NUTRITIONAL AND ENVIRONMENTAL CONDITIONS 21 



incontrovertible facts of bacteriology." Since 1948, additional infonnation 

 has appeared. Roth et al (1955), using Bacillus anthracis and Bacillus 

 globigii, determined that when cultures were initiated from a heat-shocked 

 spore inoculum, an oxygenation rate of 0.7-1.0 mM 02/L/min, was re- 

 quired for complete sporulation. whereas only 0.1-0.2 mM O^/L/m. was 

 required for subcultures initiated from a culture at the peak of vegetative 

 growth. Lower oxygenation rates markedly reduced the percentage of 

 sporulation. 



Tinelli (19551 has recently presented evidence that as a culture of B. 

 megatherium approaches the sporulation stage, the rate of endogenous oxy- 

 gen uptake progressively decreases. However, just before the cells enter the 

 prespore stage, the rate markedly increases and, after a short time at this 

 higher rate, again decreases and becomes quite low as sporulation proceeds 

 to completion. This would suggest that, as the cells sporulate, they have an 

 increased energy demand in order to sustain the endogenous changes lead- 

 ing to the formation of a spore. 



//. Chemical factors. 



(a) Carbon sources. From the literature, it is difficult to evaluate the ef- 

 fect of carbon sources on sporulation per se because there is no clear dif- 

 ferentiation between the carbon requirements for growth and for sporula- 

 tion. It is logical to assume that sufficient carbonaceous material must be 

 present for reproduction and cell growth; and it must be of the kind to 

 produce cells having sufficient energy reserve for sporogenesis. Foster and 

 Heiligman (1949b) demonstrated that B. cereus grows well, but sporulates 

 poorly in a glutamate-salts medium. The addition of 2 mg/ml of glucose in- 

 creased growth by onlv 23 percent but increased sporulation by 2500 per- 

 cent. It would appear that, in this case, glutamate alone was adequate as 

 a carbon source for vegetative growth, but that cells produced in such a 

 medium were lacking in energy reserves sufficient to cause a high degree 

 of sporulation. The addition of the small amount of glucose corrected this 

 condition without significantly increasing the level of vegetative growth. 



There has been considerable speculation on the effect of glucose per se. 

 Grelet (1951), who supports Knaysi's hypothesis that sporulation only oc- 

 curs in healthy cells facing starvation, concludes that sporulation by B. 

 megaterium does not occur, under his experimental conditions, unless the 

 glucose is exhausted or omitted from the medium. However, if the nitro- 

 gen, sulfur, iron, or zinc is limiting, sporulation occurs in the presence of 

 glucose. In his replacement cultures, the delayed addition of glucose failed 

 to stop sporogenesis. Powell (1956) demonstrated that in a complex medium 

 sporulation by B. cereus was complete even though up to 20 percent of the 

 original carboydrate was still present. We have demonstrated that Putre- 



