To begin, transfer five milliliters of an acidified culture supernatant of pseudomonas aeruginosa into a 50-milliliter polypropylene tube. Add five milliliters of chloroform methanol mixture into this tube. Invert the tube three times for 30 seconds to agitate and mix the solution.
Leave the tube without inversion for two minutes between each agitation. Centrifuge the tube for 10 minutes at 3000 G at four degrees Celsius, then transfer the organic phase into a clean tube. Leave the tube in an extraction hood until it is dried.
Next, use a 10-microliter pipette to apply five microliters of the samples onto a TLC plate. To prepare the liquid phase, mix 26 milliliters of chloroform, six milliliters of methanol, and 0.8 milliliters of 20%acetic acid. Place the solvent mix in a closed TLC chamber for at least 10 minutes to saturate it.
Transfer the TLC plate into the chamber, avoiding contact with the sample application points. Leave the TLC plate in the closed chamber until the solvent reaches one centimeter before the edge of the plate. At this point, remove the plate and let it dry.
To detect the presence of rhamnolipids, spray alpha naphthol solution onto the plate in the extraction hood, then place a sprayed plate in an oven at 85-degrees Celsius for several minutes until the pinkish mark of the rhamnolipid lipid is visible. All three pseudomonas aeruginosa strains produce different amounts of mono and di-rhamnolipids.
