Cholesterol assimilatioand the remaining cholesterol in the
spent broth was determined calorimetrically using ophthalaldehyde
method described by Rudel and Morris
(1973). One millilitre of the cell- free broth was added to
1 mL of KOH (33% wt/vol) and 2 mL of absolute ethanol,
vortexed for 1 min, followed by heating at 37 C for 15 min.
After cooling, 2 mL of distilled water and 3 mL of hexane were
added and vortexed for 1 min. One millilitre of the hexane
layer was transferred into a glass tube and evaporated in water
bath at 65 C. The residue was immediately dissolved in 2 mL
of o-phthalaldehyde reagent. After complete mixing, 0.5 mL
concentrated sulphuric acid was added and the mixture was
vortexed for 1 min. Absorbance was read at 550 nm (T80
UV/Vis spectrometer PG Instruments LDT, United Kingdom)
after 10 min. All experiments were replicated twice.