CAS agar diffusion assay for the measurement of siderophores in biological fluids

Department of Microbiology, Chosun University, Medical School, Kwanju 501-759, South Korea.
Journal of Microbiological Methods (Impact Factor: 2.03). 03/2001; 44(1):89-95. DOI: 10.1016/S0167-7012(00)00229-3
Source: PubMed


We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. 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. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C 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 the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.

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    • "Reduction of potassium nitrite was shown by a transparent color of the mixture. Siderophore production of isolates was tested on Chromazurol-S (CAS) agar media as described by Shin et al. (2001). Isolates were cultivated for 120 h at 28 • C. Siderophoreproducing strains formed a halo zone around the colony. "
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    • "After incubation at 28 0 C for 24 h orange color indicated siderophore production and the diameter was measured. Ratio of orange halo diameter on media to colony diameter shown siderophore production as described by Sung et al., (2001). "
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    • "Protease activity indicated by casein degradation was determined according to distinct zones in skim milk agar (50 ml sterilized skimmed milk mixed with 1 g agar at 55 °C) after incubation at 30 °C for 3 days. Siderophore expression was determined on CAS agar as described by Shin et al. (2001). Diversity of amplified rDNA restriction analysis For analyzing DNA fingerprint diversity of antagonistic bacteria, bacterial genomic DNA was extracted by the Mini BEST Bacterial Genomic DNA Extraction kit (TaKaRa Biotechnology Co., Ltd, Dalien, China). "
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