Quantifying Siderophore Production

Samples containing siderophores of interest were assessed using a Chrome Azurol S-based assay as described previously (12 (link)). Briefly, sterile solid medium containing the colorimetric dye (2% agar, 100 µM Chrome Azurol S, 200 µM hexadecyltrimethylammonium bromide, 10 µM FeCl₃, 0.5 M 2-(N-morpholino)ethanesulfonic acid, pH 5.5) was poured as 15 mL plates. To measure siderophore activity for conditioned medium in situ, 15 mL of CAS agar was prepared and poured as described above into a 10 cm Petri dish. After 7 days of growth, biomass was removed from the surface of the conditioned solid medium, followed by overlaying of the conditioned medium with CAS agar. These sandwiched agar plates were then incubated in the dark at room temperature for 4 hours; after which, the CAS agar layer was removed and photographed.