Quantification of indole-3-acetic acid

An overnight culture of strain MSSRFD41 grown on Luria-Bertani (LB) medium was inoculated into tubes containing 5 ml LB broth supplemented with 100 μg ml⁻¹ L-tryptophan and incubated at 28°C. Samples were collected after 72 h of incubation and the bacterial cells were removed by centrifugation at 6,500 g for 10 min. A 1 ml aliquot of the supernatant was mixed vigorously with 4 ml of Salkowski's reagent and incubated in the dark for 20 min at room temperature. IAA production was observed as the development of a pink-red color and absorbance was measured at 535 nm using a spectrophotometer. The concentration of IAA was quantified by comparison with a standard curve prepared with 5–100 μg ml⁻¹of pure IAA (Sigma Chemicals, India) (Patten and Glick, 2002).