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100% acetone solution. The samples were infiltrated with Spurr’s resin and polymerized at 60 oC 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 onto 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 JEOL 1400 (JEOL, Ltd, Tokyo, Japan). 3.6 Determination of membrane leakage Overnight cultures of the three strains were inoculated into the respective broth medium containing different concentrations of ZnO NPs (0, 12, 16, 20 mM) and allowed to sit for 10 h at 37 oC. Similarly, strains were exposed to Ag NPs (0, 1.8, 2.7, 4.6 mM) for 6 h at 37 oC. After incubation, 1 mL of the treated bacterial suspension was centrifuged at 18,200 × g for 5 min and resuspended in peptone water. The light absorbance of the suspensions was examined using a UV-visible spectrophotometer (UV-1650 PC, Suzhou Instruments Manufacturing Co. Ltd, Suzhou, China) at a wavelength of 260 nm (for DNA absorbance) and 280 nm (for protein absorbance). All experiments were replicated twice. 3.7 Viability of bacterial cells To determine the viability of the treated cells, 1 mL of the samples was centrifuged at 18,200 × g for 5 min. Cell pellets were washed with 1 mL of 0.85% NaCl and stained using the BacLightTM Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal volumes of Component A (SYTO 9 dye) and component B (propidium iodide) were mixed thoroughly and 3 μL of the dye mixture 22PDF Image | ZINC OXIDE AND SILVER NANOPARTICLES ON INTESTINAL BACTERIA
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