I am preparing CAS agar medium for testing the siderophore production of antagonistic rhizobacteria. I am following the procedure of Schwyn and Neilands 1987 which is elaborated in detail by Louden et al. 2011. The recipe says that after preparing the casamino acid solution extract trace iron with 3% 8-hydroxyquinoline in chloroform. Can anyone tell me how much volume of 3% 8-hydroxyquinoline in chloroform should be prepared and after I have prepared the solution how can I extract iron from casamino acid solution. I need the methodology.
Hi Muhammad
I reckon it is best to do it on the stock solutions so that you can keep the volumes limited. If you ggole creatively for "3% 8-hydroxyquinoline in chloroform and CAS" you get some nice links eg to a book that I cannot directly copy from: Iron Chelation in Plants and Soil Microorganisms, edited by Larry Barton.
It's a bit of old school chemistry, ideally you use a separating funnel (http://en.wikipedia.org/wiki/Separatory_funnel) but patience, a measuring cilinder and big enough pipettes should also work. Then:
Dissolve 3 g of Casamino acid in 27 ml of ddH2O. (well as much you need)
Extract with equal volume 3% 8-hydroxyquinoline in chloroform to remove any trace iron: stand at 4 degrees o/n and let phases separate. recover only the water phase (lighter than CHCl3). Wash with Chloroform till no coloured 8-hydroxyquinoline-iron complex is extracted by the chloroform.
You may want to leave it in the fumehood for a wee while to let the last traces of chorofom evaporate....
Filter sterilize.
Please let us know how it worked for you.
What are you going to use for?
Hi Henk-jan
Thanks. I did it the way you suggested me and it worked finely. I used the equal volume of 3% 8-hydroxyquinoline in chloroform for the extraction and kept it overnight. Next day I separated the casamino acid solution followed with filter sterilization.
I am going to use it for preparing CAS agar media for testing the siderophore production by rhizobacteria.
Hi Muhammad,
Do you mean you used 27 ml of 3% 8-hydroxyquinoline in chloroform? Please i need more details about this. I'm also working on the same research.
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