Hi,
I am trying to detect siderophore production in Mycobacterium using CAS (Chrome Azural) agar method.
Protocol for the preparation of blue dye is shown below.
Blue Dye:
Solution 1:
Dissolve 0.12 g of CAS (50% dye; Sigma) in 50 ml of ddH2O.
Solution 2:
Dissolve 0.0027 g of FeCl3-6 H2O in 10 ml of 10 mM HCl.
Solution 3:
Dissolve 0.073 g of HDTMA in 40 ml of ddH2O.
Mix Solution 1 with 9 ml of Solution 2. Then mix with Solution 3. Solution should now be a blue color. Autoclave and store in a plastic container/bottle.
Add 20 ml of the above solution to 200 ml of the 7H10 agar before making plate.
The problem is whenever I make the blue dye, the dye gets precipitated.
I even tried adding all components into 7H10 agar and then autoclaving (as given in one of the articles). But it didn't work out.
Please help me if anyone has experience in preparing CAS agar plate.
Thanks a ton
Good luck
Bindu
Dear Bindu,
It seems that you work after this protocol (see the foollowing link)
http://jmbe.asm.org/index.php/jmbe/article/view/249/html_106
On other part, have you tested the purity of the hygroscopic FeCl3, 6 H2O and chromazurol S. Have you cleaned your glasswar with 6M HCL (see link)
If you dissolve 10 g FeCl3. 6H2O in 100 ml dd water, the solution must be clear.
Try again after this link.
JRG
Thank you Jean Rene Grezes for the kind reply.
I will have a look at the purity of FeCl3 and chromazural.
Yes, I did clean the glasswares with 6M HCl.
Thank you
Did you take a pH of the solution? The pH is critical for the assay to work. This is a good reference to start...
Yes Kayleigh Jones, I had checked the pH.
Thank you for the link.
Have a good day
Bindu
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