This article details a protocol for quantifying bacterial colonization in germ-free adult C. elegans. The method involves introducing test bacteria, followed by a series of centrifugation and washing steps to isolate viable bacteria adhered to the intestinal epithelium.
Begin with germ-free adult C. elegans worms suspended in media.
Introduce the test bacterium and incubate.
As the worms feed, bacteria enter the intestinal lumen and adhere to the epithelium.
Centrifuge to pellet the worms and remove the non-ingested bacteria.
Resuspend in buffer, centrifuge, and remove the remaining non-ingested bacteria.
Add media containing heat-killed E. coli and incubate.
During feeding, the heat-killed E. coli displaces and expels non-adhered test bacteria from the intestinal lumen.
Centrifuge and remove the supernatant containing the expelled bacteria.
Wash with buffer, centrifuge, and remove the supernatant.
Chill the worms on ice to induce paralysis, preventing further ingestion.
Add a bleach solution and incubate to kill residual external bacteria.
Remove the bleach and add cold buffer.
Centrifuge and remove the supernatant to eliminate residual bleach.
The worms now retain only viable test bacteria adhered to the intestinal epithelium, enabling quantification of colonization.
Begin by resuspending the worm sample in one milliliter of M9TX-01 in a microcentrifuge tube. Collect the adult worms by centrifugation and discard the supernatant. Using the same centrifugation parameters, rinse the worms twice with one milliliter of M9TX-01 and once with one milliliter of M9 worm buffer to minimize the external bacteria. Then, resuspend each sample in one milliliter of S medium plus 2X heat heat-killed OP50 in a culture tube. Incubate the tubes at 25 degrees Celsius for 20 to 30 minutes to pass the non-adhered bacteria from the gut.
After incubation, rinse the purged worms twice with one milliliter of cold M9TX-01 and discard the supernatant. Put the tubes on ice for 10 minutes to paralyze the worms. Then, add one milliliter of ice-cold M9 worm buffer with unscented bleach to each tube.
Incubate the tubes on ice for a minimum of 10 minutes to kill external bacteria. Discard the bleach buffer and return the tubes to the ice to prevent the worms from pumping. Next, add one milliliter of cold M9TX-01 to each tube and centrifuge for five seconds in a mini centrifuge. Return the tubes to ice and remove the supernatant. Repeat this step once.
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