*2.4. Scanning Electron Microscopy (SEM)*

To observe the effect of ceragenins on cell membranes, selected isolates were cultured to mid-log phase and washed three times with PBS. Bacteria were re-suspended in PBS (OD600 = 0.2). CSA-131 (25 μg/mL) was then added and the mixtures were incubated at 37 ◦C for 1 h. A control was prepared by incubating the bacterial suspension without adding CSA-131. After collection via centrifugation, cells were washed with PBS three times. Gluteraldehyde (2.5% (w/v)) was added to fix the cells at 4 ◦C overnight. Resulting material was washed five times with PBS at 5000 rpm for 10 min

using a microhematocrit centrifuge (Hettich Mikro 20, Hettich, Tuttlingen, Germany) to remove the glutaraldehyde. Osmium tetroxide (0.5 mL) was used as a second fixative reagent, and samples were stored at room temperature under a protective laboratory hood system for 2–3 h. Cells were washed with PBS five times at 14,000 rpm for 8 min. A graded ethanol series including 10%, 30%, 50%, 70%, 90% (1 time), 100% (3 times) and HMDS (2 times) for 15 min each was used to dehydrate the cells. Samples were collected by centrifuge each time and the supernatant was discarded after each centrifugation. Finally, dried bacterial specimens were sputter-coated with 5–10 nm of a Gold-Palladium alloy and visualized under a scanning electron microscope (FEI Helios NanoLab 600 SEM/FIB, Hillsboro, Oregon, USA) [12].
