This article describes a method for isolating and analyzing bacteria that stably colonize the intestines of Caenorhabditis elegans. The protocol involves starving the worms, washing them to remove food bacteria, and dissecting their intestines for further analysis.
Take a culture plate with Caenorhabditis larvae harboring the bacteria of interest colonizing their intestinal lumen.
The plate contains a known bacterial strain as food. Starve the worms so they consume all the bacteria and enter a dormant stage.
Add a medium and transfer the worms to a tube.
Centrifuge and discard the supernatant.
Add a detergent solution, agitate to remove surface-adhered bacteria, centrifuge again, and discard the supernatant.
Repeat the wash, then transfer the worms to a fresh culture plate.
Allow crawling to remove loosely bound food bacteria from the surface and intestinal lumen while retaining the bacteria of interest that stably colonize the intestine.
Repeat the transfer and crawling step to ensure complete removal of food bacteria.
Add the medium, transfer the worms to a watch glass, and dissect to expose their intestines.
Dissect the intestines containing the bacteria of interest, collect them in water-filled tubes, and freeze for future use.
For starving the worms and reducing the number of OP50-1 bacteria, incubate the nematodes at 20 degrees Celsius for three to four days. Add 5 milliliters of the M9 media to the plate of recently starved worms, and then transfer the M9 media and worms to a sterile 15 millimeter centrifuge tube to spin down at 1,000 times g for 30 seconds at room temperature.
Remove the supernatant before washing the worms five times with 10 milliliters of the M9 containing 0.05% Triton X-100 on a Nutator for 20 minutes. After the last wash, remove the supernatant without disturbing the pellet in 100 microliters of the solution and transfer the M9 and the worms to an unseeded 6-centimeter NGM plate. Let the plate dry while the worms crawl around for 20 minutes to help remove OP50-1 from the cuticle and the intestine.
When the plate is dry, repeat the procedure by adding 250 microliters of the M9 and transferring the worms to a new unseeded 6-centimeter NGM plate to allow the worms to crawl while the plate dries.
After 20 minutes, add 250 microliters of the M9 to the plate and transfer 100 microliters of the M9 along with worms to a clean watch glass.
Then decapitate the nematodes using a 26-gauge syringe needle while holding the worm down with another 26-gauge syringe needle.
Once decapitated, the intestine, a granular mass, and the transparent gonad will naturally avert from the nematode body, then cut off a piece of the exposed intestine and transfer a single dissected intestine into a 0.5 milliliter PCR tube containing 10 microliters of sterile water. Repeat the procedure to collect intestines from at least five different animals in PCR tubes.
Freeze the PCR tubes at minus 80 degrees Celsius for a minimum of 5 minutes. Thaw the intestine samples before proceeding with PCR and sequencing for identification.
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