Live cell fluorescence microscopy was performed using Hoechst 33342 (Molecular Probes, 15 μg/mL) and 1 μg/mL FM4–64 membrane stain (Molecular Probes) as previously described (Fimlaid et al., 2013 (link), Fimlaid et al., 2015b (link)). Briefly, 23 hr sporulating cultures of C. difficile strains were harvested into 1 mL PBS, pelleted, and resuspended into 100 μL PBS containing the dyes listed above. Live bacterial suspensions (4 μL) were added to a freshly prepared 1% agarose pad.
DIC and fluorescence microscopy was performed using a Nikon PlanApo Vc 100x oil immersion objective (1.4 NA) on a Nikon Eclipse Ti2000 epifluorescence microscope. Multiple fields for each sample were acquired with an EXi Blue Mono camera (QImaging) with a hardware gain setting of 1.0 using the NIS-Elements software (Nikon). Images were subsequently imported into Adobe Photoshop CC 2015 for minimal adjustments in brightness/contrast levels and pseudocoloring. The percentage of sporulating cells of wildtype, ΔIID, ΔIIP, and ΔIIQ that had completed engulfment was determined from 6–10 fields from one to two biological replicates. A minimum of 200 cells were counted per strain.
DIC and fluorescence microscopy was performed using a Nikon PlanApo Vc 100x oil immersion objective (1.4 NA) on a Nikon Eclipse Ti2000 epifluorescence microscope. Multiple fields for each sample were acquired with an EXi Blue Mono camera (QImaging) with a hardware gain setting of 1.0 using the NIS-Elements software (Nikon). Images were subsequently imported into Adobe Photoshop CC 2015 for minimal adjustments in brightness/contrast levels and pseudocoloring. The percentage of sporulating cells of wildtype, ΔIID, ΔIIP, and ΔIIQ that had completed engulfment was determined from 6–10 fields from one to two biological replicates. A minimum of 200 cells were counted per strain.
