SYNCHRONOUS GROWTH FOR SPORE PRODUCTION 13 



Table II 



Degree of sporulation obtained with niedium 2 



Time (hours) pH Percent spores 



2 7.5 



3 7.5 

 41/2 7.3 



5 7.3 20 

 51/2 7.3 50 



6 7.3 77 



7 7.3 96 

 20 7.0 57 



in the process of sporulation prior to transfer to the test flask. We have 

 found that if the above happens, it delays rapid growth of the culture in 

 the test flask by five or more hours, and interferes with the subsequent 

 process of sporulation. It became apparent that, in order to avoid the above 

 complications, it was necessary to find out the approximate time at which 

 the inoculum was committed to sporulation. This problem was first ap- 

 proached by assuming that growth of a vegetative cell and sporulation were 

 separate processes. While it is known that this organism requires anaer- 

 obic conditions for growth, it is not known whether the sporulation proc- 

 ess is so restricted. We tested this by aeration with penicillin after the 

 vegetative cells were presumed to have been committed to sporulation. If 

 spores could be formed from such cells under aerobic conditions, we could 

 thus separate the process of growth from sporulation. 



Samples of the inoculated test medium were removed at different time 

 intervals and placed in aeration tubes with penicillin to give a final con- 

 centration of 100 units of peniciUin per milliliter. The tubes were then 

 aerated until the test flask showed 96 percent sporulation, as determined 

 by staining. The results are shown in Table III. 



It appears from these results that there is a gradual production of spores. 

 Aeration with peniciUin completely arrests further sporulation, but it has 

 no effect on the spores already formed; thus penicillin and aeration to- 

 gether block spore formation even after cells are committed. 



The effect of aeration and penicillin has been somewhat clarified from 

 the following experiment. Samples of the inoculated test medium were re- 

 moved from the test flask at times when the culture was undergoing sporu- 

 lation. The cells were washed free from all nutrients under anaerobic con- 

 ditions, and allowed to incubate at SOX. in distilled water also under an- 

 aerobic conditions. The culture sporulated completely although there were 



