Chrome Azurol S Assay for Siderophore Detection

A chrome azurol S (CAS) agar assay was carried out as previously described (24 (link)). To make a 100-ml CAS stock solution, 60.5 mg CAS powder (Sigma) was added to 50 ml of ddH₂O, followed by 10 ml of 1 mM FeCl₃. Then, 72.9 mg hexadecyltrimethylammonium bromide (HDTMA; Sigma) was fully dissolved in 40 ml ddH₂O. At last, the entire HDTMA solution (40 ml) was slowly poured into 60 ml of CAS solution with constantly shaking to form a 100-ml CAS stock solution. A CAS agar plate was prepared by 9 parts freshly autoclaved 1.5% agar KB plate and 1 part CAS stock solution. After the agar solidified, two circular holes were dug into CAS agar by the round end of a 1-ml sterile pipette tip. About 2 μl overnight bacterial culture (OX-yedQ and OX-yhjH strains; OD₆₀₀, 1.0) of the experiment group and control group was added into one of two holes and cultivated at 28°C. The CAS plate (without antibiotics) was photographed after 3 days against a white background. Three CAS plates were used for two strains, and the experiment was repeated with three independent bacterial cultures.