Materials Required
l
Infection trap (White trap).
l
IJ suspension.
l
G. mellonella larvae.
l
Plastic petri dish.
l
Whatman No. 1 filter paper.
Procedure
1. Adjust the IJ suspension to a concentration of 200 IJs/ml.
2. Distribute evenly 1 ml of the IJ suspension on a 10 cm diameter
piece of filter paper placed in the top of a 100 Â 15 mm
petri dish.
3. Put in ten insect larvae (G. mellonella) to the dish. The goal is
to have about 20 IJs/larva; too many IJs per larva produce few
progeny because of competition and/or contamination with
foreign bacteria.
4. Close the top with the petri bottom inverted.
5. Label and incubate the petri dish at room temperature 25
C,
in the dark.
6. Place the cadavers in a White trap after 3–5 days.
Note:
For the labeling of the dish, include the following information:
(a) Nematode species name (if known).
(b) Isolate code/name.
(c) Date of infection trap.
Note: Discard the cadavers if they smell putrid.
8.1.2 In Vitro Method
Production of EPN in Liquid
Medium
Materials Required
l
WOUTS-Agar [197]:
– Nutrient broth: 16.0 g.
– Agar: 12.0 g.
– Sunflower oil: 5.0 g.
– Distilled water: 1000 ml.
l
YS Medium [198]:
– Yeast extract: 5.0 g.
– Sodium chloride (NaCl): 5.0 g.
– Monoammonium phosphate (NH 4 H 2 PO 4 ): 0.5 g.
– Dipotassium hydrogen phosphate (K 2 HPO 4 ): 0.5 g.
– Magnesium sulfate heptahydrate (MgSO 4 ∙7H 2 O): 0.2 g.
– Distilled water: 1000 ml.
l
Egg surface sterilization solution:
l
Sodium hypochlorite (NaOCl) 12%: 0.5 ml.
104
Laboratory Techniques for Entomopathogenic Nematodes
l
Infection trap (White trap).
l
IJ suspension.
l
G. mellonella larvae.
l
Plastic petri dish.
l
Whatman No. 1 filter paper.
Procedure
1. Adjust the IJ suspension to a concentration of 200 IJs/ml.
2. Distribute evenly 1 ml of the IJ suspension on a 10 cm diameter
piece of filter paper placed in the top of a 100 Â 15 mm
petri dish.
3. Put in ten insect larvae (G. mellonella) to the dish. The goal is
to have about 20 IJs/larva; too many IJs per larva produce few
progeny because of competition and/or contamination with
foreign bacteria.
4. Close the top with the petri bottom inverted.
5. Label and incubate the petri dish at room temperature 25
C,
in the dark.
6. Place the cadavers in a White trap after 3–5 days.
Note:
For the labeling of the dish, include the following information:
(a) Nematode species name (if known).
(b) Isolate code/name.
(c) Date of infection trap.
Note: Discard the cadavers if they smell putrid.
8.1.2 In Vitro Method
Production of EPN in Liquid
Medium
Materials Required
l
WOUTS-Agar [197]:
– Nutrient broth: 16.0 g.
– Agar: 12.0 g.
– Sunflower oil: 5.0 g.
– Distilled water: 1000 ml.
l
YS Medium [198]:
– Yeast extract: 5.0 g.
– Sodium chloride (NaCl): 5.0 g.
– Monoammonium phosphate (NH 4 H 2 PO 4 ): 0.5 g.
– Dipotassium hydrogen phosphate (K 2 HPO 4 ): 0.5 g.
– Magnesium sulfate heptahydrate (MgSO 4 ∙7H 2 O): 0.2 g.
– Distilled water: 1000 ml.
l
Egg surface sterilization solution:
l
Sodium hypochlorite (NaOCl) 12%: 0.5 ml.
104
Laboratory Techniques for Entomopathogenic Nematodes