2.10. Scanning Electron Microscopy (SEM)

To visualize the effect of the selected SEO NEs on the morphology of bacterial strains and the inhibition of biofilm formation, SEM was performed. The NEs were added to selected different biofilm producers at 1 and ½ of relative MIC concentrations. A 1 mL sample from each tube was seeded onto glass slides in 24-wells culture plates and incubated for 48 h. Samples were then washed twice with PBS (pH 7.4) and suspended in 2.5% glutaraldehyde (v/v) in 0.1 M cacodylate buffer (pH 7.4). After overnight fixation at +4 °C and washing with 0.1 M cacodylate buffer, samples were post-fixed with 1% OsO₄ in 0.1 M cacodylate buffer (pH 7.4), dehydrated in ethanol–water mixture with increasing ethanol concentrations (35%, 50%, 70%, 85%, 95%, and 100%), and dried with hexamethyldisilazane (HMDS, Sigma-Aldrich, St Louis, MO, USA) to remove fluids. Dehydrated specimens were gold-sputtered and observed by ultra-high resolution field emission gun scanning electron microscopy (FEG-SEM, FEI Company, Hillsboro, OR, USA). Secondary electron images were performed with an acceleration voltage of 20 KV. The images were processed for display using Photoshop software (Adobe Systems Inc., San Jose, CA, USA).