I heard that it should be around 90-100 micrograms, but at this concentration my crude extracts are not showing activity. Should I increase the concentration? I read 5mg in one paper. Kindly help.
No, In my opnion it all depends on the concentration of the active component in your crude extract.
Therefore you can not say that a particular concentration of the crude extract is suitable for the antimicrobial assay unless and untill you isolate, puirify and test the pure active compound.
So you may increase your concentration.
I agree with Vijay. There are no hard and fast rules. for advice on this you will have to tell us what you are extracting and by what protocol. However there is one question you may wish to address.
How was the extract made? For plant extracts testing at NCI we used at least three extraction methods (eg hot water; cold 50% aqueous ethanol; hot ethanol; etc) at least that got round the problem of thermal instability.
If the extract in question is one related to traditional medicine then the clue may be in the original literature?
If you want more advice you will have to specify the origin of the material (plant parts/marine organism/ animal tissue) and your antifungal/antibacterial testing protocols.
There is no perfect answer for this question but generally in my experience concentrations above 50mg/ml are considered not active biologically.
Thankyou so much for the input .
Dr. Robert, I am working on endophytic fungi isolated from plant and extracting the metabolites in ethyl acetate and I am doing agar well diffusion method for antibacterial assay.
OK Hira - I take it from your reply that you are doing a cold EtOAc extract on the cultured mycelium of your fungus. Also that you are taking care to reduce this to dryness at a reasonable temperature.... OK so far? The next step is to decide on the resuspension method (and remember the controls) - If it were my experiment I'd run a series of dilutions starting at an effective ca 5mg/ml in buffered physiological saline (disolve extract in EtOH at very high concentration and then squirt into the aqueous solution to disperse...OK), mix on an omnimix (or similar), dilute and test.
You could also consider using a disc difusion method. This is crude but very effective for an initial assay. You dip a Whatman filter disc (typically 5mm diameter) in the crude extract (with serial dilutions if necessary) - dry the disc thoroughly at room temperature and then place it on an agar plate seeded as a bacterial lawn. What you should see is an inhibition zone round the disc. If you get a result you can move to more sofisticated methods.
In fact it depend of the stape of your experience. If it's a crude extract, you could use until 1g/ml of concentration.
I always do an approaching test. Use a range of concentrations before designing the final experiment is recommended. I would assay the following concentrations 0, 0.5, 1, 5, 10, 50, 100, 500 (example).
Ms. Hira
I am attaching some reviews on the subject of bio-activity of essential oils (not crude extracts). You may look for some useful cross references.
Good luck.
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