Journal of Methods Microbiological Journal of Microbiological Methods 37 (1999) 1–6 Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay * ˜ Adriane M.F. Milagres , Angela Machuca, Diovana Napoleao ´ Department of Biotechnology, Faculdade de Engenharia Quımica de Lorena-FAENQUIL, Lorena, CP 116, CEP 12.600-000, SP , Brazil Received 14 September 1998; received in revised form 21 December 1998; accepted 15 January 1999 Abstract A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.  1999 Elsevier Science B.V. All rights reserved. Keywords: Bacteria; Chrome azurol S; Fungi; Siderophore 1. Introduction (1997). This assay is based on a competition for iron between the ferric complex of a indicator dye, To sequester and solubilize ferric iron many chrome azurol S (CAS), and a chelator or sidero- microorganisms utilize an efficient system consisting phore produced by microorganisms. The iron is of low-molecular mass ( , 1000 Da) compounds removed from CAS by the siderophore, which with high iron affinity, termed siderophores (Hider, apparently has a higher affinity for iron (III). The 1984; Neilands, 1984, 1995). Nowadays, the most positive reaction results in a color change of CAS common detection method for siderophore product- reagent (usually from blue to orange). ion is the universal assay of Schwyn and Neilands The blue dye can be applied as a solution test, or alternatively, it can be incorporated in the solid growth medium and used for screening several *Corresponding author. Tel.: 1 55-125-533-422; fax: 1 55-125- microorganism siderophore producers by direct plat- 533-165. E-mail address: adriane@debiq.faenquil.br (A.M.F. Milagres) ing mode (Ames-Gottfred et al., 1989; Alexander 0167-7012 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0167-7012(99)00028-7