- Begin by preparing the compound to be tested for toxicity, and distribute it to the wells of an assay plate. Transfer worms of the appropriate developmental stage to each well, and immediately start a timer. Note any worms that are damaged and do not include them in your survival calculations.
After a predetermined time, count the number of worms that are still alive in each well. The bright light of a dissection microscope or gentle tapping on the plate should cause any live worms to move. Repeat this observation after each time interval, until most of the animals are dead. In the following protocol, we will observe L4 larvae survival time during exposure to paraquat, a compound that induces oxidative stress.
- In this demonstration, oxidative stress will be induced using paraquat. Prepare a fresh 100 millimolar paraquat solution as described in the accompanying protocol text. Paraquat is a hazardous and highly toxic compound, and must be handled according to appropriate guidelines.
Pipette 40 microliters of the paraquat solution to every well of a 96-well plate. Use at least 12 wells as replicates of every condition tested. Using a platinum wire worm pick, transfer five to eight L4 larvae animals to every well.
Indicate the start time and the end time for every condition tested. Place the 96-well plate at 20 degrees Celsius. An hour after the start time, score dead and alive animals using a dissecting microscope. Gently shake the plate before starting to count.
Worms that do not move even after spotting a high intensity light are scored as dead. Look at worm shape, tails, and head movement at high magnification to distinguish dead worms from live worms. Score survival every hour until most worms are dead, and repeat this assay at least three independent times.
To determine the survival percentage for each time point, first calculate the total number of animals per condition by adding the total number of animals transferred to each well. Ignore the animals that were damaged or killed upon transfer. Next, for every time point, calculate the total number of dead animals per condition. Calculate the percentage of death and the percentage of survival.