C. elegans Survival Assay: A Short-Term Toxicity Test

This protocol is an excerpt from Possik and Pause, Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates, J. Vis. Exp. (2015).

1.  Performing the Oxidative Stress Resistance Assay

1. Pipette 40 µl of the 100 mM PQ solution (prepared fresh) to every well of a 96 well plate. Use at least 12 wells as replicates of every condition (treatment, mutant, etc.). Using a platinum wire worm pick, transfer 5-8 L4 larvae animals to every well indicating the start time and the end time.
2. When starting another condition, note the start time and the end time. Place the plate at 20 °C in the incubation time between transferring the worms and scoring dead animals an hour later from the start time.
3. Using the dissecting microscope, count survival every hr. Gently shake the plate before starting to count. Indicate worms that do not move, even after spotting a high-intensity light, as dead animals. Also indicate the number of animals alive.
   NOTE: Looking at worm shape, tails and head movement at high magnification, help determine dead worms from alive.
4. Repeat this step every hour until most worms are dead.

2.  Determination of Survival Percentage at Every Time Point

1. Calculate the total number of animals per condition by adding the total number of animals transferred to every well. Ignore the animals that are damaged or killed upon transfer.
2. For every time point, calculate the total number of dead animals per condition (sum of dead animals in the 12 wells). For every time point, calculate percentage of death (number of dead animals divided by total number of animals and multiplied by 100) and percentage of survival (100 minus percent death).
3. Repeat this assay at least 3 independent times.