- To measure the average lifespan of a nematode population, start with developmentally synchronized L1 larvae in M9 buffer. Determine the density of the larvae, then add 50 to each plate. Prepare at least three replicate plates per condition, as well as backup plates.
Allow these worms to develop normally until they reach the L4 stage. Now you may add FUdR to the plates, which will prevent the worms from producing progeny. Alternatively, you will need to move the worms to new plates each day after counting, to ensure you are only measuring the lifespan of a single generation.
Next, remove any males. This will remove the variables of sex and mating from the experiment. The next day, count the number of living worms on the plate by looking for spontaneous movement or response to gentle touch. Remove any dead worms and record the numbers. Count surviving worms daily until none remain. In the example protocol, we will see a demonstration of a lifespan assay.
- Load the prepared bacteria-seeded plates with 50 L1 animals each. Set up the plates in a 20 degrees Celsius incubator until the embryos develop to the L4 stage. Normal development from L1 to L4 takes about 40 hours.
Then, to prevent progeny production, add 160 microliters of 50x FUdR to each plate. Then remove the males, as lifespan is typically measured only with hermaphrodites. Once completed, repack the plates into the 20 degrees Celsius incubator.
Until all the worms are dead, score their viability on a daily basis. To do so, gently touch each non-moving nematode on the head with a platinum wire or eyelash. Non-reactive animals are dead and must be removed from the plate. Take note of any anomalies during this routine.