we are trying to evaluate antimicrobial activity of some synthetic compounds using well diffusion method, they are water insoluble, and they were dissolved in pure DMF, but after application of compounds solutions with few seconds the liquid appears to get out from the well to the surface of the agar (as if the volume increased,although when applied they didn’t reach edges of the well), and after 24 hour there is still some liquid that didn’t diffuse remaining in the well, and there is something like precipitate around the well from inside and outside. And when inhibition zone occurred it is the same or smaller than the zone of the solvent, I tried to filter them with 0.22 µl filter, compounds gave inhibition zone similar to that of DMF, and there was less precipitate in the well. (Standard antibiotics gave good reproducible results without problems)
Should I solve the precipitation problem first to take results or is it normal for synthetic compounds and I should take it as negative result.
And I’m afraid to do broth dilution method after filtration sterilization so compound activities decrease.
To evaluate the antimicrobial activity of synthetic compounds which are insoluble in water, i suggest you that disc diffusion and agar well methods are best for only screening of antimicrobial activity. If compounds are insoluble in water then you have to used Dimethyl Sulfo oxide (DMSO) about 20-50 % concentration according to solubilty of compounds. I you have some problem in agar well method according to above said then you can use disc diffusion method because in this method, compound loaded paper discs are air dried and water containt of dissolving solution will not creat the problem on the surface of the agar. Another solvent which you can be used that is Tween 80. DMSO and Tween 80 have same property to dissolve polar and nonpolar compounds. I hope that it will be helpfull for your experiment.
Similar to what Parikshit stated, you can dissolve a very high concentration of your water insoluble compound into a polar and water-miscible solvent and add it to the first wells of your broth dilution assay at a very low volume. For example, make 50 mM Hydrophobic Drug in DMSO and add only 2 uL of this solution into 100- uL (depending on your MIC assay) broth. Then serially dilute this solution with broth to give you a dynamic range of Hydrophobic Drug. You must be careful not to exceed 0.1% DMSO into the broth as this is the maximum carrier solvent usually used in pharmaceutical delivery. Any concentration higher than that will skew your results by facilitating the activity of the drug (this is because DMSO will weaken the outer membrane of prokaryotic and eukaryotic cells. Lastly, you add 100 uL of bacterial broth at a 0.5 McFarland cell density. So for this example, you highest concentration will be 500 nuM. Hope this helped.
To enhance the stability of the suspension, two dispersants, PVP and PEG400, you can use.Disk diffusion method is best for insoluble or synthetic compound. I also suggest you shake flask method.
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