NUTRITIONAL AND ENVIRONMENTAL CONDITIONS 27 



I 2) removal of a sporulation inhibitor; (3) the synthesis of something the 

 organism requires for sporulation. 



When the culture filtrate was fortified with the dry ingredients of the 

 medium at the level initially used in preparing the medium, the spore crop 

 was increased over that of the non-refortified filtrate with the same per 

 centage of sporulation. This seems to argue against the exhaustion of nu 

 trients or the removal of inhibitors of sporulation as responsible for the in 

 creased sporulation in the culture filtrates. In light of the report by Hard 

 wick. Guirard and Foster ( 1951 ) implicating saturated fatty acids as anti 

 sporulation factors, we used their methods for removing these antisporula 

 tion factors, namely treatment of the medium with charcoal, addition o 

 soluble starch to the medium, and saponification of the dry ingredients o 

 the medium, then neutralizing the soaps to convert them to the free fatty 

 acids and extracting the material with fat solvents, in the manner described 

 by Hilditch. None of these treatments enhanced the sporulation in the 

 mediums treated. 



Inasmuch as the evidence seemed to point toward the synthesis of some 

 sporulation factor in the culture filtrates, work was directed toward char- 

 acterization of the sporulation-enhancing property of the spent medium. A 

 simple medium for growth of the cells was a broth composed of 2% tryp- 

 licase (BBLl with 0.1% sodium thioglycollate. When this medium was ex- 

 hausted, it could be refortified with the initial ingredients, and reinoculated 

 to yield good sporulation. I might point out that, after much wasted time, 

 we learned to our sorrow that "trypticase" is not always trypticase. Much of 

 our early work was done with one lot of this peptone, and when some of 

 that work was repeated at a much later time, we got different results. For 

 example, spent medium, when refortified with 2% trypticase, yielded cul- 

 tures with 70-90% sporulation and a total population of about 1 x 10^ spores 

 per ml. Four per cent fresh tryptica?e was used as a fresh medium control 

 because this concentration of peptone was equivalent to the total concen- 

 tration of trypticase used in the refortified culture filtrate. Under these con- 

 ditions, the fresh 4% trypticase yielded less than 1% spores, in spite of 

 good vegetative growth. Much later in this work, with a subsequent lot of 

 trypticase, we observed that the 4% fresh control yielded very good sporu- 

 lation, or about 10-20% of that of the spent refortified medium. This rep- 

 resented about 15% sporulation in the culture. On the advice of Dr. Vera 

 of the Baltimore Biological Company, who suggested that the most proba- 

 ble difference between the various lots of trypticase was a difference in 

 thianline content, we routinely began adding 0.1 gamma of thiamine per ml 

 of the medium. When such additions were made to the fresh 49r trypticase 

 mediums, spore crops in the order of 3 x 10''' spores per ml were obtained, 



