l
Sodium hydroxide (NaOH) 4 mol: 1.5 ml.
l
Distilled water: 10 ml.
Procedure
Pre-culture of Symbiotic
Bacteria
1. Isolate the symbiotic bacteria of EPN from 24 h infected
G. mellonella larvae and grow the bacterial colonies for 48 h
at 28
C.
2. Propagate the bacteria in a 100-ml flasks containing 30 ml YS
broth (in order to provide a homogenous bacterial inoculum
for the nematode culture) and keep culture in shaker at
180 rpm and 25
C.
3. Transfer the suspension into 2 ml Eppendorf tubes, when the
cell density reaches to 10
10 cells/ml, and then mix well with
glycerol at 15% (v/v).
4. Store at À80
C until use.
Nematode’s Egg Isolation
and Monoxenic Culture
5. Infect late instar G. mellonella larvae with IJs (100 IJs/larva) in
a moist filter paper assay method.
6. Dissect the infected cadaver in Ringer’s solution after 3 days.
7. Collect the females separately (at least 100 big adults are necessary for successful egg isolation).
8. Wash the female nematodes (until no more tissue pieces of
cadaver) and then transfer to a glass container.
9. Add sharp razor blades to the nematodes containing glass
container and vortex to disrupt the adult to damage and their
eggs to be released.
10. Allow the suspension to 53-μm sieve to filter the adult tissues
and collect the filtrate (with eggs).
11. Transfer the filtrate to Eppendorf tubes.
12. Centrifuge at 450 x g for 1 min to sediment the eggs.
13. Remove the supernatant.
14. Transfer the eggs to sterile tube and add 1 ml of surface
sterilization solution in aseptic condition.
15. Shake gently the tubes for 4 min.
16. Centrifuge at 2000 rpm for 2 min (totally 6 min surface
sterilization time).
17. Remove the supernatant and fill with sterile YS medium.
18. Centrifuge at 2000 rpm for 2 min.
19. Repeat this step once.
20. Transfer the sterile eggs to WOUTS-Agar plates.
21. Incubate at 28
C for 24 h.
Mass Production of Nematodes
105
Sodium hydroxide (NaOH) 4 mol: 1.5 ml.
l
Distilled water: 10 ml.
Procedure
Pre-culture of Symbiotic
Bacteria
1. Isolate the symbiotic bacteria of EPN from 24 h infected
G. mellonella larvae and grow the bacterial colonies for 48 h
at 28
C.
2. Propagate the bacteria in a 100-ml flasks containing 30 ml YS
broth (in order to provide a homogenous bacterial inoculum
for the nematode culture) and keep culture in shaker at
180 rpm and 25
C.
3. Transfer the suspension into 2 ml Eppendorf tubes, when the
cell density reaches to 10
10 cells/ml, and then mix well with
glycerol at 15% (v/v).
4. Store at À80
C until use.
Nematode’s Egg Isolation
and Monoxenic Culture
5. Infect late instar G. mellonella larvae with IJs (100 IJs/larva) in
a moist filter paper assay method.
6. Dissect the infected cadaver in Ringer’s solution after 3 days.
7. Collect the females separately (at least 100 big adults are necessary for successful egg isolation).
8. Wash the female nematodes (until no more tissue pieces of
cadaver) and then transfer to a glass container.
9. Add sharp razor blades to the nematodes containing glass
container and vortex to disrupt the adult to damage and their
eggs to be released.
10. Allow the suspension to 53-μm sieve to filter the adult tissues
and collect the filtrate (with eggs).
11. Transfer the filtrate to Eppendorf tubes.
12. Centrifuge at 450 x g for 1 min to sediment the eggs.
13. Remove the supernatant.
14. Transfer the eggs to sterile tube and add 1 ml of surface
sterilization solution in aseptic condition.
15. Shake gently the tubes for 4 min.
16. Centrifuge at 2000 rpm for 2 min (totally 6 min surface
sterilization time).
17. Remove the supernatant and fill with sterile YS medium.
18. Centrifuge at 2000 rpm for 2 min.
19. Repeat this step once.
20. Transfer the sterile eggs to WOUTS-Agar plates.
21. Incubate at 28
C for 24 h.
Mass Production of Nematodes
105