“ENDOTROPHIC” SPORULATION

The ability of vegetative cells to sporulate in water was first reported by Buchner ( 1890) and confirmed by Schreiber (1896) and Knaysi (1945). More recently, the replacement of a complex sporulation broth by water has proved to be a useful procedure for a variety of purposes (Grelet, 1951, 1955; Hardwick and Foster, 1952; Powell and Hunter, 1953; Foster and Perry, 1954; Perry and Foster, 1954, 1955; Murrell, 1955; Tinelli, 1955; Nakada, Matsushiro, and Miwatani, 1956; Ordal, 1957; McDonald and Wyss, 1959; Portellada, 1959a, b; Bennett and Williams, 1960; Black, Hashimoto, and Gerhardt, 1960; Hodson and Beck, 1960; Pelcher, 1961; Bmck, 1962; Keynan, Murrell, and Halvorson, 1962; Stevenson, Miller, Strothman, and Slepecky, 1962). Hardwick and Foster (1952) were among the first to exploit the “replacement technique” for a systematic study of sporogenesis and, as a result of their experiments, advanced the hypothesis that endospore development is strictly an endogenous process. Termed “endotrophic” sporulation, the metamorphosis of a vegetative cell to a spore in water was described as “occurring independently of exogenous nutrition and supported exclusively by the pre-existing makeup of the vegetative cell” (Foster and Perry, 1954). Powell and Hunter (1953), however, observed a substantial degree of lysis after transfer of vegetative cells to water and contended that sporulation of the surviving cells took place not in water but in a dilute autolysate medium. Others employing the replacement technique ( Murrell, 1955; Ordal, 1957; Pelcher, 1961) also have reported autolysis. In our experience with “endotrophic” sporulation (Black et al., 1960), we found that lysis of about 90 per cent of the population occurred when synchronously developing bacilli in the granular stage were removed from a primary culture, washed in water, and reincubated in water. We came to question, therefore, whether sporogenesis is in fact completely endogenous and whether autolysis in the population contributes to, or is required for, sporulation of the remaining cells. Using Bacillus cereus strain terminalis, we performed experiments designed to prevent lysis or interference by lytic products. Materials and Methods The test organism was grown either in baffled 2 liter Erlenmeyer flasks on a shaker or in carboy mass culture (Hashimoto, Black, and Gerhardt,