Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

1. Preparation of compost

1. Obtain compost or garden soil from any convenient source and store inside the laboratory in a standard kitchen plastic container with holes cut in the lid to let air in. Plug the holes with cotton wool to keep fruit flies and other invertebrates out (Figure 1A).
   NOTE: Five hundred grams of soil (fitting in a 1.5 gallon container, dimensions: 30 cm x 20 cm x 10 cm) will provide enough material for 12 microcosms.
2. Enrich the compost or soil with chopped produce or a mixture of different produce in a 1:2 mass ratio of produce to soil.
3. Incubate for 7-14 days at 20-25 °C, mixing once a day and adding M9 medium as necessary to maintain moisture without making it muddy.
   NOTE: Soils not enriched with produce usually do not support C. elegans growth, but which specific produce to use is up to the researcher, with many types and mixes capable of supporting worm growth. Enrichment with different produce will promote bacterial community diversification in different ways, enabling the study of gut microbiome assembly from different starting points (see the discussion section).

2. Preparation of compost microcosms

1. For each microcosm, add 10 g of enriched compost to a 30 mL glass beaker covered with tin foil and autoclave (Figure 1B).
2. To prepare microbial extract to replenish the autoclaved compost, start by adding 30 g of the same compost used in step 2.1 to each of three 50 mL tubes and fill with M9. Vortex for 1 min (Figure 1C).
   NOTE: This should provide enough bacteria for nine microcosms, each supporting the development of hundreds of nematodes.
3. Centrifuge the tubes at 560 × g for 5 min at room temperature (RT).
4. Paying attention not to disturb the pellet, remove the supernatants with a serological pipette and combine in a new 50 mL tube.
5. Concentrate the bacterial extract by centrifuging at maximum speed (2,000 × g) for 15 min at RT. Resuspend the pellets in enough M9 to have 200 µL for each microcosm and 200 µL more to add to a plate that will serve as a visible proxy of worm development inside microcosms.
   NOTE: For example, for nine microcosms, resuspend the microbial pellet in 2 mL of M9.
6. Add 200 µL of the concentrated microbial extract to each beaker of autoclaved compost, as well as to an NGM plate that will serve as the visible proxy plate.
7. Incubate microcosms and proxy plate for 24 h at 20-25 °C before addition of worms.

3. Raising worms in compost microcosms

1. Add 500-1000 eggs or L1 larvae³ to each microcosm and to the proxy plate (step 2.7) to begin the experiment (Figure 1D).
   ​NOTE: The experiments described here utilize N2 wild-type worms. However, other C. elegans strains (and potentially other nematodes) can be used.
2. Raise the worms to adulthood at 20 °C (typically 3 days).

Figure 1: Preparing compost microcosms, raising worms, and harvesting. (A) Enrich local soil or compost with produce and incubate for 2 weeks. Combine (B) autoclaved enriched soil with (C) microbial extract and (D) incubate for at least 24 h before adding synchronized L1s worms to microcosms to begin the experiment. (E, F) When ready to harvest, add compost from the microcosm into a Baermann funnel support cylinder and cover with M9. (G) After 15 min, release the filtrate into a 50 mL tube. Abbreviation: sup. = supernatant. Please click here to view a larger version of this figure.

4. Preparing a Baermann funnel for harvesting worms

1. Assemble a Baermann funnel by attaching 5-8 cm of rigid rubber tubing to the end of a plastic funnel.
2. Slide a clamp onto the tubing and clamp it shut.
3. Place in the funnel a 7 cm long, 5 cm diameter, cylindrical PVC pipe with a 1 mm nylon mesh glued at its bottom (Figure 1E). Line the cylinder with two sheets of tissue paper.
4. Place the Baermann funnel into a flask (Figure 1F).

