I think my previous post merits more explanation. In this research project, as you may have read from my very first post, we intend to complete two objectives: genetic screening for uncultivable antimicrobial production and whole-cell screening for cultivable antimicrobial production from soil samples. Both start by randomly taking soils samples out of areas such as a rainforest, salt lands, and parking lots (incredible!) in Puerto Rico. 

Genetic screening is rather simple in comparison to troublesome incubators and take-too-long-to-grow microbes. In this phase, soil samples are subjected to commercially available soil DNA isolation kits. This DNA extract is then put into a thermocycler  where it undergoes polymerase chain reaction using selective primers that amplify genes encoding for enzymes strongly associated to antibiotic production called PKS and NRPS. In the previous post, I explained that even tough amplification occurred, the primers for PKS were not being used at conditions that optimized their specificity. Hence, we obtained positive results, all samples had PKS amplifications, but further optimization will give us a better idea of the concentration of amplicons and variability within them. 

In another level, we want to search for novel antimicrobial activity from cultivable soil microorganisms. After obtaining soil samples, we suspend them in water, dilute them, plate them on solid media, and incubate them under different conditions. Once the microbial populations start to flourish, we isolate those microorganisms that present antimicrobial activity – ergo, inhibition zones – and test them against our target bacteria. From the results that I posted the other day you can observe that we focused this aspect of our experiment on two actinomycetes isolated by the grad student Vanessa Cardona. The first one, AR, did not present any antagonistic activity over Staphylococcus aureus or Pseudomonas aeruginosa. However, in a very intense week at the lab, every single culture plate belonging to my experiment with AR got contaminated. Guess who? Well Fungi of course! Not only does every microbiology laboratory has to fight against fungal contamination, but  when you live in a sunny, beautiful and immensely humid tropical island such as Puerto Rico, you are forced to take extreme caution with the purity and sterility of the incubators, tools, etc. Well, during Mr. Fungi’s visit to my AR cultures, we were surprised to see that AR was able to inhibit microbial growth after all, just that it was not bacteria. It was all the fungus in the plate. We decided to isolate both funguses present in the plate and run further tests on AR and the other actinomycete AV against them. We dubbed the funguses H1 and H2. As we can see from the image in the previous post, AR was able to inhibit growth in both funguses, as opposed to AV and our control of course. That means that AR produces an antifungal substance. Now we look forward in doing further experiments to characterize H1 and H2 and to see if we can isolate the antifungal. 

Now it was a whole different story with AV. On the first test of antimicrobial activity of AV against our microbial targets, we observed something that looked like an inhibition zone in the S. aureus culture. Upon repetition of this assay we confirmed that indeed there was a 0.5 – 0.7 cm inhibition zone between S. aureus and the yellow pigment secreted by the actinomycete. Nothing against P. aeruginosa though. Therefore, it is safe to say that AV is secreting an antimicrobial agent against S. aureus, yet we would like to test the antimicrobial potential of AV against other gram positive-bacteria to verify its specificity.