22. In addition to that, inoculate the pre cultured symbiotic bacteria in the egg containing petri plates.
23. Meanwhile, symbiotic bacteria are freshly propagated at 25
C
in YS broth medium.
24. Inoculate YS bacterial culture into the J1 stage nematodes
which are present in the WOUTS-Agar plates and incubate at
25
C until harvest at 15 days after IJ inoculation.
25. Separate active IJs from immobile IJs and adults by suspending
the IJs in 30-μm sieve in water and collect IJs which have
passed through the sieve after 4 h in sterile Ringer’s solution.
26. Store IJs at 4
C until use.
Note: Perform every step under sterile condition.
Mass Production of EPN
in Liquid Medium [199]
Materials Required
Preparation of BSA Medium
Nutrient broth:
10.00 g
Trypcase-soy broth:
10.00 g
Yeast extract:
5.00 g
Peptone:
5.00 g
Sodium chloride:
5.00 g
Potassium chloride:
0.35 g
Calcium chloride:
0.21 g
Distilled water:
1000 ml
Sunflower oil:
3%
Procedure
1. Prepare BSA medium.
2. Aliquot 50 ml of the medium into 250-ml conical flasks.
3. Add 100–200 IJs from seed culture medium.
4. Keep in shaker (110 rpm) at temperature ranging from 25
C.
5. Harvest the IJs from the medium by using filtration method
after 6 weeks of incubation period.
6. Store at 20
C (Fig. 15).
106
Laboratory Techniques for Entomopathogenic Nematodes
23. Meanwhile, symbiotic bacteria are freshly propagated at 25
C
in YS broth medium.
24. Inoculate YS bacterial culture into the J1 stage nematodes
which are present in the WOUTS-Agar plates and incubate at
25
C until harvest at 15 days after IJ inoculation.
25. Separate active IJs from immobile IJs and adults by suspending
the IJs in 30-μm sieve in water and collect IJs which have
passed through the sieve after 4 h in sterile Ringer’s solution.
26. Store IJs at 4
C until use.
Note: Perform every step under sterile condition.
Mass Production of EPN
in Liquid Medium [199]
Materials Required
Preparation of BSA Medium
Nutrient broth:
10.00 g
Trypcase-soy broth:
10.00 g
Yeast extract:
5.00 g
Peptone:
5.00 g
Sodium chloride:
5.00 g
Potassium chloride:
0.35 g
Calcium chloride:
0.21 g
Distilled water:
1000 ml
Sunflower oil:
3%
Procedure
1. Prepare BSA medium.
2. Aliquot 50 ml of the medium into 250-ml conical flasks.
3. Add 100–200 IJs from seed culture medium.
4. Keep in shaker (110 rpm) at temperature ranging from 25
C.
5. Harvest the IJs from the medium by using filtration method
after 6 weeks of incubation period.
6. Store at 20
C (Fig. 15).
106
Laboratory Techniques for Entomopathogenic Nematodes