Scanning Electron Micrograph-Energy Dispersive Spectroscopy (SEM-EDS) and Transmission Electron Microscope (TEM)
Morphology of bacterial cells before and after arsenic treatment was examined using SEM (JSM 6490, JOEL, USA). The NR5 were raised in normal and 25 mg L-1 As amended nutrient broth. Cells were harvested by centrifugation at 3000 rpm for 10 minutes and collected in sample size of 2-4mm. Fixation of bacterial cells was carried out using 2.5% glutaraldehyde solution for 2-6 hours at 4°C, and subsequently washed with 0.1M phosphate buffer. Post fixation was done by treating samples with 1% osmium tetroxide for 2 hours at 4°C, and again, samples were washed with 0.1M phosphate buffer to remove unreacted fixative. Specimens were dehydrated using increasing concentrations of ethanol to remove water. Specimens were dried and mounted on aluminium stubs with carbon tape. Sputter coater used for coating and to make samples conductive. Finally, samples were visualized under SEM. Content of As in control and arsenic treatment NR5 was estimated using energy dispersive spectroscopy (EDS). To perform TEM analysis of control and arsenic loaded bacterium, cells initially fixed in 2.5% glutaraldehyde and later with osmium tetroxide in sodium cacodylate buffer. Samples were dehydrated, thin sections were prepared using microtome, and visualized under (TEM Jeol 1011, Japan, 80KVA.)