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Spore formation is a controlled process of differentiation which has been arbitrarily divided into seven stages: the axial chromatin stage, septation, engulfment of the spore protoplast, cortex formation, coat formation, maturation, and the free spore stage [P. C. Fitz-James, Colloq. Intern. Centre Natl. Rech. Sci. (Paris) 124: 529, 1965; P. Schaeffer et al., Colloq. Intern. Centre Natl. Rech. Sci. (Paris) 124:553, 1965]. For studies of the biochemical events related to spore formation and the development of the spore structures, the time at which sporulation commences and the degree of sporulation synchrony must be known at the beginning of the experiment. Otherwise, particular treatments under study may be commenced at the wrong stage, or time-consuming biochemical analyses may subsequently prove worthless because sporulation was not well synchronized. The time of the initial event in spore formation, to, has been arbitrarily defined as the end of exponential growth (P. Schaeffer, Ph.D. Thesis, Paris, 1961; J. Szulmajster and P. Schaeffer, Compt. Rend. 252:220, 1961); it is evident only some time after it occurs. Spore septum formation ("septation"), the beginning of stage II, would be just as useful a temporal point on which to base the sporulation time scale. Septation can be detected by negative staining with phosphotungstate (A. Ryter, P. Schaeffer, and H. Ionesco, Ann. Inst. Pasteur 110:305, 1966), but this incurs a delay of at least a few minutes, and an electron microscope is required. Thin sectioning involves considerably more delay, and both methods severely limit the sample size. During studies on the biochemical events during spore formation in Bacillus cereus T, we wished to add antibiotics at definite stages in spore formation, e.g., at the beginning of stage III. It was found that the early stages, from the beginning of septation to the end of stage III, were observed most conveniently and simply by mounting the cell suspension in an equal volume of 0.03% (w/v) aqueous crystal violet. The cells were not fixed or dried in any way, as this breaks the permeability barrier, thus allowing uptake of too much stain and obscuring membrane development. Mounting the live cells in dilute crystal violet resulted in an immediate uptake of stain by the invaginated membranes, and, with the aid of light microscopy, septation and engulfment of the spore protoplast were delicately revealed (Fig. 1). In samples of hundreds of cells, the stage of sporulation was ascertained within seconds, and the degree of sporulation synchrony was estimated rapidly. Cells held at 0 C for 2 to 3 days stained satisfactorily. Accurate counts were made to determine whether 95% of the cells were in the process of septation (synchrony index of 0.95). Similarly, counts made at any later stage could determine whether the synchrony index remained constant throughout sporulation. When the cells were viewed microscopically, there was evidence of fine gradations both in the uptake of stain by the membranes and in the permeability of different cells, especially when the stain was not well mixed with the cells. These gradations were very difficult to record photographically. The exact concentration of stain required varied with cell concentration and may need to be varied with different organisms and conditions of growth, as the concentration of stain revealing most detail was rather critical: a concentration too low failed to show the membranes, and one too high completely obscured them. These early membrane developments were not visible under phase-contrast. However, during and after stage IV it was preferable to view under phase-contrast. Similar observations have been made with the larger Bacillus species, e.g., B. megaterium and B. coagulans. The cells of B. subtilis are too small to permit the membrane rearrangements to be satisfactorily detected.

Simple method of detecting spore septum formation and synchrony of sporulation