5. Harvesting worms from microcosms and collecting the respective soil samples

1. Add 20 mL of M9 to the microcosm in which the worms were raised, agitate the mixture, and then pour the mixture from the beaker into the tissue paper-lined cylinder in the Baermann funnel setup. Add more M9 to completely submerge the compost in the funnel.
   NOTE: C. elegans (N2) populations reach adulthood in compost microcosms at a similar rate as on standard agar plates seeded with Escherichia coli. For further accuracy in harvesting worms at a specific developmental stage, refer to the proxy plate from step 3.3.
2. After 30 min, unfasten the clamp to release the filtrate containing the harvested worms into a 50 mL tube (Figure 1G).
3. Add more M9 to the cylinder and repeat for a second round to harvest more worms, and once more if additional worms are required.
   NOTE: When repeating harvesting rounds, be careful not to compromise the tissue paper's integrity, which could allow soil particles to pass through. A maximum of four harvest rounds should isolate at least 50% of the worms originally added to the microcosm without compromising tissue paper integrity.
4. Concentrate the worms by centrifuging at 560 × g for 2 min (RT). Remove 35 mL of the supernatant with a serological pipette.
5. Transfer the remaining 15 mL to a 15 mL tube and centrifuge again at 560 × g for 1 min to further concentrate the worms. Remove 14 mL of the supernatant with a serological pipette.
6. In parallel, collect 1 g of the remaining microcosm soil into a 1.5 mL tube. Process the soil samples containing the environmental bacterial community immediately or store them at −20 °C for later extraction of nucleic acids, as described below for worm samples.

6. Washing and surface sterilization of harvested worms

1. Transfer 1 mL of the concentrated, harvested worms from step 5.5 to a 1.5 mL tube using a glass pipette. Incubate for 2 min to allow the worms to settle at the bottom of the tube. Remove the supernatant, leaving the bottom 100 µL undisturbed.
2. Wash 6x with 1.5 mL of M9+T (0.025% Triton-X in M9), allowing the worms to settle at the bottom each time.
3. Transfer the washed worms in a volume of 100 µL to a new 1.5 mL tube using a glass pipette.
   NOTE: At this point in the protocol, roughly 30 min should have passed since the first wash (step 6.2). Allowing the worms to remain without food for at least 1 h before the addition of levamisole is recommended to allow the excretion of transient bacteria and the full digestion of food bacteria.
4. Add 100 µL of 25 mM levamisole hydrochloride to paralyze the worms. Incubate for 5 min at RT.
5. Add 200 µL of 4% bleach solution. Incubate for 2 min.
6. Remove the supernatant, leaving the bottom-most 150 µL undisturbed, and wash 3x with M9+T, as above.
   NOTE: To minimize contamination of the samples, it is recommended to use filtered M9+T (through a 0.2 µm filter) and to wear gloves from this point onward.
7. Following the washes, take 50 µL of the remaining 150 µL from the last wash and plate on an LB plate, incubating at 25 °C for 48 h to confirm the effective removal of external bacteria.
   NOTE: Observing up to 30 colonies in the last wash (representing 60 external bacterial cells total remaining in the sample) is permitted and expected to have only a marginal contribution to the analyzed microbiome composition, given the thousands of bacteria that typically colonize each adult worm¹⁵. When more are observed, sample integrity might be compromised.
8. Use the surface-sterilized worms immediately (e.g., to extract live bacteria for culturing [CFU counts]) or store at −20 °C for the subsequent extraction of nucleic acids.

7. DNA extraction

NOTE: The following steps describe DNA extraction of the harvested worms using a commercial kit designed for the extraction of microbial DNA from soil (see Table of Materials), with modifications described below to facilitate the extraction of microbial DNA from worms.

1. Transfer the sample containing surface-sterilized worms (defrost at RT if necessary) to the tubes provided by the kit, replacing the provided glass beads with roughly 30-50 1 mm diameter zirconia beads (see Table of Materials).
   NOTE: The observed DNA yields are higher with zirconia beads than with the glass beads provided by the kit. Although the reason behind this observation remains undetermined, zirconia beads may break open the nematode cuticle more efficiently, releasing more bacteria.
2. For compost samples, add roughly 250 mg of the collected soil to the kit's tubes without replacing the glass beads.
3. After the addition of the buffer solution provided by the kit to all the samples, homogenize with a power homogenizer at RT (see Table of Materials) for two rounds of 2,000 rpm for 30 s each, pausing for 30 s in between.
4. Complete the remaining DNA purification steps according to the kit protocol. Elute the worm samples in no more than 50 µL of elution buffer to ensure DNA concentrations that are high enough for sequencing.