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 molecularweight 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 and other 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.
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). The siderophores 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 of iron, 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 able to
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 as CAS 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 is able 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 to successful 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 buffer was used to
substitute as a detergent. This has been a much more successful method for growth and
testing for ecological microbial samples.
Liquid CAS solution: The change in color of the blue dye- chrome azurol sulphonate
(CAS) assay solution to orange indicating the presence of siderophore (Iron chelating
compounds) production by a marine bacterium. (National Institute of Oceanography)
CAS agar plate: Siderophore production comparison between the SO1377 wild-type
strain DSP-10 and mutant WG3 on a CAS siderophore testing agar. The chelator-iron
(III) complex tints the agar with a rich blue background. The orange halo surrounding the
colony indicates the excretion of siderophore and its dimension approximates the amount
of siderphore excreted. (5)
For the liquid assay solution one only needs to observe the color change that occurs or
more rigorous turbidity measurements can be taken. The diameter of discoloration on the
CAS media is directly positively correlated to ability to mobilize iron. Since the diameter
of discoloration area and efficacy of iron mobilization are correlated, CAS tests are
inexpensive and simple methods for screening bacterial isolates for the ability to mobilize
iron. Using this method only visual observation of bacterial growth and measurement of
the diameter of discolored area are needed for analysis.
Literature cited
1. Alexander, D.B. and Zuberer, D.A. 1991. Use of chrome azurol S reagents to
evaluate siderophore production by rhizosphere bacteria. Biology and Fertility of
Soils 12: 39-45.
2. Calvaruso, C, Turpault, M.P., Leclerc, E and Frey-Klett, P. 2007. Impact of
Ectomycorrhizosphere on the functional diversity of soil bacterial and fungal
communities from a forest stand in relation to nutrient mobilization processes.
Microbial Ecology 54: 567-577.
3. Duval, B.D. and Hungate, B.A. 2008. The long road to nitrogen fixation. Nature
Geoscience 1: 213-214.
4. Frey-Klett, P., Chavatte, M, Clausse, M.L., Courrier, S, Le Roux, C, Raaijmakers, J,
Martinotti, M.G., Pierrat, J.C. and Garbaye, J. 2005. Ectomycorrhizal symbiosis
affects functional diversity of rhizosphere fluorescent pseudomonads. New
phytologist 165: 317-328.
5. Gao, W, Liu, Y, Giometti, C.S., Tollaksen, S.L., Khare, T, Wu, L, Klingeman, D.M.,
Fields, M.W. and Zhou, J. 2006. Knock-out of S01377 gene, which encodes the
member of a conserved hypothetical bacterial protein family COG2268, results in
alteration of iron metabolism, increased spontaneous mutation and hydrogen
peroxide sensitivity in Shewanella oneidensis MR-1. BMC Genomics 7:76.
6. Neilands, J.B. 1995. Siderophores: Structure and function of microbial iron
transport compounds. The Journal of Biological Chemistry 45: 26723-26726.
7. Schwyn, B. and Neilands, J.B. 1987. Universal Chemical Assay for the
Detection and Determination of Siderophores. Analytical Biochemistry 160:
47-56.