Selective Cleaning of Caenorhabditis Worms for Enrichment of Intestinal Microbiota

Abstract

Source: Morgan, E., et al. Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria. J. Vis. Exp. (2021)

This video demonstrates a protocol for isolating intestinal bacteria of interest from wild Caenorhabditis worms. The worms are first starved and washed to eliminate residual feeding bacteria from their surface and intestinal lumen while preserving the target microbes. After surface cleaning and multiple crawling steps, the worms are dissected to extract intestines containing the bacteria of interest, which are then collected and frozen for storage.

Protocol

1. 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 (Escherichia coli strain 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, fluorescence in situ Hybridization (FISH), or immunohistochemistry.

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 (polymerase chain reaction) 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Fisher Scientific	BP1356
10% SDS	Invitrogen	AM9822
BD PrecisionGlide Needle - 26 G	Fisher Scientific	305115
KH₂PO₄	Fisher Scientific	P-286
NaCl	Fisher Scientific	S-671
NH₄Cl	Fisher Scientific	A-661
Streptomycin	Millipore-Sigma	S6501-50G
Tetracyclin	Millipore-Sigma	T7660-5G
Triton X-100	Fisher Scientific	BP-151
Watch glasses	VWR	470144-850