The formation of endospores in bacteria is generally studied in media which do not support growth: the exhausted growth medium or a solution of mineral salts into which washed growing bacteria are transferred.", 2 Under carefully adjusted cultural conditions, a reasonably synchronous sporulation can thus be obtained in the majority of the population. There seems therefore to be no need, and even no possibility, for a change in the conventional methods used to study sporulation.An entirely new approach became available in 1961, however, when it was observed that spores are constantly being formed in cultures of Bacillus rnegateriuin growing exponentially in a mineral-glucose medium.3 Similar results have recently been obtained with B. subtilis.' Relative to the total viable population, the number of spores first reaches a limit value, and then increases with time at a constant rate, equal to the growth rate of the total population.3The present work investigates the influence of various factors on the size of the sporulated fraction (hereafter called p) in growing cultures of B. subtilis. The interpretation of the results is that the probability for a cell in a growth medium to become committed to sporulate" I must be determined by the intracellular concentration of at least one nitrogen-containing catabolite repressing, directly or indirectly, the expression of all the sporulation genes. A preliminary account of this work has been included in recent reviews;6 further evidence supporting the proposed interpretation will be published elsewhere.8' I Experimental Procedure.-The wild Marburg strain of Bacillus subtilis has been used. A common mineral base for all media was prepared from the following three solutions (all figures in gm/liter); solution I: K2HP04, 10.5; KH2PO4, 3.5; solution II: MIgSO4, 7H20, 5.0; FeSO4, 7H20, 0.5; solution III: CaCl2, 5.0; MnCl2, 4H20, 0.5; solutions I and III were autoclaved, solution II sterile-filtered. The base medium is made of 100 vol of solution I for 1 vol each of solutions II and III. Various substances are then added as carbon or as nitrogen source; the complete medium is thus fully described with the mention of the nature of each source, followed, in gm/liter, by its concentration [e.g., NH4Cl(0.5)-glucose (2)].

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Fig. 1. Bacteria were first grown in nutrient broth (17), and protoplasted in SMM, the sucrose-magnesium-maleate buffer of Wyrick and Rogers (13), to which 5 ,ug/ml of DNase I (Worthington Biochem. Corp.) had been added (SMMD). Protoplasts were made to revert to bacillary forms by plating on RDR, a rich regeneration agar medium of high tonicity (13), to which 5 /g/ml each of DNase I and rifamycin (Lepetit Labs, Milano) were added. Prototrophic clones within the film of growth that appeared on RDR plates after incubation were selected out by replica plating onto variously supplemented SDR medium. This is a nonhypertonic minimal medium (14), to which 20 ,M MnCl2, 5 gg/ml of DNase, 1 Ag/ml of rifamycin, and 15 g/liter of (Difco) agar have been added. It was used as a selection medium, both unsupplemented (SDR) and supplemented (see Table 2).Procedure Adopted for Fusion Experiments. Overnight precultures of both parental strains in nutrient broth at 300 were inoculated, before growth ceased, into 20 ml of broth, to give an initial optical density (OD570) = 0.05. These cultures were incubated with shaking at 370 until an OD of 0.4 was reached. From each culture, 15 ml were centrifuged, the pellets were taken up in 3 ml of SMMD (OD570 = 2, or about 4 X 108 colony forming units/ml), and lysozyme was added to a concentration of 200,ug/ml. Complete protoplast formation was usually seen after 10 min of gentle shaking at 420, but exposure to lysozyme was continued for 20 more minutes. Samples (0.1 ml) of each suspension were then plated on ordinary nutrient agar. The plates usually remained sterile, and indicated that the frequency of osmotic shock resistant forms was below 2.5 X 10-8.One milliliter samples from each of the two suspensions were mixed in a third tube, the three tubes were centrifuged, and each pellet was resuspended in 0.2 ml of SMMD. To one tube, 1.8 ml of a 40% (wt/vol) solution of polyethylene glycol (PEG)t in SMM was added and the suspension immediately homogenized by shaking. After a 1 min exposure to PEG, either at 200 or at 00, several 0.05 ml samples were spread on the surface of duplicate RDR plates and used to make 10-1 and 10-2 dilutions in SMMD, from which, in turn, further reversion plates and also tThe molecular weight is not critical; PEG 6000 from Merck was usually used.

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