Cas powder

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.

Chrome azurol S (CAS) was developed by Schwyn and Neilands to determine siderophore activity41 (link). It is a universal method that detects the mobilization of iron, thus assaying siderphore production. To prepare 100 ml CAS dye, 60.5 mg CAS powder (Sigma) was dissolved in 50 ml distilled water and mixed with 10 ml of 1 mM iron solution (anhydration FeCl_3, Alfa Aesar). Then, 40 ml of 72.9 mg HDTMA (Sigma) was added slowly to 60 ml CAS solution with FeCl₃ and autoclaved to sterilize. After the CAS cooled down and could be hand-held, one-tenth of the CAS solution was mixed with LP agar medium and immediately poured into plates.
CAS plates were used to demonstrate the activity of purified pyoverdine. Different concentrations of purified pyoverdine were injected into the hole (5 mm) of CAS plates. Plates were incubated at 28 °C for 6 h or until they had a yellow halo appearance.