To begin, separate 200 L4 stage, fog-2 Caenorhabditis elegans virgin females one day prior to pheromone extraction. Distribute the females onto three separate six centimeter OP50 NGM plates. On the extraction day, transfer 100 virgin one-day-old fog-2 females into a micro centrifuge tube containing one milliliter of M9 buffer.
After the last wash, incubate females in 100 microliters of M9 buffer for six hours at 20 degrees Celsius to allow pheromone production. Similarly, isolate 15 to 20 L4 stage Caenorhabditis remanei females onto three OP50 NGM plates. After washing, incubate five virgin females in 100 microliters of M9 buffer for six hours at 25 degrees Celsius.
Centrifuge the worms at 15, 000 G for 30 to 60 seconds. Carefully transfer the supernatant containing pheromones to a clean tube. Discard the tube containing the worm pellet.
Store the collected volatile sex pheromone extract at minus 80 degrees Celsius.
