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Chemostat cultures of Bacillus subtilis can be obtained in which a large proportion of the cells are initiated to sporulation. In a steady-state culture the fraction of cells which exhibit any particular stage of spore development is a function of the time taken for a cell to reach that stage after having been initiated to spore formation, and of the time required for the cell to pass through that stage. Equations describing this relationship have been derived. From these equations and from values for the fractions of cells exhibiting the morphological stages of spore development seen in electron micrographs, a time scale for the morphological events occurring during sporulation has been constructed.
By observing the effect of a sudden decrease in flow rate of a continuous flow system initially at steady state it was possible to time the occurrence of specific events, biochemical or physical, which were connected with the formation of spores. By this means, refractility was shown to be associated with stage V.