Nematode Synchronization: A Method to Obtain Populations of Worms in Identical Stages of Development
Transcript
-Begin by placing self-fertilizing worms in their last larval stage on plates seeded with a bacterial food source. Allow the larvae to develop into mature adults until there's an abundance of eggs within worms and laid on the plates. Wash the plates with buffer to collect the eggs and worms.
Next, replace the buffer with bleach solution which dissolves adult worms. Protected by the eggshell, embryos will survive this treatment, though they will be at different stages of development. Then replace the bleach with buffer and allow the embryos to develop. As larvae hatch from their eggshells in the absence of food, development will pause at the first larval stage.
Transfer the larvae to plates with food to resume development. This will stimulate synchronous progression through the remaining larval stages.
When worms reach the last larval stage, move them to plates with food and FUdR, a DNA synthesis inhibitor that prevents the production of offspring. Continued development in the presence of FUdR produces a population of sterile adult worms. In this experiment, we will synchronize a population of the nematode C. elegans.
-To begin, transfer L4 larvae of desired genetic backgrounds onto a freshly seeded lawn of Escherichia coli on nematode growth media or NGM plates. Use at least two 100 millimeter or three 60 millimeter plates for each strain.
Incubate the worms at the appropriate growth temperature for three to four days or until plates are concentrated with a large number of eggs and gravid worms. Wash the eggs and worms off the plates using approximately six milliliters of M9 buffer per 100 millimeter plate of nematodes. Using glass Pasteur pipettes, transfer the eggs and worms into individual 15 milliliter centrifuge tubes for each strain. Spin these tubes down for three minutes at 6,180 g.
Following centrifugation, aspirate out the M9 buffer retaining just the animal and egg pellet. Add three to four milliliters of bleach solution to each tube and intermittently vortex for six minutes.
Then add M9 buffer to fill each tube and spin at 6,180 g for one minute. Aspirate the supernatant and repeat this wash with M9 three times.
Following the wash, move the egg pellet to a fresh 15 milliliter tube containing approximately nine milliliters of M9 buffer. Synchronize the freshly hatched worms by nutating at 20 degrees Celsius for 16 to 48 hours.
After spinning these tubes down at 6,180 g for 1.5 minutes, put the synchronized L1 animals down on a freshly seeded lawn of OP50 on individual NGM plates. Keep the plates at 20 degrees Celsius until the animals reach the L4 larval stage after approximately 42 hours. At this time, use a platinum pick to move the L4 larvae to OP50 seeded NGM plates containing 0.5 milligrams per milliliter of FUdR to prevent them from producing progeny.
