The effects of pre-conditioned culture medium on the germination state of B. anthracis spores. DMEM (A, B) or RPMI (B) were pre-conditioned by incubating with monolayers of RAW264.7 (A, B) or MH-S cells (B) at 37°C and under 5% CO2, in the absence (A) or presence (MOI 10) (B) of B. anthracis spores. (A, B). After 4 h (white bars) or 24 h (black bars) (A), or after 1 (white bars) and 4 h (black bars) (B), the medium was removed from the monolayers, filter sterilized, and then incubated with B. anthracis spores in 96-well plates at 37°C and with rotary agitation. Germination and outgrowth of spores were monitored at indicated times by measuring O.D.600 nm. The results are rendered as the O.D.600 nm of the spore suspension at the indicated time relative to the original O.D.600 nm of the spore suspension at time = 0 of the 37°C incubation. P-values were calculated to evaluate the statistical significance of the differences between O.D.600 nm values at the initial time point and O.D. O.D.600 nm values at the indicated times. For (B), BHI (gray bars) was used as a positive control for germination and outgrowth. (C) An equal number of B. anthracis spores were incubated at 37°C and under 5% CO2 in DMEM (no FBS) in the absence (white bars) or presence (black bars) of RAW264.7 cells (MOI 10). At indicated times, aliquots of culture medium were removed, and spores were evaluated for heat resistance. The results are rendered as the number of heat resistant spores relative to spores incubated in DMEM alone, which were normalized to 1.0. P-values were calculated to evaluate the statistical significance of the differences in heat resistant spores between those incubated in the presence or absence of RAW264.7 cells. The data in (A-C) are combined from 3 independent experiments conducted in triplicate with error bars indicating standard deviations.