28 Z. JOHN ORDAL 



and variability between lots was removed. The percentage of sporulation 

 was difficult to evaluate because of a concomitant lysis of the vegetative 

 cells. 



When thiamine was added to the spent medium or to the refortified spent 

 medium, no increase in percentage of sporulation was observed. When 2% 

 trypticase broth was enriched with up to 10 gamma of thiamine per ml 

 of the medium, almost 100% incipient sporulation occurred, as is demon- 

 strated by the fact that, at the end of 24 hours of incubation, almost every 

 cell in the culture, as observed microscopically, was in the swollen prespore 

 stage. These swollen prespores, however, never went on to form the retrac- 

 tile mature spores, but after 72 hrs. these forms disappeared and only vege- 

 tative cells could be seen. This inability to observe refractile spores in the 

 2% trypticase medium containing thiamine indicated that this medium was 

 still deficient for sporulation. Efforts to overcome the deficiency by the ad- 

 dition of Mg, Mn, Fe, Co, Ca, vitamins, nucleic acid bases, glucose, and 

 short-chain fatty acids were unsuccessful in improving the sporulation. 



The sporogenic effect of the culture filtrates was not restricted to PA 

 3679-h, either with regard to production or response. Clostridium hotulinum 

 62A and CI. aerofoetiduin ATCC 4894 grew with enhanced sporulation in 

 the refortified spent medium from PA 3679-h. The culture filtrates from 

 CI. hotulinum 62A, CI. aerojoetidum, PA 3679. Bacillus cercus, and Pseu- 

 domonas fluorescens enhance, to varying degrees, the sporulation of PA 

 3679-h. PA 3679 sporulated very poorly in all culture filtrates studied. 



When the replacement technique of Hardwick and Foster was used and 

 PA 3679-h was grown in refortified spent medium, it became committed 

 to sporulation, so that when the growth medium was replaced by a non- 

 sporogenic medium such as 2% trypticase broth, before incipient sporula- 

 tion could be observed, sporulation occurred in the replacement medium. 

 No sporulation occurred when the growth medium was replaced by buffered 

 thioglycollate solution or by a thioglycollate and glucose solution. This 

 failure to sporulate in the absence of the peptone might not have been 

 caused by a nutritional inadequacy; it might have been the result of an 

 inadequate poising of the replacement solution in the absence of the pep- 

 tone. This problem was not studied further. 



Upon dialysis of the culture filtrates of PA 3679-h. onl> the diahsate 

 was sporogenic when refortified with trypticase. The activity in the whole 

 spent medium was stable to lyophilization and to heat. The activity could 

 be removed from the whole spent medium by treatment with charcoal and 

 by absorption on both strong anion (IR 400) and strong cation lIR 120) 

 ion exchange resins, but the activity could not be recovered from either the 



