Hi..I would like to detect siderophore production by rhizobacteria. I have rhizosphere soil samples to test and have used a CAS overlay method that hasn't worked since the original colour was red orange so a change to red orange cannot be seen. I am thinking of using an assay involving HDTMA but some Shweyn and Neilands say that gram positive bacteria are sensitive to this detergent. Please advice on the best method to use if testing from raw soil samples.
hexadecyltrimethylammonium bromide (HDTMA) as indicator. HDTMA has a moderate health and contact rating. Appropriate personal protective equipment should be used. The CAS/ HDTMA complexes tightly bind with ferric iron to produce a blue color. When a strong iron chelator such as a siderophore removes iron from the dye complex, the color changes from blue to orange. ts a gud option try t.
Siderophore production on the Chrome azurol S (CAS) agar medium. CAS agar plates were prepared and divided into two equal part and spot inoculated with test organism and incubated at 28± 2oC for 3-6 days. Development of yellow–orange halo zone around the spot due to remove iron from dye complex by siderophore. it was considered as positive for siderophore production and measure colony diameter.
I think that you had problem with the pH in the CAS medium. The pH value is the critical point when you are going to prepare the CAS medium. If I am correct, HDTMA is also in composition of CAS. I suggest you to prepare Petri dishes that contain CAS and a common solid medium, for instance KB.
Firstly you should pour the common solid medium. Once it is solid, you should cut and remove from the Petri dish a hal part of the medium. Then you will pour melted CAS medium in this empty section of the Petri dish.
You will streak the bacteria that you want to evaluate on the common solid medium. The siderophore will move in the solid and determin the change in the colour of CAS.
I hope it helps.
you can also use CAS diffusion assay as method described in "CAS agar diffusion assay for the measurement of siderophores in biological fluids".
Detection of siderophores by inoculated the bacteria on the Chrome azurol S (CAS) agar medium. The change in color of the CAS reagent was due to the fact that siderophores, present in the supernatant, chelate the iron from the CAS reagent, which results in color change from blue to orange-red.
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