4.4. Preparation of Bacterial Samples for Scanning Electron Microscopy (SEM)

SEM assessed the morphological changes of bacteria after ZnO-NP treatment. Briefly, 1 mL of KPC suspension (10⁸ CFU/mL) was used for mixing with 1 mL of ZnO-NPs to reach a final concentration of 0.5 mg/L, followed by cultivation overnight at 37 °C. Two controls (negative control with only KPC with medium and positive control with KPC treated with the antibiotic impenem) were prepared in salt-free lysogeny broth medium alone. After the incubation, the samples were washed with normal saline by centrifugation at 1500× g and then fixed with 2.5% glutaraldehyde at 4 °C for 2 h, followed by washing with phosphate buffer (pH = 7.2). The samples were post-fixed in 1% osmium tetroxide, followed by dehydration through an ascending ethanol series, critical point drying, and coating with Au–Pd (80:20) using a Polaron E5000 sputter coater, Quorum Technologies, Laughton, UK. The samples were checked at an accelerating voltage of 25 kV in FEI Quanta 250 using an SE detector [17,18].