Isolation and Characterization of Arsenic Tolerant Bacteria …
235
characterize arsenic tolerant bacteria from arsenic polluted areas and to analyse the
arsenic removal potency.
2 Materials and Method
2.1 Sampling and Preparation of Metal Solutions
The present study selected two industrial zones where arsenic is used for manufacturing their products. Two soil samples were collected from the industrial waste
disposal area (5–6 cm) depth. Control sample was collected from region free
of any arsenic contamination. The simulated arsenite and arsenate solution was
prepared from the salts sodium arsenate and sodium arsenite, respectively, in 1 mM
concentration.
2.2 Isolation of Metal (Arsenic) Resistant Bacteria,
Screening and Characterization
The metal resistant bacteria were isolated from the collected soil sample by spread
plate method. The media used was minimal media [M9 salts: 20 ml, MgSO 4 (1 mM)–
200 µl, CaCl 2 (1 mM)–10 µl] and TSB agar media supplemented with 0.5 mM of
arsenite solution. Pure culture was done by streak plate method.
The MIC at which no colony growth occurred for arsenite solution was performed
by broth dilution method. Minimal media was prepared supplemented with different
concentrations of arsenite solution (0.1–150 mM/L). The tubes were inoculated aseptically and incubated at 35 °C for 48 h. Minimum concentration of arsenite allowing
growth of the isolates was an indication of positive tolerance.
The bacterial isolates that could tolerate the arsenic concentration were selected
and characterized physically, biochemically and molecular. Gram nature was
observed, silver nitrate test was performed as described by Simeonova et al. (2004).
Biochemical tests were performed, the MRVP test, catalase test, starch hydrolysis,
TSI, respectively, to characterize the bacteria isolated. Molecular characterization
was done by isolation of plasmid DNA and confirming that the bacterial isolates
contain plasmid.
235
characterize arsenic tolerant bacteria from arsenic polluted areas and to analyse the
arsenic removal potency.
2 Materials and Method
2.1 Sampling and Preparation of Metal Solutions
The present study selected two industrial zones where arsenic is used for manufacturing their products. Two soil samples were collected from the industrial waste
disposal area (5–6 cm) depth. Control sample was collected from region free
of any arsenic contamination. The simulated arsenite and arsenate solution was
prepared from the salts sodium arsenate and sodium arsenite, respectively, in 1 mM
concentration.
2.2 Isolation of Metal (Arsenic) Resistant Bacteria,
Screening and Characterization
The metal resistant bacteria were isolated from the collected soil sample by spread
plate method. The media used was minimal media [M9 salts: 20 ml, MgSO 4 (1 mM)–
200 µl, CaCl 2 (1 mM)–10 µl] and TSB agar media supplemented with 0.5 mM of
arsenite solution. Pure culture was done by streak plate method.
The MIC at which no colony growth occurred for arsenite solution was performed
by broth dilution method. Minimal media was prepared supplemented with different
concentrations of arsenite solution (0.1–150 mM/L). The tubes were inoculated aseptically and incubated at 35 °C for 48 h. Minimum concentration of arsenite allowing
growth of the isolates was an indication of positive tolerance.
The bacterial isolates that could tolerate the arsenic concentration were selected
and characterized physically, biochemically and molecular. Gram nature was
observed, silver nitrate test was performed as described by Simeonova et al. (2004).
Biochemical tests were performed, the MRVP test, catalase test, starch hydrolysis,
TSI, respectively, to characterize the bacteria isolated. Molecular characterization
was done by isolation of plasmid DNA and confirming that the bacterial isolates
contain plasmid.