An Approach to Synchronous Growth for 

 Spore Production in Clostridium roseuni 



Robert E. Collier 



Department of Bacteriology, University of Illinois, Urbana, Illinois 



THE production of large amounts of anaerobic spores free from vege- 

 tative cells is not possible with conventional methods, but means to 

 do this must be found if an adequate study of the physiology of 

 these spores is to be made. Culture methods used heretofore result in a 

 mixture of all possible cell forms: young vegetative cells, cells in various 

 stages of sporulation, free spores, and spores in various stages of germi- 

 nation. It is virtually impossible to harvest clean spores from such a mix- 

 ture. 



We have been able to solve this problem by using methods that give a 

 pseudosynchronous growth of the vegetative cells. By transferring a very 

 heavy inoculum of organisms in the log phase of growth into a suitable 

 sporulation medium, a culture is produced in which sporulation is initiated 

 in every cell, after only a few generations, and every cell produces a spore 

 at about the same time. If the growth medium is properly balanced with 

 nutrients, those required for germination are practically exhausted during 

 growth, and the spores which are formed cannot germinate in the original 

 medium. 



The culture may need to stand for some time before the spores are set 

 free, but as long as the spores do not germinate in the growth medium, 

 the final result is a suspension of relatively clean spores that can be har- 

 vested with little difficulty. 



Materials and Methods 



Preparation of inoculum for pseudosynchronous groivth. A stock spore sus- 

 pension of Clostridium roseum was prepared by growing the organism in 

 1.5 percent trypticase medium for 48 hours at 30°C. Six-ounce screw-cap 

 bottles each contained 100 cc. of this medium; and a volume of the spore 

 suspension equal to 10 percent of the medium to be inoculated was desig- 

 nated as the 10 percent inoculum. The spore suspension was subjected to heat 

 shock at 65°C. for 30 minutes, after which a 10 percent inoculum was trans- 

 ferred to fresh trypticase medium contained in the bottles every three hours. 

 This was repeated three times. On the fourth transfer the inoculation was 

 made into the test flask in which the spores were to be produced. 



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