Procedure
1. Place 2000 IJs in 100 μl on filter paper disk in a petri dish.
2. Remove excess moisture through vacuum filter.
3. Place disk on a petri dish (50 mm diameter) and transfer to
standard desiccators.
4. Expose to different relative humidity (RH) levels generated
using saturate salt solutions, KCl or K 2 SO 4 .
5. Incubate for different hours (24, 48, or 72 h) at 25
C.
6. Rehydrate the nematodes after 72 h immersion into petri
dishes containing 5/10 ml of distilled tap water for 24 h.
7. Determine nematode viability by observing motility.
8. Consider dead, if not responding to probing with a fine needle.
9. Count the number of dead and live IJs under a stereomicroscope by taking samples for each plate.
10. Calculate survival percentage.
7.2.8 Heat Tolerance
[193]
The nematode species/strains have diverse temperature optimums
and ranges that disturb their longevity and hence their motility
[194]. Nematodes lose their motility at low temperatures
(30–40
C). Survival in nematodes at various temperatures contrasts to species habitats and strains. Prolonged exposure to temperature extremes (below 0
C or over 40
C) is fatal for most types
of EPNs. In soil conditions, IJs are usually buffered from intense
temperatures. At higher temperatures, the IJs increased their metabolic activity and exhaust their energy stores, thus curbing their
life span.
Petri Dish Assay Method
Materials Required
l
IJ suspension.
l
Petri dishes.
l
Sterilized sand.
l
Parafilm.
l
Baermann funnels.
l
Stereomicroscope.
Procedure
1. Fill petri dish (50 mm diameter) with 8 g of sterilized sand
(15% water w/w).
2. Place 100 IJs in 100 μl suspension on the bottom.
3. Seal the dish with parafilm.
4. Incubate in the dark at different temperatures (25–40
C) for
different hours (e.g., 2–24 h).
5. Transfer the dishes after exposure to different temperatures to
Baermann funnels.
6. Count the living nematodes using a stereomicroscope.
100
Laboratory Techniques for Entomopathogenic Nematodes
1. Place 2000 IJs in 100 μl on filter paper disk in a petri dish.
2. Remove excess moisture through vacuum filter.
3. Place disk on a petri dish (50 mm diameter) and transfer to
standard desiccators.
4. Expose to different relative humidity (RH) levels generated
using saturate salt solutions, KCl or K 2 SO 4 .
5. Incubate for different hours (24, 48, or 72 h) at 25
C.
6. Rehydrate the nematodes after 72 h immersion into petri
dishes containing 5/10 ml of distilled tap water for 24 h.
7. Determine nematode viability by observing motility.
8. Consider dead, if not responding to probing with a fine needle.
9. Count the number of dead and live IJs under a stereomicroscope by taking samples for each plate.
10. Calculate survival percentage.
7.2.8 Heat Tolerance
[193]
The nematode species/strains have diverse temperature optimums
and ranges that disturb their longevity and hence their motility
[194]. Nematodes lose their motility at low temperatures
(30–40
C). Survival in nematodes at various temperatures contrasts to species habitats and strains. Prolonged exposure to temperature extremes (below 0
C or over 40
C) is fatal for most types
of EPNs. In soil conditions, IJs are usually buffered from intense
temperatures. At higher temperatures, the IJs increased their metabolic activity and exhaust their energy stores, thus curbing their
life span.
Petri Dish Assay Method
Materials Required
l
IJ suspension.
l
Petri dishes.
l
Sterilized sand.
l
Parafilm.
l
Baermann funnels.
l
Stereomicroscope.
Procedure
1. Fill petri dish (50 mm diameter) with 8 g of sterilized sand
(15% water w/w).
2. Place 100 IJs in 100 μl suspension on the bottom.
3. Seal the dish with parafilm.
4. Incubate in the dark at different temperatures (25–40
C) for
different hours (e.g., 2–24 h).
5. Transfer the dishes after exposure to different temperatures to
Baermann funnels.
6. Count the living nematodes using a stereomicroscope.
100
Laboratory Techniques for Entomopathogenic Nematodes