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. Prepare 24 milliliters of diluted stock of P. aeruginosa in S Basal at an OD 600 equal to 0.09, then add 21 milliliters of liquid SK media, and, using a multichannel pipette, transfer 45 microliters of bacteria and media to each well of a 384 well plate. Wash the worms from their source into a 50 milliliter conical tube, and allow them to settle under gravitational force.

Aspirate the supernatant to 5 milliliters, then use a total of 50 milliliters of S Basal to resuspend the worms, and repeat the wash twice more. Using a worm sorter, sort approximately 22 worms into each well of a 384 well plate, according to the text protocol, then use a gas-permeable film to seal the plate and incubate it at 25 degrees Celsius for 24 to 48 hours. At the desired time, use a microplate washer and S Basal to wash the 384 well plate a total of 5 times.

One of the easiest mistakes to make is to aspirate the 384 well plate before worms have completely settled. It takes practice to be able to accurately see whether the worms have settled completely. After the final wash, aspirate the supernatant down to 20 microliters.

Then, add 50 microliters of 0.98 micromolar nucleic acid stain per well, for a final concentration of 0.7 micromolar. Incubate the plate at room temperature for 12 to 16 hours. After the desired incubation period, use the microplate washer to wash the plates a minimum of three times.

For a data acquisition use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence.