Quantitative Siderophore Detection in M9 Medium

The relative quantitative siderophores in chelated M9 minimal medium (c-M9) was determined as described previously (Payne, 1994 (link)). Briefly, the siderophore assay solution was made by 50 ml of 1.2 mM hexadecyltrimethylammonium bromide, 1.5 ml of 1 mM FeCl₃ · 6H₂O, 7.5 ml of 2 mM chrome azurol S (CAS), and 1.37 M piperazine (pH=5.6). 100 ul of each sample was added to wells of a flat-bottom 96-well plate, and then 100 ul of siderophores assay solution with 2% 0.2 M 5-sulfosalicyclic acid was added. The reaction mixture was incubated for 30 min, and were measured at A680 nm. The c-M9 was plus siderophores assay solution plus 5-sulfosalicyclic acid solution was used as a reference (r). The sample (s) should have a lower absorbance than the reference. Siderophore units (Su) are defined as [(Ar - As)/Ar] × 100 = % siderophore units. Each assay was performed in duplicate and repeated three times independently.
Siderophore production was also detected by the CAS agar plate assay (Schwyn and Neilands, 1987 (link)). The stationary-phase iron-cheated cultures (3μl) were dropped on CAS plates, and siderophore production was determined by the orange halos after incubation for 48 h at 37°C.