What number of filter paper used? How much use? Are they allowed to dry or wet placed on agar?
I tried and I do not succeed, I do not get halo.
hi, Marielsa, first let me know the thickness of the filter paper you are using. On an average you can place 5 discs (10mm) in 100mm dish.
Usually if you use methanol or ethanol as solvent then it is possible to impregnate the disc with 30ul (0.9mm thick) at a time and if you need higher concentration (concentration gradient assay for particular extract) then you should add 20ul at the first time then air dry the disc ( make sure it got detached from the surface easily) and add another 20ul and dry again. This is how you can make a disc with higher concentration. Place the disc on culture plate with a sterile needle.
Make sure that your extract is clear enough and no debris or solid particle is in the solution. That will give rise some error with the concentration and even the diffusion will be uneven.
Hope this will do for you. Let me know if you need further clarification.
Best
Taufiq
Hello Marielsa, I usually allow my extracts to absorb into the discs for 2-3 hours at 37'C. If there is no halo, it could be that your natural substance has no inhibitory effects on your bacteria.
Thank Khan and Nurul your answers have been very helpful. The substance I'm using is ethanol extract of propolis using the technique of minimum inhibitory concentration in wines has been very effective but have been unable to obtain disk diffusion halos because I think it was not using the correct paper.
How to make sure that the paper discs are fully soaked by the solution?
Whatman #3 discs with absorption of ethanolic solution of materials works pretty good, key point is Whatman #1 can be a too thin material for certain materials, checkout the attached reference.(http://www.allresearchjournal.com/archives/2015/vol1issue11/PartI/1-10-152.pdf)
I will grateful if I can an article in which blank discs were impregnated with antiseptics (or natural products) using the advise you have put on this forum
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