infected larvae display gray color, and genus Heterorhabditisinfected larvae display brick red color.
3.3.2 Modified Baiting
Technique [161]
Materials Required
l
Soil samples.
l
Tap water.
l
Petri dish.
l
Mesh metal screen.
l
Stereoscopic microscope.
Procedure
1. Take 0.5 kg of soil from each soil sample.
2. Moisten the soil with 200 ml of tap water to attain field
potential 3 days preceding the nematode traps placement.
3. The traps consist of 5 cm diameter petri dishes shielded by
150 mm metal mesh panel and having two final instar larvae
of the G. mellonella.
4. Bury one nematode trap in every sample of soil.
5. Incubate the samples at 25
C.
6. Remove the larvae after 5–6 days from the soil.
7. Dissect the dead larvae.
8. Quantify the nematodes (infective juvenile, fourth stage juvenile, or adult stage) under stereoscopic microscope.
3.4 Collection
of Nematode Infective
Juveniles from
Infected Cadavers
3.4.1 White Trap Method
[162]
Materials Required
l
Petri dish.
l
Plastic container.
l
Sterile distilled water.
l
Whatman filter paper.
l
Beaker.
l
Tissue culture flask.
l
BOD incubator.
Procedure
1. Place the bottom of an inverted petri dish inside a large
plastic jar.
2. Pour 200 ml of sterile distilled water into the plastic jar.
3. Set a circular Whatman filter paper inside the petri dish.
4. Allow the periphery of filter paper to come in contact with
water in the plastic jar.
5. Place cadavers (about 2–4 per trap) on the filter paper of the
petri dish.
6. Close the plastic container with the lid.
7. Incubate the container at 20–27
C.
62
Laboratory Techniques for Entomopathogenic Nematodes
3.3.2 Modified Baiting
Technique [161]
Materials Required
l
Soil samples.
l
Tap water.
l
Petri dish.
l
Mesh metal screen.
l
Stereoscopic microscope.
Procedure
1. Take 0.5 kg of soil from each soil sample.
2. Moisten the soil with 200 ml of tap water to attain field
potential 3 days preceding the nematode traps placement.
3. The traps consist of 5 cm diameter petri dishes shielded by
150 mm metal mesh panel and having two final instar larvae
of the G. mellonella.
4. Bury one nematode trap in every sample of soil.
5. Incubate the samples at 25
C.
6. Remove the larvae after 5–6 days from the soil.
7. Dissect the dead larvae.
8. Quantify the nematodes (infective juvenile, fourth stage juvenile, or adult stage) under stereoscopic microscope.
3.4 Collection
of Nematode Infective
Juveniles from
Infected Cadavers
3.4.1 White Trap Method
[162]
Materials Required
l
Petri dish.
l
Plastic container.
l
Sterile distilled water.
l
Whatman filter paper.
l
Beaker.
l
Tissue culture flask.
l
BOD incubator.
Procedure
1. Place the bottom of an inverted petri dish inside a large
plastic jar.
2. Pour 200 ml of sterile distilled water into the plastic jar.
3. Set a circular Whatman filter paper inside the petri dish.
4. Allow the periphery of filter paper to come in contact with
water in the plastic jar.
5. Place cadavers (about 2–4 per trap) on the filter paper of the
petri dish.
6. Close the plastic container with the lid.
7. Incubate the container at 20–27
C.
62
Laboratory Techniques for Entomopathogenic Nematodes