The method was developed to facilitate the enrichment and identification of a novel microbe of interest found in wild Caenorhabditis nematodes, allowing further investigation into host microbe interactions. The main advantage of the technique is that it enables researchers to enrich for non-culturable pathogens and microbiome bacteria directly in the intestine of wild nematodes. Begin by obtaining the wild Caenorhabditis strain of nematodes with an unculturable microbe of interest and growing the worms on a standard nematode growth medium or NGM plates containing OP51 Escherichia coli as a food source.
Incubate the nematodes for four to five days at 20 degrees Celsius until all the OP51 Escherichia coli is consumed and the majority of the worms have reached the Dauer stage. To wash the nematodes at the Dauer stage, add five milliliters of M9 minimal salts media to a six centimeter plate of starved worms and use a sterile glass pipette and bulb to pipette up the M9 media and worms from the plate and transfer them to a sterile 15 milliliter centrifuge tube. Spin down the worms at 1, 000 times G for 30 seconds at room temperature, then remove and discard the M9 supernatant above the pellet of worms using a sterile 15 milliliter pipette without disturbing the live worms by leaving approximately 50 microliters of M9 above the worms.
To the same centrifuge tube, add 10 milliliters of the M9 media containing 0.05%of Triton X-100 and adequately tighten the tube's cap before placing the tube on a Nutator at room temperature to remove the external microbes. After 20 minutes of incubation on the Nutator, spin down the worms at 1, 000 times G for 30 seconds at room temperature, then remove and discard the M9 supernatant as demonstrated and repeat the washing procedure three times with the M9 media containing 0.05%of Triton X-100. Next, incubate the tube containing nematodes overnight on a Nutator at room temperature by adding the freshly prepared antibiotic and SDS solution.
After the treatment with antibiotic, spin down the worms in the tube at 1, 000 times G for one minute at room temperature. Remove the supernatant before adding 10 milliliters of M9 containing 0.05%Triton X-100 and adequately tighten the cap to incubate the tube on a Nutator for 20 minutes at room temperature. Once done, spin down the worms at 1, 000 times G for one minute and repeat this procedure three times.
After the fourth wash with the M9 containing 0.05%Triton X-100, leave the worm pellet undisturbed in 100 microliters of the solution and discard the rest. Transfer 100 microliters of the supernatant and the pellet to the center of a 10 centimeter NGM plate seeded with OP51 Escherichia coli. Allow the plate to dry undisturbed while the Dauers crawl out of the center and through the OP51 lawn for 5 to 10 minutes.
Carefully pick up one single Dauer and plate it onto a six centimeter NGM plate seeded with OP51. In the same way, plate at least 10 Dauers in individual six centimeter NGM plates seeded with OP51. Incubate the plates for four to five days at 20 degrees Celsius until Dauers have grown and the following generation F1s have reached the adult stage, then visually check for contamination on all the plates.
For each clean plate, verify the propagation of the microbe of interest via Nomarski or fluorescent microscopy depending on the phenotype of interest. For starving the worms and reducing the number of OP51 bacteria, incubate the nematodes at 20 degrees Celsius for three to four days. Add five 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 six centimeter NGM plate. Let the plate dry while the worms crawl around for 20 minutes to help remove OP51 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 six 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 five minutes. Thaw the intestine samples before proceeding with PCR and sequencing for identification.
A wild Caenorhabditis tropicalis strain JU1848 was found to have thin microbes that colonized the lumen of the intestine in a directional manner. The wild strain JU1848 propagated noticeable microbial growth on standard six centimeter NGM plates seeded with Escherichia coli OP51 bacteria. After cleaning, a plate of F1 progeny from a single Dauer showed no visible microbial contamination after four days of incubation.
In the Nomarski image of a decapitated JU1848 animal, the colonizing bacteria were visible in the lumen of a piece of the intestine that is outside of the nematode body. Some Caenorhabditis species may require a lower SDS concentration to prevent Dauer death. To propagate a clean nematode strain, pick Dauers that crawled furthest from the center as crawling helps remove surviving contaminants.
