Modeling Aeromonas Pathogenesis in C. elegans Using a Survival Assay

Begin with a culture of an Aeromonas strain, a bacterial pathogen, grown in a nutrient-rich medium.

Measure the optical density of the culture and adjust the bacterial concentration to ensure a consistent infection load.

Plate the culture onto nematode growth medium or NGM agar and incubate to form a uniform bacterial layer.

Prepare developmentally synchronized C. elegans larvae-a bacterivorous nematode maintained on an enriched medium.

Transfer the larvae onto the bacterial layer and incubate to allow oral ingestion of Aeromonas and enable intestinal colonization.

Inside the host, the bacteria multiply and release cytotoxins that damage intestinal epithelial cells, and lead to mortality.

Transfer surviving nematodes onto fresh bacterial plates and count the number of live, dead, and unresponsive worms daily until the last worm dies.

Finally, generate a survival curve to assess host viability over time.

First, select a single colony of each of the four Aeromonas strains and culture them according to the text protocol. Then, measure the OD₆₀₀ absorbance of the bacterial broth and adjust it until the absorbance is equal to 2.0.

Next spot and spread 30 microliters of bacterial broth from each strain into NGM plates.

Randomly select and transfer the previously prepared L4 worms to the NGM plates with the Aeromonas strains. Then, incubate the plates at 20 degrees Celsius until the assay is complete.

Every day transfer all of the living worms to a new NGM plate with bacteria. Count the numbers of living, dead, and sensor worms every day until the last worm is dead.