2.10. Chrome Azurol S (CAS) Assay

Detection of secreted siderophore was performed by a previously published protocol [42] with some modifications. Briefly, corynebacterial cells grown overnight in HI broth at 30 °C were washed in a semi-defined low-iron medium (mPGT) and resuspended in fresh mPGT supplemented with or without 10 μM FeCl₃ to inoculate new cultures with a starting OD₆₀₀ of 0.05. After 20 h of growing at 30 °C, supernatants obtained by centrifugation were used in a colorimetric assay with chrome azurol S (CAS) to estimate siderophore production according to a published procedure [43]. In brief, obtained supernatants were mixed with CAS solution (3:1 ratio) and incubated at room temperature for 3 h, and absorbance at 630 nm of the resulting solutions were measured using a microplate reader (Tecan M1000). Data from three independent experiments performed in triplicates were expressed as mean with standard deviations. Siderophore production (SP) was estimated by the following equation: SP = (A_r − A_s) × 100/A_r, where A_r is absorbance of reference (CAS solution and un-inoculated broth) and A_s is absorbance of sample (CAS solution and culture supernatant).