2.4. Morphological Examination of the Bacterial Cells

Scanning electron microscopy (SEM) was used to examine the morphological changes of the bacterial cells before and after treating with ZnO NPs and Ag NPs. The three bacterial strains treated or untreated with NPs were fixed with a primary fixative (2.5 glutaraldehyde, 2% paraformaldehyde in 0.1 M Na-cacodylate buffer, pH 7.4). The samples were then rinsed three times with ultrapure water, followed by dehydration with a series of ethanol solutions (10%, 30%, 50%, 70%, 90%, and 100%). The dehydrated samples were immediately dried by a critical point dryer (Auto-Samdri 815 Automatic Critical Point Dryer; Tousimis, Rockville, MD, USA), mounted on SEM stubs and coated with a thin layer of carbon using a sputter coater (K575X Turbo Sputter Coater; Emitech, Ltd., Kent, UK). The coated samples were observed under SEM (FEI Quanta 600, FEI Company, Hillsboro, OR, USA) [30].

Energy dispersive spectroscopy (EDS) coupled with SEM was used for elemental analysis and was effective in locating and identifying NPs that were attached to the cells. Untreated cells and NPs attached to treated cells were spotted to analyze the elements. Three spots were selected in each sample to analyze the elements (Figures 4e, 5e, 6e, 7e, 8e and 9e).

Transmission electron microscopy (TEM) was employed to characterize the size of the NPs and to observe the morphology of the bacterial cells following treatment with ZnO and Ag NPs. Three samples of bacterial cells treated with and without NPs were fixed with a primary fixative and microwaved under vacuum conditions in a Pelco Biowave (Ted Pella, Inc., Redding, CA, USA) at 120 W. The samples were rinsed with 0.1 M cacodylate buffer and embedded in histogel, followed by a secondary microwave fixation with a buffered (0.1 M cacodylate, 0.01 M of 2-mercaptoethanol, and 0.13 M of sucrose) 1% osmium tetroxide. The samples were then quickly rinsed three times with 0.1 M 2-mercaptoethanol and 0.13 M of sucrose and then rinsed three times with ultrapure water. Then, samples were dehydrated with ethanol solutions (20, 50, 70, 90, and 100%) and 100% acetone solution. The samples were infiltrated with Spurr’s resin and polymerized at 60 °C for 24 h. The sample blocks were processed in 85 nm thin sections with Leica Ultracut UCT ultramicrotomes (Leica Microsystems GmbH, Wetzlar, Germany). The sections were placed on 200 mesh thin bar grids and post-stained for 20 min with 5% uranyl acetate and 10 min with Sato’s triple lead stain. Stained samples were then observed in a JEOL 1400 (JEOL, Ltd., Tokyo, Japan) transmission electron microscope [30].