FBS in cell culture media promotes germination and outgrowth of B. anthracis spores. B. anthracis spores were incubated in 96-well plates at 37°C and with rotary agitation in 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 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. For BHI, DMEM, RPMI, and MEMα, initial decreases in O.D.600 nm reflect the loss of spore refractility that occurs subsequent to germination initiation, while the increases in O.D.600 nm measured at later time points (1 and 4 h) reflects bacterial replication. For PBS, the modest increases in O.D.600 nm are due to time-dependent medium evaporation. 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) Spore heat sensitivity as a function of medium conditions. Aliquots from 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 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.