26 Z. JOHN ORDAL 



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Discussion 



Arnold J. Lund 



In my role as discusser of Dr. Ordal's paper, I appreciate that he in- 

 cluded a category he termed "other factors," because this gives me the op- 

 portunity to describe a phenomenon we have been studying. I shall limit 

 my remarks to describing our work. 



In searching for a method of culturing the Clostridium species, known as 

 Putrefactive Anaerobe (PA 3679), so as to obtain massed crops of "clean 

 spores," we observed that culture filtrates of the organism had sporogenic 

 activity. 



The organism we used was an isolate from an agar plate colony of PA 

 3679 (ATCC 7955). It varied from the parent culture only insofar as it 

 sporulated more freely than the parent culture, and on some mediums its 

 spores were slightly smaller. Except for these variations, it has been cul- 

 turally indistinguishable from the original strain. We have designated it 

 PA 3679-h. 



The sporogenic activity of the culture filtrates was first observed when 

 using Difco Brain-Heart Infusion Broth with added 0.1% sodium thiogly- 

 coUate as a culture medium for the organism. When the medium was inoc- 

 ulated at the level of 0.1% by volume with a spore suspension containing 

 1 x 10^ spores per ml., then incubated at 37°C. for 5 days, the culture de- 

 veloped less than 1% spores for a total spore crop of less than 1 x 10^ spores 

 per ml. When, however, the cells were removed from the culture after only 

 18-20 hr. incubation and the medium was reinoculated at the original level 

 and again incubated, the second cell crop yielded 70-90% spores, with a 

 total population in excess of 1 x 10^ spores per ml. This indicated to us that 

 the growth of the first crop of cells had made the medium sporogenic. This 

 sporogenic character of the culture filtrate must have resulted from one of 

 the following: (1) exhaustion of essential nutrient for vegetative growth; 



