Bacillus subtilis vs. homegrown penicillin

This is an update post on the quest to isolate a Penicillium strain from citrus fruit, in the hopes of at some point along the journey finding some penicillin.  A video was recently uploaded to our youtube channel that briefly explained the experimental conditions.  Basically, three different liquid cultures (not pure or sterile- they were grown in coffee cups) were grown for a few weeks in the hope that some would produce pencillin, and we could test for its presence by a simple disc diffusion assay.  Much simpler said than done, as is often the case in science!

The video covers how the experiment was setup, but does not cover the results, which didn’t boast well for our novel coffee cup penicillin cultivation technology.



As you can see, none of the experimental homemade disc diffusion assays showed any signs of inhibition at 5, 10, 15, 20 uL concentrations against Bacillus subtilus – the only gram positive bacterial strain we could get our hands on.   The “experimental” discs had to be hand cut from new filter paper, so weren’t perfect circles, but rather an assortment of rhomboids created by a pair of scissors.

The one semi-perplexing thing was that even the control plate showed no rings of inhibition with the 10 unit penicillin discs.  The reasons for that could be; bad penicillin, resistant bacterial strain, the fact that it took me 3 days to check the plates after the initial plating.  If I had to make an educated guess, I’d go with #3.  Plates are usually checked daily, and the fact that it took 3 days before the plates could be checked probably meant whatever penicillin had been present had been “used up” in a sense, allowing the bacteria to colonize the area after the initial 10units were gone.

After doing some googling, I came across this pretty nifty image, which showed just how innefective pencillin is against B. subtilis. (Not my photo! Thanks docpilgrim!) Notice how the P10 disc shows the smallest ring of inhibition, and that’s the same P10 disc we were using in our control.

So back to the drawing board!  For the time being our goal is to use our ITS primers to try to ID the strains we did isolate.  On the upside, this has been a pretty pleasant mold cultivation project, as everything smells like lemons.

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