3.4.2 Modified White
Trap Method [163]
Materials Required
l
Plastic container.
l
Petri dish.
l
Distilled water.
l
Whatmann filter paper.
l
Beaker.
l
Tissue culture flask.
l
BOD incubator.
Procedure
1. Modified white trap consists a plastic jar (9 Â 9 Â 6 cm
3 ),
containing distilled water up to a depth of 1 cm.
2. Place the underside of an inverted petri dish (about
5 Â 3.5 cm
2 depending on plastic container) in the jar.
3. Place a piece of filter paper (about 7 cm) on the petri dish.
4. Allow the periphery of the filter paper to touch the water in
the jar.
5. Keep the dead larvae at the midpoint of the filter paper on the
petri dish.
6. Close the plastic jar with the lid.
7. Incubate the white traps at corresponding infection temperature until nematode offspring emerges.
8. Contingent on nematodes species, infective juveniles begin to
leave the cadaver 8–20 days post infection.
9. Harvest the virulent juveniles that move down through the
filter paper and reach the water after 2–3 days.
Harvesting
1. Pour the water comprising virulent juveniles in white trap into
100-ml beaker containing distilled water.
2. Leave the nematodes to sink and settle for about 30 min at the
underside of the dish.
3. Repeat the steps thrice until the water turns out to be clear.
4. Take a tissue culture flask (250 ml) and transfer the nematodes.
5. Hoard the flask with nematode suspension in BOD incubator
between 10 and 15
C (Fig. 9).
The entomopathogenicity of the nematodes should be verified
by Koch’s postulates.
3.5 Koch’s
Postulates [163]
The harvested juveniles after their emergence are subjected to fresh
G. mellonella larvae to ratify pathogenicity of the nematodes by
Koch’s postulates.
Steps to confirm involve:
64
Laboratory Techniques for Entomopathogenic Nematodes
Trap Method [163]
Materials Required
l
Plastic container.
l
Petri dish.
l
Distilled water.
l
Whatmann filter paper.
l
Beaker.
l
Tissue culture flask.
l
BOD incubator.
Procedure
1. Modified white trap consists a plastic jar (9 Â 9 Â 6 cm
3 ),
containing distilled water up to a depth of 1 cm.
2. Place the underside of an inverted petri dish (about
5 Â 3.5 cm
2 depending on plastic container) in the jar.
3. Place a piece of filter paper (about 7 cm) on the petri dish.
4. Allow the periphery of the filter paper to touch the water in
the jar.
5. Keep the dead larvae at the midpoint of the filter paper on the
petri dish.
6. Close the plastic jar with the lid.
7. Incubate the white traps at corresponding infection temperature until nematode offspring emerges.
8. Contingent on nematodes species, infective juveniles begin to
leave the cadaver 8–20 days post infection.
9. Harvest the virulent juveniles that move down through the
filter paper and reach the water after 2–3 days.
Harvesting
1. Pour the water comprising virulent juveniles in white trap into
100-ml beaker containing distilled water.
2. Leave the nematodes to sink and settle for about 30 min at the
underside of the dish.
3. Repeat the steps thrice until the water turns out to be clear.
4. Take a tissue culture flask (250 ml) and transfer the nematodes.
5. Hoard the flask with nematode suspension in BOD incubator
between 10 and 15
C (Fig. 9).
The entomopathogenicity of the nematodes should be verified
by Koch’s postulates.
3.5 Koch’s
Postulates [163]
The harvested juveniles after their emergence are subjected to fresh
G. mellonella larvae to ratify pathogenicity of the nematodes by
Koch’s postulates.
Steps to confirm involve:
64
Laboratory Techniques for Entomopathogenic Nematodes