1. Soil Sample Collection

1. Consider covering a minimum area of 2 – 4 m² for each sampling site.
2. Collect soil samples at a depth of at least 15 cm (Figure 4).
3. Take at least 5 random samples within this area.
4. Take 3 subsamples per sample. Depending on the objective of the study, either combine the subsamples or keep them separate.
5. Place each sample in a plastic bag. Consider double-bagging to avoid leaking of samples (Figure 4).
6. Label samples with a waterproof marker. Include the following information from each sampling site: Site information (including locality/area/site name and GPS coordinate information), date, habitat, associated vegetation, temperature, elevation, etc.
   NOTE: If applicable, collect and/or record the presence of insects or other invertebrates collected with the sample for subsequent identification. They may represent potential natural EPN hosts.
7. Clean collecting tools between samples by thoroughly washing with water and/or disinfecting them with 70% ethanol or 0.5% bleach solution.
8. Keep samples in a cooler (8 – 15 °C) during transport to the laboratory.
9. Take a portion of the soil sample for analysis to obtain information on soil composition, texture, moisture, electrical conductivity or other desired soil parameters.

2. Nematode Isolation from Soil Samples: Insect-baiting Technique

1. Remove any debris (i.e., rocks, pieces of wood or bark, leaves, etc.) collected with your samples to avoid contamination with saprobic microorganisms.
2. Add water to moisten the soil and facilitate the movement of nematodes.
   NOTE: Use a spray bottle to slowly increase the moisture level of the soil.
3. Place approximately 200 to 250 ml of moist soil in a clean plastic container with a lid.
4. Add insect baits. Consider 5 to 10 insects to the soil surface of each sample (Figure 5).
5. Cover the container with a lid and turn containers upside down.
6. Maintain containers in the dark and at RT (usually 22 – 25 °C).
7. Check containers every 2 – 3 days and remove dead insects.
   NOTE: add additional healthy insects to each container for further baiting of the soil sample.
8. Remove dead insects from the containers. NOTE: Cadavers with a brown or ochre coloration are usually parasitized by steinernematids, whereas brick red to dark purple cadavers are parasitized by heterorhabditids.
9. Rinse cadavers in sterile water.
10. Place cadavers in a modified White trap for recovery of nematode progeny (see procedure below).

3. Nematode Recovery from Infected Cadavers: Modified White Trap

1. Place the top of a 50 (or 60) mm diam. Petri dish inside a larger dish (100 mm).
2. Set one single circular filter paper (Whatman #1) inside the smaller dish.
3. Place cadavers on the filter paper of the smaller dish making sure cadavers do not touch each other to avoid any contamination.
4. Fill the outer (larger) Petri dish with ca. 20 ml of sterile distilled water. Do not add water in the dish that holds the cadavers.
5. Cover the large Petri dish and its contents with the lid (Figure 6).
6. Label dishes accordingly. Add the following information: Nematode name (species/isolate), infection date (date infection was set) and trap date (this is the date the trap is set up).
7. Keep trap at RT until emergence of infective juvenile stages (IJs) occurs. This process can take between 10 – 25 days depending on the nematode species and/or strain considered.
8. Harvest water with IJs by removing the larger dish of the trap and pouring water with nematodes into a beaker.
9. Allow nematodes to decant to the bottom of the beaker. This process may take a few minutes.
10. Pour water carefully, making sure nematodes remain in the bottom of the beaker.
11. Rinse nematodes by adding more water and allowing nematodes to decant. This step can be repeated 2 – 3 times until water is clean.
12. Place nematode suspension in a tissue culture flask (250 ml). Keep concentration of the suspension to 1,000 – 3,000 nematodes/ml.
13. Store flask with nematode suspension in a cold room or in incubator between 10 – 20 °C.
    NOTE: check stored flasks periodically as shelf life of EPN is variable. Usually steinernematids can be stored for 6 – 12 months without the need of subculturing, whereas heterorhabditids may require more periodic check-ups.