February 5, 2014 in Posts
Pictured below is the fungal endophyte we isolated from a Ginko tree fermenting in 300mL of Sabouraud dextrose broth. We performed an ethyl acetate extraction on the liquid partition of the broth, after filtering out the mycelial mass through cheesecloth. We dried the combined ethyl acetate partitions over sodium sulfate and concentrated on a rotovap. Due to an analytical balance malfunction, we were not able to get an accurate mass of recovered crude extract, but by eye it appeared to be a few mg.
The extract was brought up in 200uL DMSO and a small analytical sample was taken to run on an LCMS. (Chromatograms to be posted soon)
We wanted to do a quick disc-diffusion test to see if there were any compounds we had extracted in the fermentation broth that had antibacterial properties, so we plated some E. Coli and Staph. Aureus and put 2,10, and 20uL respectively on filter paper discs and let incubate at 37C overnight. Much to our excitement, we saw a concentration dependent zone of inhibition around the 10uL and 20uL discs for E. Coli.
This was a really quick and really crude disc diffusion test (we know we were missing the concentration of our original extract) but we still wanted to be able to prove to ourselves that we could isolate an endophyte, subculture it, liquid culture it, and extract the broth and test for bio-active compounds.
Notice on the 20uL disc that the DMSO appeared to “drip down” when the plate was flipped, probably because not enough time was given for it to soak into the agar, leading to a skewed portion of inhibited growth. Nevertheless, it is clear that as the concentration of the doped discs increases, so to does the ring of inhibition. Now comes the hard part…figuring out what compound(s) are responsible for the activity in a mixture of many!
Another interesting observation is that around the Staph A. discs, there appears to be a ring of where the DMSO diffused, with perhaps a little less growth of the organism, but not clear inhibition.