Deltavision core microscope system

Overnight cultures of B. subtilis harbouring GFP-SpoIVA and variants were induced to sporulate by the resuspension method48 (link) in medium containing 1 μg ml⁻¹ of the fluorescent membrane dye FM4-64 (Life Technologies). Cells were harvested and prepared for microscopy using a 1% agarose pad made with distilled water and viewed with a DeltaVision Core microscope system (Applied Precision)49 (link). Images were captured with a Photometrics Coolsnap HQ2 camera and deconvolved using SoftWorx software (Applied Precision). ImageJ was used to quantify the fluorescence located in the cells and forespores. For microscopy of SSLBs, 5-μl suspensions were placed on a glass bottom culture dish (Mattek Corp.) and covered by the agarose pad as described above. Thirty planes were acquired every 0.2 μm at room temperature; the data were deconvolved using SoftWorx software. The fluorescence intensities were then projected onto a single plane, quantified using SoftWorx software and reported as fluorescence micron⁻² of SSLB surface area.

S. aureus cultures were grown overnight at 37°C in TSB and subsequently diluted 1:10 in fresh medium. Cultures were then grown to mid-log phase (OD₆₀₀ equal to 0.5) at 37°C and treated with 2 μg mL⁻¹ gentamicin, where indicated, for a period of 2 h. Following the growth period, 1-mL aliquots of the cultures were washed in 1× PBS, and then cell pellets were resuspended in 100 μL of 1× PBS. Cells were stained with 1 μg mL⁻¹ FM4-64 in order to visualize the cell membrane. Five microliters of sample was transferred to a glass-bottom culture dish (MatTek) and then covered with a 1% agarose pad. All imaging was conducted at room temperature using a DeltaVision Core microscope system (Applied Precision/GE Healthcare) equipped with an environmental chamber. All images were captured using a Photometrics Coolsnap HQ2 camera, and data were deconvolved using SoftWorx software supplied by the microscope manufacturer.

Florescence microscopy was carried out as described previously (62 (link)). Overnight cultures of B. subtilis strains in LB liquid medium, or S. aureus strains in TSB supplemented with 10 μg/mL chloramphenicol (pCL15 backbone) or 5 μg/mL erythromycin (pJB67 backbone), were diluted to OD₆₀₀ of 0.1 and grown at 37°C to mid-logarithmic phase (OD₆₀₀ of 0.4 to 0.6). Then, for B. subtilis cultures, where needed, 1 mM IPTG or 1% xylose was added to the cultures and grown for an additional 3 h. For S. aureus, 1 mM IPTG (pCL15) or 1.25 μM CdCl₂ (pJB67) was added to the cultures (except GFP-TarG where no inducer was added) and where needed, 20 μg/mL tunicamycin or 8 μg/mL targocil was also added. Cells were then grown for an additional 1 h. Following incubation, 1-mL aliquots were then pelleted and resuspended in 100 μL PBS and 1 μg/mL SynaptoRed fluorescent dye to allow for visualization of the cell membranes. A culture aliquot (5 μL) was then transferred to a glass bottom dish (Mattek) and covered with a 1% agarose pad. Samples were imaged on a DeltaVision Core microscope system (Applied Precision/GE Healthcare) equipped with a Photometrics CoolSnap HQ2 camera and an environmental chamber. Seventeen planes were acquired every 200 nm, and the data were deconvolved using SoftWorx software. Cell diameter was measured using ImageJ and analysis completed in GraphPad Prism 9.