To begin, using a pipette, spot five microliters of each extract two centimeters apart on the dots marked on the bottom line of the thin-layer chromatography, or TLC, plate. Let the spots dry on the TLC plate and repeat until about five milligrams of each extract are loaded onto the plate. Prepare a 1:2 solution of dichloromethane and methanol in a TLC developing tank.
Put the TLC plate into the developing tank and develop it, until the solvent reaches the line marked two centimeters from the top of the plate. Once developed, remove the TLC plate from the tank. Immediately, spot the positive controls on the dots above the solvent line.
Place the plate in an ethanol-sterilized TLC plate box before all solvent evaporates. Scrape the mycelial mat of the five pathogen plates using a sterile metal spatula. Then, transfer it into a 50-milliliter centrifuge tube.
After adding 25 milliliters of Potato Dextrose Broth amended with agar, add sterile glass beads to the tube, and vortex for five minutes to break up the mycelial mat. After assembling the chromatography sprayer in the hood, attach the tubing. Then, attach the flow meter to the setup.
Transfer the mycelial suspension to the chromatography sprayer using a sterile syringe with a needle tip to ensure large pieces of mycelia do not clog the sprayer. Place the previously developed TLC plates on sterile paper towels in the laminar flow hood to reduce hand contact with the plate, while applying the media suspension. Connect the pathogen sample to the assembled sprayer and apply three coats of the suspension to the TLC plate, allowing the plate to dry completely between applications.
Next, prepare the incubation setup by adding 50 milliliters of sterile water into a sterilized plastic plate box. Place four Petri plates in it for the TLC plate to sit on. Also, place sterilized folded cellulose sheets or filter paper on each side of the box to hold moisture.
Place the completed assay plate onto four empty Petri dishes in the box. Incubate the assay for three days to one week or until an even layer of mycelia grows across the plate, except for around the positive controls and zone of inhibition. Then, using a metal spoon, scrape the silica off the inhibition zones and place it into microcentrifuge tubes.
Add 500 microliters of methanol to each tube and vortex to extract the metabolites from the silica. Centrifuge the tubes to pellet the silica and transfer the supernatant to liquid chromatography vials for analysis. Imaging under ultraviolet light showed the separation of metabolites on the TLC plate.
After the incubation, the pathogen appeared to grow evenly across the entire plate, except over the positive controls and the inhibition zones.
