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.)