To begin pipette 1.2 milliliters of a 24-hour culture of Pseudomonas aeruginosa into a 1.5 milliliter centrifuge tube. Centrifuge the tube at 3000 G for four degrees Celsius for three minutes. Collect the supernatant in a clean tube.
Pipette 333 microliters of the supernatant into a clean tube. Then add one milliliter of ether into the tube. Mix the solution in a vortex for 30 seconds.
After all the tubes have been vortexed, centrifuge them at 3000 G at four degrees Celsius for two minutes. Then collect the organic phase and transfer it into a clean tube tube. Place the solution in an extraction hood until it is dried.
Next place a clean flask covered with foil paper in ice for five minutes to prepare a 60%sulfuric acid solution. Use this sulfuric acid solution to prepare the orcinol reagent. Add 100 microliters of each Rhamnolipid sample to a glass tube and add 900 microliters of orcinol to the samples.
After mixing the solutions together, incubate the sample in a water bath at 80 degrees Celsius for 30 minutes. When the tube cools to room temperature, transfer a sample to a quart cell and place it in the spectrophotometer to read the absorbance at 421 nanometers. The strain PA14 produces the highest amount of rhamnose equivalents while strain PA7 produces the lowest amount.
