Fluorescence Microscopy of B. subtilis Sporulation

Fluorescence microscopic images of WT and mutant B. subtilis were taken as previously described (Ebmeier et al., 2012 (link)). Briefly, overnight cultures of B. subtilis grown in casein hydrolysate (CH) media at 22°C were diluted 1:20 into 20 ml CH and grown at 37°C for 2 hr. Sporulation was induced via resuspension method (Sterlini and Mandelstam, 1969 (link)) in A+B media supplemented with 80 μg/ml threonine (Sigma) at 37°C. After 3.5 hr, cells were harvested and resuspended in PBS (KD Medical) containing 1 µg/ml FM4-64 (Invitrogen) to visualize membranes, then placed on lysine-coated glass bottom dish (MatTek Corp.) under a 1% agarose pad. Cells were viewed with a DeltaVision Core microscope system (Applied Precision) equipped with an environmental control chamber. Images were captured with a Photometrics CoolSnap HQ2 camera. Seventeen planes were acquired every 0.2 μm at 22°C, and the data were deconvolved using SoftWorx software (GE Healthcare). At the sporulation time points that we examined, phase bright forespores had not yet developed; thus, the autofluorescence of forespores was not higher than background fluorescence. Additionally, control experiments with sporulating strains that did not harbor a gfp fusion indicated that the level of GFP fluorescence from fusions to SpoIVA was well above the limited background fluorescence of the cells.