Assessing Pathogenic Bacteria–Induced Mortality in C. elegans Larvae

Begin with a pathogenic bacterium cultured on agar.

Scrape the bacteria, suspend them in a buffer, and add a medium to maintain bacterial viability.

Dispense the suspension into a microplate.

Next, take separate strains of C. elegans larvae, either with increased susceptibility to pathogens or with enhanced resistance, and allow them to settle.

Remove the supernatant, resuspend the larvae in a fresh medium, and transfer them into the microplate containing the bacteria.

Seal the microplate with a gas-permeable membrane and incubate. The bacteria secrete toxic metabolites that activate stress pathways and lead to cell death.

Wash the wells to eliminate the bacteria, add a cell-impermeant fluorescent dye, and incubate.

The dye penetrates only the dead larval cells with disrupted membranes and binds to their nucleic acids.

Wash to remove excess dye and image the fluorescently stained worms to assess mortality in the susceptible and resistant strains.

To carry out a liquid killing assay use a cell scraper to remove the P. aeruginosa from an SK plate and resuspend the bacteria in approximately 5 milliliters of S Basal.

Using a spectrophotometer measure the OD 600 of the bacterial suspension. Prepa