4.2.7 Lipid Agar Plate
Method
Materials Required
l
Nutrient broth: 8 g.
l
Agar: 15 g.
l
Yeast extract: 5 g.
l
D.dH 2 O: 890 ml.
l
MgCl 2 ∙6H 2 O: 0.2 g/ml, 10 ml.
l
Corn oil: 4 ml.
l
Corn syrup mix (7 ml corn syrup in 89 ml H 2 O): 96 ml.
Preparation of Media
1. Mix together nutrient broth, agar, and yeast extract. Add
double-distilled H 2 O.
2. Autoclave for 15 min at 121
C.
3. Add sterile mixture of corn oil and corn syrup and mix.
4. Stir vigorously making sure oil is dispersed evenly.
Note: The oil will not dissolve into the liquid, but ensure it is in
tiny droplets. Ensure an even distribution of oil and volume to each
plate, since variation in agar thickness or oil distribution can lead to
conflicting nematode development between plates. If this is not an
issue, simply ensure the oil is dispersed evenly while pouring from
the flask/bottle.
Procedure
1. Use 5-cm petri dishes for this purpose.
2. Inoculate plates with Phase I bacteria isolated from the nematode strain or it can also be the species from which the female
partner in the cross is obtained.
3. Leave the plates at 25
C overnight.
4. For every individual cross, place ten virgin females and ten
males of the suitable strain or species on each of six lipid agar
plates.
5. Check plates under a microscope on a daily basis for the presence of outcross progeny and record observations.
6. After 3 days and if males have died and/or no egg development
is observed in the females, add ten more males to individual
plate to confirm that viable males are still able to fertilize the
females.
7. Continue to visualize the plates under a microscope daily for
three additional days.
Note: In successful crosses, progeny will be visible after
2–3 days.
8. Accumulate IJs after 2 weeks from these crosses.
9. Relocate to fresh lipid agar plates to allow a hybrid line to be
formed.
Morphological Characterization
85
Method
Materials Required
l
Nutrient broth: 8 g.
l
Agar: 15 g.
l
Yeast extract: 5 g.
l
D.dH 2 O: 890 ml.
l
MgCl 2 ∙6H 2 O: 0.2 g/ml, 10 ml.
l
Corn oil: 4 ml.
l
Corn syrup mix (7 ml corn syrup in 89 ml H 2 O): 96 ml.
Preparation of Media
1. Mix together nutrient broth, agar, and yeast extract. Add
double-distilled H 2 O.
2. Autoclave for 15 min at 121
C.
3. Add sterile mixture of corn oil and corn syrup and mix.
4. Stir vigorously making sure oil is dispersed evenly.
Note: The oil will not dissolve into the liquid, but ensure it is in
tiny droplets. Ensure an even distribution of oil and volume to each
plate, since variation in agar thickness or oil distribution can lead to
conflicting nematode development between plates. If this is not an
issue, simply ensure the oil is dispersed evenly while pouring from
the flask/bottle.
Procedure
1. Use 5-cm petri dishes for this purpose.
2. Inoculate plates with Phase I bacteria isolated from the nematode strain or it can also be the species from which the female
partner in the cross is obtained.
3. Leave the plates at 25
C overnight.
4. For every individual cross, place ten virgin females and ten
males of the suitable strain or species on each of six lipid agar
plates.
5. Check plates under a microscope on a daily basis for the presence of outcross progeny and record observations.
6. After 3 days and if males have died and/or no egg development
is observed in the females, add ten more males to individual
plate to confirm that viable males are still able to fertilize the
females.
7. Continue to visualize the plates under a microscope daily for
three additional days.
Note: In successful crosses, progeny will be visible after
2–3 days.
8. Accumulate IJs after 2 weeks from these crosses.
9. Relocate to fresh lipid agar plates to allow a hybrid line to be
formed.
Morphological Characterization
85