I've been using agar plug method for antimicrobial primary screening against E.coli. There is a fungus that shows activity against the E.coli. This means that the fungus has the potential to be further studied by the fermentation method. I am still looking for the appropriate extraction method to obtain a secondary metabolite of the fungus.
I recommend adding Ethyl acetate (EtOAc) and homogenize with a blender (you had mentioned that the fungi was grown on agar) to break up the agar and micellium. I usually add 1:1 EtOAc to broth, but in the case of a solid-state extraction 2:1 or higher might be better. After homogenization, let the EtOAc and agar chunks sit, undisturbed, to let the mixture settle. If an emulsion was created during the blending step then sonicate the sample briefly to remove the trapped air. This should be performed in an Erlenmeyer flask as the flask shape is conducive for effective decanting. After decanting, add fresh EtOAc, swirl, and let sit again. In the case of extracting liquid culture add EtOAc (1:1), homogenize, briefly sonicate to remove emulsion, and let it sit. Repeat this for a total of 3 extraction.
I have often found zones of inhibition on agar plates for both bacteria and fungi and have had some success and translating the solid-state growth to liquid culture, which often results in a higher yield of the secondary metabolites.
The use of volatile organics allows for the evaporation of solvent without high heat (which can destroy certain compounds). Methanol would probably work for the solid-state agar growths, but is not acceptable for liquid extractions. We use rotovapors, speed vacs, and V10 for evaporation and recover the EtOAc for later use. Evaporating the organic solvent results in the crude extract, which can then be resuspended in MeOH for LCMS and DMSO at ~10 mg/ml for bioactivity assay.
Thanks Bakkiyaraj Dhamodharan, Shaban Ahmed Ali Abdel-Raheem and Carter Mitchell for giving me information and ideas. I will try the method given and hopefully get good results. Thanks again.
I agree with Bakkiyaraj Dhamodharan, first find out if its secreated or not.
Then take a look at this paper by Bucar, Wube and Schmid 2013
http://pubs.rsc.org/en/content/articlepdf/2013/np/c3np20106f
Yoram
i got contamination while growing the fungus in broth media, plse suggest any remedy for the same
Vandana Vandana If the broth is properly sterilized I don't think there should be any contamination and please check the primary inoculum may be there is some contam present there, try to use fresh primary inoculum!
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