B. anthracis spore germination and outgrowth in FBS-free cell culture media. B. anthracis spores were incubated in 96-well plates at 37°C and with rotary agitation within the indicated medium. Germination and outgrowth of spores were monitored at the indicated times. Medium conditions are listed at the top of the figure, and are applicable to (A-C). (A) Optical determination of germination and outgrowth. The data are rendered as the O.D.600 nm of the spore suspension at the indicated times relative to the original O.D.600 nm of the spore suspension at time = 0 of the 37°C incubation. Error bars indicate standard deviations. For each medium tested, the P-values were calculated to evaluate the statistical significance of the differences between O.D.600 nm values at the indicated times and O.D.600 nm values at the initial time point. (B) Spores heat sensitivity as a function of medium conditions. Aliquots from the spore cultures were removed at indicated times, incubated for 30 min at either at 65°C or on ice, diluted 101- or 102-fold (PBS pH 7.2), spotted (10 μL) on LB plates, and incubated at 25°C. After 18 h, the plates were photographed. (C) Visual determination of B. anthracis spore outgrowth as a function of cell culture medium. Aliquots from the spore cultures were removed at indicated times and analyzed for outgrowth using DIC microscopy. The bars indicate a length of 6.5 μm. The data in (A) are combined from 3 independent experiments. The data in (B) and (C) are from a single experiment and are representative of 3 independent experiments.