SUMMARY: Sporulation of Bacillus sphaericus, NCTC 7582, in a complex medium was strongly stimulated by growth in mixed culture with a sporing strain of B. cereus and with sporogenous and asporogenous strains of B. subtilis, NCTC 85. There was a similar, but less pronounced, effect with a variety of Gram-positive and Gramnegative organisms. The degree of sporulation of B. sphaericus in these mixed cultures depended on ( a ) an external stimulus from the medium and (b) the previous cultural history of the B. sphaericus inoculum upon which depended its ability to respond to the external stimulus. Sporulation of B. sphaericus in pure culture was strongly stimulated by bicarbonate and ketoglutarate, suggesting that increased carbon dioxide production was the stimulating factor in mixed cultures.The stimulatory effect of one organism upon the growth of another has been observed in bacteria and in fungi (e.g. Hawker, 1939). This report describes a similar phenomenon in the genus Bacillus and suggests a mechanism for the effect.
ORGANISMS AND METHODSA culture of Bacillus sphaericus, NCTC 7582, grown for 24 hr. at 37" on casein hydrolysate yeast extract (CCY) agar (Gladstone & Fildes, 1940) was suspended in 50 ml. glucose serum broth (Fry & Greaves, 1951) and freeze-dried in 5 ml. batches. Starting with one of these freeze-dried batches, a series of sporulation tests was made, during which time the organism was maintained by subculturing every 48 hr. on CCY agar, and on potato-extract agar (Robinow, 1951) enriched with 1/10 vol. of CCY medium (Pot. CCY).The strongly sporing strain of Bacillus cereus isolated from air and the sporogenous and asporogenous strains of Bacillus subtilis, NCTC 85, were maintained on CCY agar. The other organisms used were subcultured at least twice in Pot. CCY medium before the mixed culture tests.For the sporulation tests, which were set up in duplicate, 10 ml. batches of liquid Pot. CCY medium in 50 ml. conical flasks were inoculated with 0.5 ml. of a just visibly turbid suspension of organisms and shaken at 37" for periods up to 36 hr.
RESULTSThere was rapid growth and remarkably vigorous sporulation of Bacillus cereus and B. subtilis in the test medium, which was selected for this reason. The first signs of sporulation appeared at 14 hr., and at 17 hr. almost every