7. Check live nematodes for natural movement or respond to
probing with a fine needle.
Well Plate Method
Materials Required
l
G. mellonella larva.
l
Well tissue culture plates.
l
IJ suspension.
Procedure
1. Place 0.5 g of sterilized sand and one G. mellonella larva on
each well of tissue culture plates.
2. Add the IJs.
3. Seal the plates with parafilm.
4. Incubate at different temperature (low to high range temperature, i.e., 5–40
C).
5. Check the well plates after 24 h for larval mortality.
6. Remove cadavers from wells and place in petri dish with filter
paper.
7. Incubate at 25
C.
8. Count the number of nematodes which come out from
cadaver.
9. Consider each well as one replicate for each temperature and
replicate thrice.
7.2.9 Soil pH [195]
The pH of soil does not have a robust influence on infective juvenile
survival. The consequence of pH on the survival, pathogenicity, and
progeny of nematodes species is evaluated under laboratory
condition.
Materials Required
l
IJ suspension.
l
Soil sample.
l
Sodium hydroxide.
l
Hydrochloric acid.
Procedure
1. Adjust the selected pH range from pH 4 to 8 for the experiment with sodium hydroxide (NaOH) for alkaline and hydrochloric acid (HCl) for acidic condition.
2. Incubate the tissue culture flasks containing 500 IJs in distilled
water with soil at room temperature 25 Æ 2
C.
3. Record the survival of IJs at weekly intervals for 28 days.
7.2.10 Soil Moisture/
Water Content [196]
Moisture is significant because nematodes require water film in the
interstitial spaces of soil for operative propulsion. When this water
film becomes too thin (i.e., in dry soil) or the interstitial spaces are
Ecological Characterization
101
probing with a fine needle.
Well Plate Method
Materials Required
l
G. mellonella larva.
l
Well tissue culture plates.
l
IJ suspension.
Procedure
1. Place 0.5 g of sterilized sand and one G. mellonella larva on
each well of tissue culture plates.
2. Add the IJs.
3. Seal the plates with parafilm.
4. Incubate at different temperature (low to high range temperature, i.e., 5–40
C).
5. Check the well plates after 24 h for larval mortality.
6. Remove cadavers from wells and place in petri dish with filter
paper.
7. Incubate at 25
C.
8. Count the number of nematodes which come out from
cadaver.
9. Consider each well as one replicate for each temperature and
replicate thrice.
7.2.9 Soil pH [195]
The pH of soil does not have a robust influence on infective juvenile
survival. The consequence of pH on the survival, pathogenicity, and
progeny of nematodes species is evaluated under laboratory
condition.
Materials Required
l
IJ suspension.
l
Soil sample.
l
Sodium hydroxide.
l
Hydrochloric acid.
Procedure
1. Adjust the selected pH range from pH 4 to 8 for the experiment with sodium hydroxide (NaOH) for alkaline and hydrochloric acid (HCl) for acidic condition.
2. Incubate the tissue culture flasks containing 500 IJs in distilled
water with soil at room temperature 25 Æ 2
C.
3. Record the survival of IJs at weekly intervals for 28 days.
7.2.10 Soil Moisture/
Water Content [196]
Moisture is significant because nematodes require water film in the
interstitial spaces of soil for operative propulsion. When this water
film becomes too thin (i.e., in dry soil) or the interstitial spaces are
Ecological Characterization
101