Chrome azurol

Chrome azurol S (CAS) assay

Chrome azurol S (CAS) is a method that can be used to detect the mobilization of iron. In many soils iron is not often in an abundant form that plants can readily uptake and use. Siderophores are produced by rhizosphere bacteria to help enhance plant growth by increasing the availability of iron near the root (1). A siderophore is a low molecular-weight Fe(III)specific ligand that is a chelator. Chelators form multiple bonds with metal ions and in the case of siderophores they assist in acquiring useable iron. Siderophore in greek literally means iron carrier. The role of siderophores is to scavenge iron from the environment and make the mineral available to the cell (6). Siderophores are not only used in the process of plants acquiring iron andother metals, they also are found on bacteria in the body on cells and in some cases the siderophores rob our own blood cells of iron. Escherichia coli strains have been used in CAS assays to demonstrate the production of siderophores (7). In an ecological study CAS was used to detect mobilization of iron by testing for siderophore presence in soil samples gathered from the study site (2). Thus,CAS can be useful in determining if siderophores are being produced in ecological samples as well as medical samples.

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Under metal-limiting conditions, the bacterium A. vinelandii secretes metal-scavenging compounds (siderophores; S) (1). These siderophores scavenge the metals molybdenum and vanadium from unavailable complexes with clay, soil organic matter or other elements (2). Thesiderophores compete with siderophores produced by other organisms such as fungi for these metals (3). The bacterium or plant roots readily take up the siderophore-metal complexes (4). Within the bacterium, the metal is incorporated into the enzyme nitrogenase (5), to allow the fixation of atmospheric nitrogen (N2) that would otherwise be unusable to the bacterium. (3)

To test mobilization ofiron, chrome azurol S (CAS) media can be used. On CAS, iron mobilization is done via the production of complexing acids or siderophores. Since siderophore production increases availability of iron, measuring the production of these gives us a measure of iron mobilization ability of organisms. Alexander and Zuberer (1991) demonstrated that CAS agar effectively differentiated bacteria that were ableto excrete large amounts of siderophore. Frey-Klett et al. (2005) used this same method however the CAS media was modified. Consequently, it seems that many bacteria fail to grow on CAS agar because of a large presence of HDTMA which is a cationic detergent that is added to CAS agar to stabilize the Fe-CAS indicator and gives the medium a blue color (1). CAS can be prepared in a solution or asCAS agar plates.

Specific chemical components of the CAS media can all be found in the study performed by Schwyn and Neilands (1987). To perform CAS analysis a strong ligand (e.g. siderophore) is added to a highly colored iron dye complex. When the iron ligand complex is formed the release of the free dye is accompanied with a color change (7). Since CAS assay is high in sensitivity it isable to be used on agar plates. The Fe(III) gives the agar a rich blue color and concentration of siderophores excreted by iron starved organisms results in a color change to orange. After inoculating it takes only a short time before a color change will occur usually no more that 6 hours (7, 1).

HDTMA is used as a detergent as mentioned above and the concentration of HDTMA is crucial tosuccessful growth and testing of bacteria. If the concentration is too low the blue dye will precipitate and if it is too high the bacteria will die. This does present selectivity issues when culturing ecological samples especially. However, Alexander and Zuberer (1991) developed a modification to circumvent the problem of HDTMA toxicity. The amount of HDTMA used was reduced significantly and MES...