12 ROBERT E, COLLIER 



Table I 

 Degree of sporulation obtained with medium 1 



Time (hours) Percent spores 



2 



3 



4 



5 

 51/2 45 



6 45 



7 64 

 8I/2 75 



21 96 (lysed sporangia) 



ulum was chilled to 10°C. for thirty minutes. Then the inoculum was placed 

 in the test flask, which was held at 30^C., whereupon sporulation time and 

 percentage sporulation were followed. This approach did not give complete 

 and simultaneous sporulation, and furthermore the beginning of sporula- 

 tion was delayed for thirty minutes. 



Clostridium roseum, like other members of the butyric acid group of an- 

 aerobes, produces a large amount of acid. It occurred to us that the actively 

 growing culture might be lowering the pH sufficiently to interfere with the 

 production of spores. Accordingly, the pH of the original test medium was 

 determined at different intervals prior to and including the period of actual 

 sporulation. The drop in pH during the eight-hour period was from 7.2 to 

 6.0, which has been shown to be inhibitory to total sporulation. After test- 

 ing various combinations of buffer, the above medium was modified to 

 maintain the pH as close to neutrality as possible. The new medium is re- 

 ferred to as the modified tris test medium. It was our hope that by modifying 

 the medium to maintain a constant pH, it would be possible to obtain 100 

 percent simultaneous sporulation in the five and one-half hour period. Table 

 II shows the results which were obtained using the modified tris test 

 medium. 



It can be seen from this table that, although the pH was maintained around 

 neutrality, not all cells produced spores at the same time. Further incuba- 

 tion nevertheless did give almost 100 percent sporulation in a seven-hour 

 period. Table II also shows that after seven hours the percentage of spores 

 dropped. This is presumably due to germination of some of the spores in 

 the growth medium. 



During the preparation of the inoculum for use in the test flask, the cul- 

 ture should not be allowed to overincubate. That is, the culture must not be 



