CAS agar diffusion assay for the measurement of siderophores in biologicalfluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluid s. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Hol es with 5-mm-diameter were punched on the CAS agar plate. Each hole was add ed by 35 mul of the test fluids containing Desferal that was twofold serial ly diluted. After incubating at 37 degreesC or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all th e biological fluids tested in this study. CASAD assay showed consistent res ults in wide pH range from 5 to 9. Addition of iron to the test fluids cont aining Desferal decreased the size of orange haloes in a dose-dependent man ner, which suggests that the CASAD assay detects only iron non-bound sidero phore. These results suggest that CASAD assay would serve as a simple, stab le, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids. (C) 2001 Elsevier Science B.V. All rights r eserved.