I am running an experiment where I try to detect the siderophores in a soil sample. I want to use a CAS assay to achieve this, but I am running in some problems when making the CAS reagent. I opted to use the protecol of Schwyn, B., & Neilands, J. B. because a lot of studies reffered to this article in there methods. Only a couple of things are changed like the MM9 salts are replaced and in stead of piperazine I think I can use pipes instead.

The choice of pipes instead of piperazine is made because the pipes is already available for me to use and this is used as a buffer for the CAS agar plates in the same article of shcwyn, B & Neilands, J. B.

but when I add my 5-sulfosalicylic to the mixture the color changes from blue to orange. and I don't really know why this is happening.

Thank you in advance and I am curious if others come across this problem.