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 find more 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. Table 2 Germination and outgrowth of B. anthracis spores as a function of FBS concentration a . outgrowth e medium b FBS (%) c germination d 1 h 4 h DMEM 0.0 – - – 0.1 – - – 0.5 – - – 1.0 BMS-777607 concentration + – + 5.0 + + + 10.0 + + + a Three independent experiments were performed with three different spore preparations, each conducted in triplicate. b Spores prepared from B. anthracis Sterne 7702 were incubated in DMEM. c Indicates the concentration of FBS used in the DMEM. d Spores were scored positive (+) for germination O-methylated flavonoid if the OD600 nm of the suspended spores decreased by more than
5% after 30 min incubation in the indicated medium. e Using DIC microscopy, spores were scored positive (+) for outgrowth if the spores bodies were visibly larger at 1 h, and had developed into vegetative bacteria by 4 h. In the absence of FBS, several media were discovered to induce germination initiation and outgrowth of B. anthracis spores (Table 1). Germination initiation (30-60 min) and outgrowth were detected when spores were incubated in brain heart infusion (BHI) broth (Table 1, Figure 2), modified minimum essential medium alpha modification (MEMα) (Table 1, Figure 2), CO2-independent media (CIM) (Table 1), or McCoy’s 5A (M5A) (Table 1). Each of these cell culture formulations contains all 20 amino acids, is enriched particularly in the known germinant L-alanine (15-20 mg/L), and also contains non-specified nucleotides. Notably, some nucleotides function as germinants [35, 44, 45].