- 

 sisted in adding sterile paraffin oil to agar that had been cooled, 

 inoculated and poured. Oil was added to the level of the rim 

 of the plate. This avoided the use of the anaerobic jar and 

 proved very effective. No spreading colonies were observed, 

 and colonies were as well isolated as on an aerobic plate. The 

 plates may be removed from the incubator and examined for 

 growth at any time. This is a decided advantage over the 

 anaerobic jar method. The oil may be poured off the plate and 

 the colonies exposed for further study. 



The organism isolated by this method was a diplo-bacillus. 

 It would not grow on nutrient agar under aerobic or anaerobic 

 conditions. It grew very well on urea agar under aerobic and 

 anaerobic conditions, although it had been isolated purely by 

 anaerobic technique. 



Cultures of this organism wete inoculated into 100 cc, respec- 

 tively of urea bouillon (no peptone), urea solution (glucose, 10 

 per cent, K2HPO4 5 per cent, MgS04 0.05 per cent, urea 1 per 

 cent), and Dunham solution. These inoculated flasks were 

 placed under aerobic and anaerobic conditions. 



Experiment V. Ammonificaiion in solution by pure culture. Ammonia in 

 milligrams per 100 cc. of solution. 



In this experiment urea bouillon seemed to be the best me- 

 dium. Ammonification with this material proceeded better under 

 anaerobic conditions. The reaction did not take place with 

 Dunham's solution. It did not proceed as well with the urea 

 solution as with urea bouillon, although the process went on 

 under aerobic and anaerobic conditions. 



