Antibiotic Properties of Isolated Endophytic Fungi Extracts from Davidson County Community College Campus Flora
These collective data are from projects conducted by the first group of undergraduate research fellows and their advisor at Davidson County Community College (DCCC) in Thomasville, North Carolina. All projects were conducted within a framework of endophytic fungi isolation and testing of organic extracts for antibiotic properties. Individual plant specimens representing 10 species were collected on the college campus. Various tissues were surface sterilized, and then plated on either potato dextrose or yeast-malt extract agar containing kanamycin (50 µg/mL) to inhibit bacterial growth followed by storage at room temperature. Plates were checked regularly for fungal growth and further subsampled and re-plated in an attempt to isolate endophytic fungi. Once a fungus appeared to be isolated, sterilized scalpels were used to remove small strips of agar from a plate and were placed in sterile 15mL conical centrifuge tubes containing sterile water or 250mL flasks containing various type of broth. The 15mL tubes are permanent water stock cultures and are currently stored on the DCCC campus. The 250 mL flasks were used for initial fermentation cultures that were allowed to grow under ambient room conditions with occasional mixing before transfer into 1,000 mL flasks containing more broth. Those flasks were allowed to grow for several weeks with occasional mixing under ambient room conditions before being subjected to organic extraction. Organic extraction consisted of separation of organic and aqueous phases with ethyl acetate. The resulting organic phase was placed in a modified distillation apparatus as there was no access to a rotary evaporator. The ethyl acetate was evaporated off and recollected in a receiving flask and the organic residue remaining from the extraction was collected and stored in methanol in a laboratory freezer until being tested for antibacterial properties. Extracts were tested against four species of bacteria (Escherichia coli, Staphylococcus epidermidis, Bacillus subtilis, and Klebsiella (Enterobacter) aerogenes) using the agar diffusion method in 1:10 potato dextrose: nutrient agar. Plates were inoculated by submerging sterile cotton swabs in fresh broth cultures of a species and subsequent swabbing of agar’s surface. Wells were constructed using sterilized 10mm diameter cuvettes and then filled with 25 µL of extract, or of control (+ kanamycin, - methanol) solutions. Plates were incubated at species appropriate temperatures for 24 hours followed by removal and the measurement (mm) of the zones of inhibition around each well. All testing was done in triplicate. Due to limited time and space, only five organic extractions were completed. Of the five, subsequent statistical analyses indicated four extracts had inhibitory effects against two species, B. subtilis and S. epidermidis while no inhibitory effects against E. coli or K. aerogenes were observed.
Steps to reproduce
Most methods followed or modified can be found in Bascom-Slack CA, Arnold AE, Strobel SA. 2012. Supplementary Materials for Student-Directed Discovery of the Plant Microbiome and Its Products. Science. 338 (October): Supplementary Materials. doi:10.1126/science1215227.