$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Feeding worms on live bacteria in liquid culture
NOTE: This protocol is used to colonize worms with laboratory-grown bacteria in well-mixed conditions in liquid culture. Worms can be colonized with individual isolates from pure culture (e.g., pathogens such as Enterococcus faecium) or mixtures of isolates (e.g., minimal microbiome communities).
- Start with sucrose-washed synchronized adult worms in a 15 mL conical tube. Wash the worms once in 12 mL of S buffer and discard the supernatant.
- Resuspend the washed worms in the volume of S medium needed for the experiment. Consider the volume of experimental conditions, the number of conditions over which worms will be split, and the final concentrations of worms and bacteria.
NOTE: Feeding in worms varies with bacterial availability and worms can be stressed by crowding. For colonization in liquid culture, <1000 worms/mL and >107 CFU/mL are recommended; 1011 CFU/mL is considered "ad libitum" feeding density on E. coli. - Spin down bacterial cultures. Pour off the supernatant; aspiration or pipetting can be used to remove the supernatant for bacteria that form loose pellets.
NOTE: For cultures >5 mL, transfer to 15 mL tubes and spin at ~2800 x g in a large tabletop centrifuge for 8-10 min. Cultures <5 mL can be transferred to 1.5 mL tubes and centrifuged at 9000 x g for 1-2 min in a small tabletop centrifuge. Highly motile bacteria (e.g., many species of Pseudomonas) may need to be chilled at 4 °C for 10-15 min to facilitate formation of a stable pellet, and it may be better to centrifuge at 4 °C. - Resuspend bacterial cultures in one volume of S buffer and centrifuge again to pellet. Remove and discard the supernatant as before.
- Resuspend bacterial cultures in S medium at the desired density for the experiment, plus any antibiotics for selection. The antibiotics to be used, if any, will depend on the resistance profile of the bacteria used for colonization.
- Using a pipette tip coated in M9 worm buffer supplemented with 0.01% Triton X-100 (M9TX-01), pipette worms gently up and down until worms are thoroughly resuspended in S medium, then transfer to tubes or plate wells for bacterial colonization.
- Add bacterial suspension to each worm culture to reach the desired bacterial concentration and final volume.
- If using a multi-well plate for colonization, cover the plate with a sterile 96-well gas-permeable sealing membrane.
- Incubate with shaking at 200 RPM to prevent bacteria from settling during incubation.
2. Mechanical disruption of individual worms in a 96-well format
NOTE: This section describes a 96-well plate format protocol for mechanical disruption of individual bacterially colonized C. elegans. The first steps in the protocol describe a method for purging non-adhered bacteria from the worm intestine and cleaning the exterior of the worms using cold paralysis and surface-bleaching. These steps will produce clean, live adult worms that can be mechanically disrupted for quantification of bacterial contents or used for further experiments. This protocol can be adapted to quantify bacteria in worms colonized in liquid culture, on agar plates, or from natural or microcosm soil.
- Place an aliquot of M9TX-01 on ice to chill (4-5x the number of samples in mL).
- Prepare an aliquot of M9TX-01 + bleach (6% sodium hypochlorite, 1:1000 or 1:2000 v/v, 1 mL per sample + 1 mL extra) and place on ice to chill.
- Resuspend each worm sample in 1 mL of M9TX-01 in a 1.5 mL microcentrifuge tube.
- Spin tubes briefly (2-3 s) in a low-speed minicentrifuge (2,000 x g) at 25 °C to pellet adults. Pipette off the supernatant and discard, being sure not to disturb the worm pellet.
- Using centrifugation, rinse worms twice with 1 mL of M9TX-01, then once with 1 mL of M9 worm buffer, to reduce external bacteria.
- Purge non-adhered bacteria from the worm intestine.
- Resuspend each sample of worms in 1 mL of S medium + 2x heat-killed OP50 in a culture tube.
- Incubate at 25 °C for 20-30 min to allow passage of any non-adhered bacteria from the gut.
NOTE: This will also purge any extracellular fluorescent protein from the lumen and allow clearer visualization of labeled bacteria adhered to the intestinal epithelium, particularly when acid-fast fluorophores (e.g., mCherry, dsRed) are used.
- Surface bleach worms to clear external bacteria.
- Rinse purged worms twice with 1 mL of cold M9TX-01 and discard the supernatant.
- Allow tubes to chill for 10 min on ice (preferred) or at 4 °C. This will paralyze worms and prevent ingestion of bleach.
NOTE: Other protocols use a chemical paralysis agent such as levamisole; this is an established approach that requires the addition of a hazardous waste stream. - Add 1 mL of ice-cold M9 worm buffer + unscented bleach (8.25% sodium hydroxide, 1:1000 or 1:2000 v/v) to each tube. Allow tubes to sit on ice (preferred) or at 4 °C for at least 10 min to kill external bacteria.
NOTE: Do not exceed 1:1000 concentration of bleach. Even in paralyzed worms, mortality can result. - Pipette off bleach buffer and discard; return tubes to ice to ensure worms do not resume pumping until bleach is cleared.
- Add 1 mL of cold M9TX-01 to each tube. Spin for ~5 s in a minicentrifuge (2,000 x g at 25 °C); return tubes to ice. Remove the supernatant and discard.
- Repeat this rinse step with another 1 mL of cold M9TX-01, discarding as much of the supernatant as possible.