Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells

A fraction of Bacillus subtilis 168 spores, purified by Urografin isopycnic density centrifugation, heat activated, and inoculated in nutrient broth plus glucose, germinated and outgrew very synchronously. Synchrony was documented by nuclear staining and fluorescence microscopy, and by determining the variation of the buoyant density of the cells during outgrowth. Cell mass increased at constant rates and the passage from one rate to the next was dependent upon deoxyribonucleic acid (DNA) synthesis. DNA synthesis inhibitors induced the formation of anucleated sister cells in a vast majority of the population, indicating that septation was programmed and became independent from DNA synthesis very early in the cell cycle.