Assessment of Intestinal Permeability in C. elegans Following Bacterial Exposure

Take C. elegans worms cultured on nutrient-rich agar plates containing E. coli for the control condition and Pseudomonas aeruginosa for the pathogen condition.

During incubation, the worms feed on bacteria that colonize the intestinal lumen.

In the control, the intestinal epithelium remains intact, while the pathogen-released toxins cause epithelial damage.

Wash the worms with a buffer, and transfer to plates supplemented with fluorescent tracers and heat-inactivated bacteria as a food source.

In the control, ingested fluorescent tracers remain in the gut, while in the pathogen-infected worms, they cross the damaged epithelium and enter the body cavity.

Wash the worms again and place them on fresh plates. Then transfer them to a multiwell assay plate containing a fixative to preserve the cellular structure.

Remove the fixative. Then add a mounting medium for imaging.

Observe using a fluorescence imaging system.

Increased fluorescence in the body cavity of P. aeruginosa-exposed worms indicates intestinal permeability induced by the pathogen.

Wash the age-synchronized L4 larvae with S-buffer, and transfer approximately 500 worms to the NGM plates containing different bacteria and chemicals. Then, incubate the plates at 20 degrees Celsius for 48 hours.

Prepare the FITC-dextran-supplemented plates by mixing 2 milliliters of heat-inactivated E. coli with four milligrams of FITC-dextran. Add 100 microliters of the mixture to each of 20 fresh NGM agar plates, and allow the plates to dry for 1 hour on a clean lab bench. After 48 hours of treatment, wash the worms with S-buffer, transfer them to FITC-dextran-supplemented plates and to NGM plates without FITC-dextran, and incubate the plates overnight.

The next day, wash the worms with S-buffer, and allow them to crawl in the fresh NGM agar plate for 1 hour. Add 50 microliters of 4% formaldehyde to each well of a black, 96-well, flat-bottomed plate, and transfer approximately 50 worms into each well. After 1 to 2 minutes, remove all the formaldehyde, and add 100 microliters of mounting medium into each well.