Quantifying Indole Compounds in Bacterial Cultures

Bacterial strains from glycerol stocks were streaked onto either an LB agar plate or a TSA plate, depending on the medium of their original isolation and grown at 28°C. For each strain, a single colony was used to inoculate 6 mL of liquid LB medium, with and without 5 mM L-tryptophan. For DAB 33B and DAB 39B, liquid TSB (with and without 5 mM L-tryptophan) was used instead due to difficulty growing these two strains on LB medium. After 48 h of growing at 28°C with shaking at 240 rpm, 1 mL of culture was centrifuged for 5 min at 14,000 rpm to collect the supernatant. The original Salkowski assay based on the Gordon and Weber protocol was adapted for a 96-well format (Gordon and Weber, 1951 (link)). In a Corning 96-well clear bottom white plate, 100 μL of the supernatant was added to 200 μL of Salkowski reagent (10 mM FeCl₃, 97% reagent grade, and 34.3% perchloric acid, ACS grade) in duplicate. After incubating samples with the Salkowski reagent at room temperature for 30 min, the color change was recorded. A BioTek Synergy HT microplate reader was used to determine the absorbance (O.D.) at a single wavelength of 530 nm. To estimate the amount of indole related compounds at 530 nm, an IAA standard curve was generated by suspending IAA (Gibco Laboratories, Life Technologies, Inc., New York, USA) in 100% acetonitrile at a concentration of 1 mg/mL and diluting in LB medium or TSB to a concentration of 100, 50, 20, 10, 5, and 0 μg/mL. Sterile LB medium (with and without 5 mM L-tryptophan) and sterile TSB (with and without 5 mM L-tryptophan) were used as controls. The concentration of indole related compounds at 530 nm of the sterile control sample, either LB or TSB depending on the bacterial medium used, was subtracted from the concentration of indole related compounds at 530 nm of the bacterial samples to obtain a background subtracted concentration.
A full spectrum analysis from 440 to 600 nm, using a 1 nm interval, was performed to identify the wavelength of maximum absorbance. Full spectrum analysis was performed on bacterial samples grown in liquid LB medium supplemented with 5 mM L-tryptophan whereas free indole and free IAA (as references) were suspended in 100% acetonitrile. The wavelength of maximum absorbance (λ_max) was calculated between 460 and 600 nm due to the high background signal observed at wavelengths shorter than 460 nm from addition of the Salkowski reagent to LB medium.
For determining the specificity of the Salkowski reagent, we tested IAA, indole-acetamide (IAM), indole-3-pyruvatic acid (IPA), ILA, indole-3-butyric acid (IBA), indole, indoxyl sulfate, tryptophol, and tryptophan. The compounds were suspended in 100% acetonitrile, HPLC grade, before diluting in LB medium, which did not contain 5 mM L-tryptophan due to the high absorbance background tryptophan generates when performing a spectrum analysis from 440 to 600 nm wavelength.