Rapid Isolation of Wild Nematodes by Baermann Funnel

Caenorhabditis nematodes are real animals, that live in nature. Collecting wild nematodes, allows us to study genes, and genomes in their natural context revealing functions, that may be obscured by the artificial conditions of the lab. This technique, extracts worms, from bacteria-rich organic substrates in the field, and creates clean cultures on Petri dishes, capturing a snapshot of each population, at the time it was collected.

These populations are well suited to ecology, population genetics, or meta genetics questions. Wild strain cultures, are also valuable, for quantitative genetic studies. This technique is simple, and easy to execute, even for a field research novice.

However, a successful collecting trip, requires thoughtful preparation of materials, before traveling. Identify a bacteria-rich substrate in the field, such as rotting fruit, flowers, fungi, stems of herbaceous plants, soil, or leaf litter. Place a small volume of the substrate, into a labeled plastic bag.

If rotting material is rare in the field side of interest, place some fruit as bait. Leave it to rock, then retrieve it, and any worms that have colonized it. Record the sample ID, latitude, longitude, date and description of the substrate.

Record any other local environmental measurements, relevant to the experiment, such as ambient and substrate temperature, substrate condition, time of collection, and presence of substrate associated macro invertebrates. Use scissors to cut a three centimeter segment, of rubber tubing from each funnel. Fit the tubing over the end of a plastic funnel.

Slide a tubing clamp over the rubber tubing, and clamp it shut. To make a funnel holder, cut circular holes, in the center of an unfolded cardboard fly vile tray. Invert the cardboard, fold the sides up once, and tape them together.

This will create legs, that elevate the holes above the bench. Ensure that the tubing clamps, are in the closed position, and place funnels in the holes. Pour sterile water into each funnel, filling it about three centimeters below the rim.

Tap the funnel, to release any trapped air bubbles in the tubing. Fold a lint-free wipe in half, to make a square, and place it over the funnel. Then, press the wipe down, to submerge it in the water.

Manually break up any large solid pieces, of the natural substrate. Gently place some of the sample onto the wipe in the funnel. Make sure not to puncture the wipe, or leave any sample protruding above the rim.

Carefully fold the corners of the wipe, over the sample, to prevent water from wicking out, over the edge of the funnel. Label the funnel with the sample ID, corresponding to field collection notes. Pick out any active insects, or other animals, that may travel and cross-contaminate samples.

Add more water to each funnel, to submerge the entire sample. While incubating at room temperature overnight, active nematodes will wiggle through the tissue, and sink to the bottom of the funnel. Write the sample ID of a funnel, on the bottom of a six centimeter NGM worm plate, seated with a spot of OP50 E.coli bacteria.

Remove the lid from the plate, remove the funnel, containing the sample from the funnel stand, and hold it upright, above the open worm plate, with one hand. Release pressure on the tubing clamp with the other hand, and allow one or two drops of water, to fall from the tubing, onto the worm plate next to the bacterial lawn. As soon as the water drops from the funnel, quickly clamp it shut again, to prevent flooding the NGM plate.

To clean up, throw out the contents of the funnels, and wash them with hot water, for subsequent reuse. Observe the isolated nematodes under the stereo microscope. In addition to nematodes, there will occasionally be small, Oligo kit annelids, Tardigrades, rotifers and small crustaceans.

To establish ISO hermaphrodite, or ISO female lines, transfer each L4 hermaphrodite or mated adult female, to a separate 3.5 centimeter NGM plate, seated with OP50. Sterilize the worm pick, before and after each transfer. Finally, wrap plates thoroughly for travel, with a paraffin film.

On Barro Colorado island Panama, in 2018, 131 substrates were processed by this method. 99%of which yielded nematodes. 34%yielded Caenorhabditis nematodes.

In the Chernobyl Exclusion Zone Ukraine, in 2019, 170 substrates were processed by this method. 56%of which yielded nematodes, none of which were Caenorhabditis. This method works because the nematodes swim through the wipe.

but the substrate and other animals stay on top. Many aspects of a protocol can be modified, to make use of whatever resources are available in the field. Samples collected using this method, allow researchers to address a wide array of population biology questions into established cultures for subsequent laboratory studies.