1. Inducing dauer formation for wild nematodes on NGM plates

1. Grow the wild Caenorhabditis strain on standard NGM plates with OP50-1 E. coli as a food source after obtaining the worms with an unculturable microbe of interest. Incubate the nematodes at 20 °C.
   NOTE: The standard temperature to grow Caenorhabditis nematodes is between 15-25 °C, but should be determined empirically to prevent loss of the microbe of interest.
2. Starve the plate of animals at 20 °C for 4-5 days until all the OP50-1 is consumed and the majority have reached the dauer stage.
   ​NOTE: Dauers are long-lived larvae that develop due to the absence of nutrition and have protective cuticles.

2. Washing the nematodes

1. Add 5 mL of M9 minimal salts media (42 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 19 mM NH₄Cl) to a 6 cm plate of starved worms.
2. Using a sterile glass pipette and bulb, pipette up the M9 and worms from the plate and transfer them to a sterile 15 mL centrifuge tube.
3. Using a clinical centrifuge, spin down the worms in the tube at 1000 x g for 30 s at room temperature.
4. Using a sterile 15 mL pipette, remove and discard the M9 supernatant above the pellet of worms. Do not disturb the live worms at the bottom of the tube by leaving approximately 50 µL of M9 above the worms.
5. Add 10 mL of M9 + 0.05% of Triton X-100 to the same centrifuge tube and adequately tighten the tube's cap.
6. Incubate the tube on a nutator for 20 min at room temperature to remove the external microbes. Remove the tube from the nutator and spin down the worms at 1000 x g for 30 s.
7. Using a sterile pipette, remove and discard the M9 supernatant above the pellet of worms. Do not disturb the live worms at the bottom of the tube by leaving approximately 50 µL of M9 above the worms.
8. Follow and repeat steps 2.5-2.8 three more times.

3. Disinfecting the nematodes

1. Prepare an antibiotic and SDS solution in 10 mL of M9 buffer by adding 0.25% SDS (250 µL of 10% SDS), 50 µg/mL of carbenicillin, 25 µg/mL of kanamycin, 12.5 µg/mL of tetracycline, 100 µg/mL of gentamycin, 50 µg/mL of streptomycin, 37.5 µg/mL of chloramphenicol, and 200 µg/mL of cefotaxime (see Table of Materials).
2. Incubate the tube containing nematodes in the antibiotic and SDS solution on a nutator at room temperature overnight.
   ​NOTE: All non-dauer worms and embryos will die, but many of the dauer worms will survive this cleaning process.

4. Removing the antibiotic and SDS solution

1. Spin down the worms in the tube at 1000 x g for 1 min at room temperature. Using a sterile pipette, remove the supernatant from the centrifuge tube without disturbing the worms at the bottom of the tube.
2. Add 10 mL of M9 + 0.05% Triton X-100 and adequately tighten the cap of the tube. Incubate the tube on a nutator for 20 min at room temperature.
3. Remove the tube from the nutator and spin down the worms at 1000 x g for 1 min. Follow and repeat steps 4.2-4.3 three times.
4. After the last wash, leave the worm pellet undisturbed at the bottom in 100 µL of the solution and discard the rest.

5. Propagate a clean nematode strain

1. Using a sterile glass pipette, transfer 100 µL of the supernatant and the pellet to the center of a 10 cm NGM plate seeded with OP50-1. Allow the plate to dry without disturbing the liquid at the center.
2. Allow the dauers to crawl out of the center and through the OP50-1 lawn for 5-10 min.
3. Carefully pick up one single dauer and plate it onto a 6 cm NGM seeded plate. In total, pick at least 10 dauers and plate them in individual 6 cm NGM plates seeded with OP50-1 (10 plates total).
4. Incubate the plates for 4-5 days at 20 °C until dauers have grown and the following generation (F₁) has reached the adult stage. Visually check for contamination on all the plates, seen as non-OP50-1 microbial growth.
5. For each clean plate, verify the propagation of the microbe of interest via Normarski or fluorescent microscopy, such as fluorescent in situ hybridization (FISH)¹², depending on the phenotype of interest.

6. Intestinal dissection and PCR identification of microbial species

1. Grow animals at 20 °C for 3-4 days to allow them to starve and reduce the amount of OP50-1 bacteria. Add 5 mL of M9 media to the plate of starved worms.
2. Using a sterile glass pipette and bulb, pipette up the M9 + worms from the plate and transfer them to a sterile 15 mL centrifuge tube.
3. Using a clinical centrifuge, spin down the worms in the tube at 1000 x g for 30 s at room temperature.
4. Using a sterile 15 mL pipette, remove the supernatant from the centrifuge tube without disturbing the live worms at the bottom of the tube.
5. Add 10 mL of M9 + 0.05% Triton X-100 and incubate the tube on a nutator for 20 min to remove external microbes. Repeat four times to wash the worms.
6. After the last wash, using a sterile pipette tip, remove the supernatant without disturbing the pellet at the bottom in 100 µL of the solution.
7. Using a sterile pipette tip, transfer M9 and the worms to an unseeded NGM plate and let the plate dry while the worms crawl around for 20 min to help remove OP50-1 from the cuticle and the intestine.
8. Add 250 µL of M9 to the dried plate and use a glass pipette to transfer M9 + worms to a new unseeded NGM plate. Again, let that plate dry and allow the worms to crawl around for 20 min.
9. Add 250 µL of M9 to the plate and transfer 100 µL of the M9 + worms to a clean watch glass.
10. Using two sterile 26 G syringe needles, decapitate the nematodes by holding the worm down with one needle and using the other needle to cut the worm. After decapitation, the intestine (granular) and the gonad (transparent) from the body will naturally come out of the body.
11. Cut off a piece of the exposed intestine by holding down the intestine with one needle and cutting it with the other.
    NOTE: If possible, verify that the microbe of interest is still present in everted intestines using Normarski microscopy, FISH, or immunohistochemistry²³.
12. Transfer a single dissected intestine into a 0.5 mL PCR tube containing 10 µL of sterile water. Repeat for a total of at least 5 PCR tubes containing intestines from different animals.
13. Freeze the PCR tubes at -80 °C for a minimum of 5 min. Remove the PCR tubes from the freezer and thaw out the samples.
14. Pipette the liquid up and down a few times to separate the bacteria from the intestine.
15. Conduct PCR using universal primer pairs against the DNA sequence of the small ribosomal subunit of bacteria, yeast, or microsporidia, depending on the suspected type of microbe.
    NOTE: For example, a sample protocol using universal bacterial 16S primers is presented in Table 1.
16. Purify the amplicon using a filter-based DNA cleaning and concentration kit (see Table of Materials) and perform Sanger sequencing.