Problem with well diffusion method?

[deleted-575427]

I was testing the effects of honey against e coli k12 using the well diffusion method but there was no range of inhibition at all! The bacteria actually grew over the samples, what did i do wrong! I started off by spreading the culture across the agar plate using a cotton bud. I used 6mm filter paper from a hole punch to test the different honey samples. i used a 50% solution of 6 different honeys, as i've read from over 15 pubmed sources that almost all honeys are really antibacterial at 50% dilution. I know there's something wrong as i used manuka honey 20+ which can kill superbugs like MRSA at 10% dilution!


Once i mixed the honey and the deionised water to make the 50% solution i used a pipette to transfer the solution to the 6mm hole punch paper, i then used tweezers to put the 6mm paper with the samples onto inoculated agar plate.

[deleted-547689]

I'm not too familiar with your well technique, but thy not try disc diffusion? Spread your bacteria over an agar plate, then place small filter paper discs (I use a standard hole punch when I do this) that have been soaked in different concentrations of honey. This wy you you can cover a wide range of honey concentrations on a single or even two plates, and you can easily measure zones of inhibition if there are any. And don't forget that negative results are results. You can say in your conclusions that further work need to be done in this area to be able to be confident in other researchers conclusions. BTW, what bacteria are you working with and where did you get them?