Neon sem

MRSE 35984 and MSSE 12228 cells were inoculated 0.5% from an overnight culture and grown at 35 °C with shaking. Each strain of bacteria was grown in four separate conditions: BPEI, oxacillin, combination (BPEI + oxacillin), and control. The OD₆₀₀ was monitored, and growth was stopped at late-lag phase (OD₆₀₀ = 0.20–0.25). Samples were fixed with Karnovsky fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer) for 30 min. The cells were then fixed with 1% OsO₄ for 30 min in the dark. The cells were washed with water three times. A couple drops of each sample were placed on clean, poly-L-lysine coated coverslips and air-dried for 30 min. To dehydrate, the samples went through a series of ethanol solutions (20%, 35%, 50%, 70%, and 95%), spending 15 min in each solution. Afterward, the samples were dried with hexamethyldisilazane (HMDS) and sputter-coated with AuPd. A Zeiss NEON SEM was used to image the samples at 5 kV accelerating voltage. Size analysis was performed on ImageJ.

P. aeruginosa BAA-47 cells were inoculated from an overnight culture (5 × 10⁵ CFU/mL) and grown at 35 °C with shaking. The bacteria were grown in four separate sublethal treatments: 600 Da BPEI (4 μg/mL), piperacillin (1 μg/mL), combination (4 μg/mL 600 Da BPEI + 1 μg/mL piperacillin), and untreated control. Growth was stopped at late-lag phase. Samples were collected by centrifugation and fixed with Karnovsky fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer) for 30 min. The cells were then fixed with 1% OsO₄ for 30 min in the dark. The cells were washed with water three times. A couple drops of each sample were placed on clean, poly-l-lysine coated coverslips and air-dried for 30 min. The samples were dehydrated by going through a series of ethanol solutions (20%, 35%, 50%, 70%, and 95%), spending 15 min in each solution. Afterward, the samples were dried with hexamethyldisilazane (HMDS). They were then mounted on aluminum stubs with carbon tape and sputter coated with AuPd. A Zeiss NEON SEM was used to image the samples at 5 kV accelerating voltage.