Bioremediation of Heavy Metals and Toxic Chemicals …
71
3.3 Hydrocarbon Overlay Agar Method
The hydrocarbon overlay agar test was performed to determine the ability of bacteria
to utilize hydrocarbon as the carbon source. Nutrient medium was poured onto sterile
petriplates and allowed for solidification. 50 µl of Crude oil was spread on the nutrient
agar medium. Spread plate technique was used to overlay the selected organism
to the top of crude oil layer. The plates were incubated at 37
◦ C for 24–72 h. A
colony surrounded by emulsified halo was considered as positive for the biosurfactant
production.
3.4 Emulsification Index (E24)
Emulsification activity of the isolates were determined by emulsification index (E24)
for crude oil. 1.5 ml of crude oil was added to a clean test tube, to this equal volume of
cell free broth was added and vortexed at high speed for about 2–3 min. The content
was allowed to stand for 24 h to examine the emulsification activity. The percentage
of emulsification activity was calculated by the following equation: (Sarubbo et al.
2007): E24 = Height of emulsion formed × 100/total height of the solution.
3.5 Drop Collapse Test
To determine the biosurfactant activity, 1 drop of crude oil was placed onto the clean
cavity slide followed by 1 drop of cell free extract. After 1 min, the shape of drop
becomes flat after adding supernatant indicating ability of biosurfactant production.
A negative control was maintained by adding 1 drop of water to the crude oil sample.
3.6 Thin Layer Chromatography (TLC)
Screening of biosurfactant activity was evaluated by Thin layer chromatography technique. Silica gel plates were prepared, samples were spotted by using Pasteur pipette.
It was allowed to run in the solvent system consisting of chloroform: methanol:
water in (90:10:0.05) ratio respectively. Anthrone reagent was sprayed to detect the
presence of glycolipid type of biosurfactant as yellow spot.
71
3.3 Hydrocarbon Overlay Agar Method
The hydrocarbon overlay agar test was performed to determine the ability of bacteria
to utilize hydrocarbon as the carbon source. Nutrient medium was poured onto sterile
petriplates and allowed for solidification. 50 µl of Crude oil was spread on the nutrient
agar medium. Spread plate technique was used to overlay the selected organism
to the top of crude oil layer. The plates were incubated at 37
◦ C for 24–72 h. A
colony surrounded by emulsified halo was considered as positive for the biosurfactant
production.
3.4 Emulsification Index (E24)
Emulsification activity of the isolates were determined by emulsification index (E24)
for crude oil. 1.5 ml of crude oil was added to a clean test tube, to this equal volume of
cell free broth was added and vortexed at high speed for about 2–3 min. The content
was allowed to stand for 24 h to examine the emulsification activity. The percentage
of emulsification activity was calculated by the following equation: (Sarubbo et al.
2007): E24 = Height of emulsion formed × 100/total height of the solution.
3.5 Drop Collapse Test
To determine the biosurfactant activity, 1 drop of crude oil was placed onto the clean
cavity slide followed by 1 drop of cell free extract. After 1 min, the shape of drop
becomes flat after adding supernatant indicating ability of biosurfactant production.
A negative control was maintained by adding 1 drop of water to the crude oil sample.
3.6 Thin Layer Chromatography (TLC)
Screening of biosurfactant activity was evaluated by Thin layer chromatography technique. Silica gel plates were prepared, samples were spotted by using Pasteur pipette.
It was allowed to run in the solvent system consisting of chloroform: methanol:
water in (90:10:0.05) ratio respectively. Anthrone reagent was sprayed to detect the
presence of glycolipid type of biosurfactant as yellow spot.