6. Add 100 ml of 20% NaCl to the soil in the centrifuge tube
and mix.
7. Centrifuge at 1000 rpm for 5 min and collect all the
supernatant.
8. Repeat the salt-flotation step once again.
9. Pour the pooled supernatants through 240-mm mesh wire
sieve, and collect nematodes into a counting tray.
3.3 Indirect Method
3.3.1 Insect-Baiting
Technique [160]
In this technique, Galleria mellonella, the highly vulnerable larvae
of the greater wax moth, is used.
Materials Required
l
Insect.
l
Soil samples.
l
Petri dishes.
l
Plastic boxes.
l
Whatmann filter paper.
l
Tissue culture flask.
l
BOD incubator.
l
Dissecting microscope.
Procedure
1. Remove any debris from the collected soil samples.
2. If required, adjust the soil moisture by adding water.
3. Place the soil sample in petri dishes (90 mm  15 cm diameter)
or in plastic boxes with a lid.
4. Place ten final instar larvae of G. mellonella on the surface of
each soil sample.
5. Cover the petri dishes/the plastic boxes with a lid and invert
these containers.
6. Incubate petri dishes or plastic boxes in darkness at 25
C with
55% relative humidity (RH).
7. Remove the lifeless larvae with distinctive signs and indications
of entomopathogenic nematode contagion after 7 days from
the soil.
8. Place cadavers on white trap, to accumulate the emerging
nematode infective juveniles.
(In case of negative results, repeat the isolation twofold to
confirm results of the experiment.)
Identification of entomopathogenic nematode is established on
the characteristics of color of the dead larvae. Genus SteinernemaIsolation and Storage of Soil Nematodes
61
and mix.
7. Centrifuge at 1000 rpm for 5 min and collect all the
supernatant.
8. Repeat the salt-flotation step once again.
9. Pour the pooled supernatants through 240-mm mesh wire
sieve, and collect nematodes into a counting tray.
3.3 Indirect Method
3.3.1 Insect-Baiting
Technique [160]
In this technique, Galleria mellonella, the highly vulnerable larvae
of the greater wax moth, is used.
Materials Required
l
Insect.
l
Soil samples.
l
Petri dishes.
l
Plastic boxes.
l
Whatmann filter paper.
l
Tissue culture flask.
l
BOD incubator.
l
Dissecting microscope.
Procedure
1. Remove any debris from the collected soil samples.
2. If required, adjust the soil moisture by adding water.
3. Place the soil sample in petri dishes (90 mm  15 cm diameter)
or in plastic boxes with a lid.
4. Place ten final instar larvae of G. mellonella on the surface of
each soil sample.
5. Cover the petri dishes/the plastic boxes with a lid and invert
these containers.
6. Incubate petri dishes or plastic boxes in darkness at 25
C with
55% relative humidity (RH).
7. Remove the lifeless larvae with distinctive signs and indications
of entomopathogenic nematode contagion after 7 days from
the soil.
8. Place cadavers on white trap, to accumulate the emerging
nematode infective juveniles.
(In case of negative results, repeat the isolation twofold to
confirm results of the experiment.)
Identification of entomopathogenic nematode is established on
the characteristics of color of the dead larvae. Genus SteinernemaIsolation and Storage of Soil Nematodes
61