Bacterial Cell Morphology and Propagation Characterization via SEM

Scanning electron microscopy (SEM) was used to characterize bacterial cell morphology and μSiM-CA bacterial propagation as previously described (Masters et al., 2019a (link), 2020 (link)). For cell morphology characterization, S. aureus cultures were grown overnight, and then subcultured and seeded onto poly-L-lysine-coated glass coverslips for 6 h before rinsing bacterial cells and fixating with 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 M cacodylate buffer overnight. Similarly, μSiM-CA membranes were incubated for 6 h, as described above, and fixed with 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 M cacodylate buffer overnight. Samples were postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol to 100%, and critical point dried in a Tousimis CPD (Rockville, MD, United States). Samples were sputter coated with gold and imaged using a Zeiss Auriga Field Emission SEM (Jena, Germany) for quantification of cell diameters or qualitative assessment of bacterial propagation. ImageJ, specifically Fiji (Schindelin et al., 2012 (link)), was used to measure the maximum cell diameter across six separate SEM images per cell type, where a minimum of 20 cells were measured in each image.