20 



Z. JOHN ORDAL 





AFTER 48 HOURS OF SHAKING 



I I CELLS SURVIVED LYSIS 



^^S SPORE YIELD 



w 



^^ 



ph of replacement medium 

 Figure 2. 



defined (a modified Campbell and Williams [1953] medium). Sporulation 

 was evaluated on stained smears from a 6-day old slant incubated at 45°C. 

 It is apparent from data in Fig. 1 that maximum sporulation occurred at 

 pH values close to 6.5, even though the organism appeared to grow equally 

 well over the pH range 5 to 7.5. Fig. 1 shows also the effect of added 

 manganese. 



Fig. 2 shows the effect of pH on cells sporulated in replacement culture. 

 The vegetative cells were obtained from a 10-hour thermoacidurans broth 

 shake culture. After washing, the cells were suspended in M/20 phosphate 

 buffer adjusted to the indicated pH level and placed on a rotary- shaker at 

 45°C. In this case the degree of sporulation is represented as the "spore 

 yield" in order to compensate for the lysis which invariably took place in 

 replacement cultures with this organism. Minimum lysis and maximum 

 sporulation occurred at pH 7.0. In our studies with Putrefactive Anaerobe 

 3679, maximum sporulation always occurred when the growth and sporu- 

 lation media were adjusted to the pH range of 7.0 to 7.6. pH levels out- 

 side this range reduced sporulation even at levels where the vegetative 

 growth was still good. 



fc) Oxygen requirements. Knaysi in 1948 believed that oxygen was an 

 absolute necessity for sporulation of the Bacilli. While admitting the lack 

 of conclusive proof, he nevertheless stated that the need of oxygen in the 

 quick and efficient formation of large numbers of spores is "one of the 



