The O-CAS (overalay CAS) assay tests for the production of siderophores, which are small organic molecules that some bacteria secrete to chelate (bind to) metals. Either to take them in to use (such as in low iron situations) or to remove them (such as removing toxic heavy metals, which is good for them and plants!)
You plate bacteria on media that has been deferrated (had iron removed, in our case by pre-treating the media with Chelex-100 resin, which binds to cations like iron). This iron starvation promotes the formation of siderophores by siderophore-producing strains. You let them grow on the plates for 3 days, then overlay the plates with a detection reagent that will change color in the presence of siderophores. The reason is that the overlay contains chemicals - CAS (Chrome azurol S) and HDTMA (hexadecyltrimetyl ammonium bromide) that form a complex with iron (provided in the form of iron chloride). The CAS/HDTMA/iron complex looks blue. When a siderophore steals the iron, the complex changes color. The overlay also contains agarose to make it all gelly and PIPES to keep the pH where you want it. Depending on the type of siderophore produced, the dye can changes color from blue to orange (hydroxymates), yellow (carboxylates), or purple (catechols).
There are a lot of CAS-based assays and their pros & cons are well-described in this good (and open access, so free for all to read!) review article on various siderophore assays and their pluses and minuses: Gomes, A. F. R., Sousa, E., & Resende, D. I. S. P. (2024). A Practical Toolkit for the Detection, Isolation, Quantification, and Characterization of Siderophores and Metallophores in Microorganisms. ACS omega, 9(25), 26863-26877. doi.org/10.1021/acsomega.4c03042
Here’s the paper that set off the craze: Schwyn, B., & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical biochemistry, 160(1), 47-56. doi.org/10.1016/0003-2697(87)...
And this is the O-CAS one we based off of: Pérez-Miranda, S., Cabirol, N., George-Téllez, R., Zamudio-Rivera, L. S., & Fernández, F. J. (2007). O-CAS, a fast and universal method for siderophore detection. Journal of microbiological methods, 70(1), 127-131. doi.org/10.1016/j.mimet.2007....
These couple were good for planning things out prep-wise:
Louden, B. C., Haarmann, D., & Lynne, A. M. (2011). Use of Blue Agar CAS Assay for Siderophore Detection. Journal of microbiology & biology education, 12(1), 51-53. doi.org/10.1128/jmbe.v12i1.249
This awesome undergrad dissertation
Elgazzar R. (2017) Screening of siderophore producers from Soil dc.etsu.edu/cgi/viewcontent.c...
Huge props and thanks to Rory Abraham for these awesome results!
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