Step 2: Processing
Nematodes to Glycerin
[166]
Materials Required
l
5% glycerin.
l
Distilled water.
l
Incubator/desiccator.
Procedure
1. Take as much fixative as conceivable without losing nematodes,
dispense it into a solution with 5% glycerin in distilled water
contained in a vial up to 5 mm high or in a cavity block up to
the brim.
2. Incubate the vial or block at 35–40
C and keep it concealed
with a small opening for slow evaporation.
3. Allow it to stay until an extensive amount of water has
evaporated.
4. Replenish with 5% glycerin, and then leave the vial for least two
more days in the incubator. Ensure that all the water has
evaporated.
5. Continuously monitor the amount of dehydration by shifting
two or three specimens to a pure glycerin drop.
6. Their cuticle will collapse, if they are not completely dehydrated. Then return the contents to the incubator for one
more day or two.
7. The whole TAF evaporation technique completes in 4–5 days
in an incubator or desiccator and results in completely fixed
nematodes.
Step 3: Mounting
Nematodes
Materials Required
l
Concave glass slide.
l
Nematode pick.
l
Glass wool (5 mm in length).
l
Cover glass (18 mm wide).
l
Ethanol.
l
Glycerol.
l
Distilled water.
l
Forceps/needle.
l
Filter paper.
l
Nail polish.
l
Small brush.
l
Dissecting microscope.
l
Phase contrast microscope.
Procedure
1. Dispense a little of the fixative in the midpoint of a clean glass
slide.
72
Laboratory Techniques for Entomopathogenic Nematodes
Nematodes to Glycerin
[166]
Materials Required
l
5% glycerin.
l
Distilled water.
l
Incubator/desiccator.
Procedure
1. Take as much fixative as conceivable without losing nematodes,
dispense it into a solution with 5% glycerin in distilled water
contained in a vial up to 5 mm high or in a cavity block up to
the brim.
2. Incubate the vial or block at 35–40
C and keep it concealed
with a small opening for slow evaporation.
3. Allow it to stay until an extensive amount of water has
evaporated.
4. Replenish with 5% glycerin, and then leave the vial for least two
more days in the incubator. Ensure that all the water has
evaporated.
5. Continuously monitor the amount of dehydration by shifting
two or three specimens to a pure glycerin drop.
6. Their cuticle will collapse, if they are not completely dehydrated. Then return the contents to the incubator for one
more day or two.
7. The whole TAF evaporation technique completes in 4–5 days
in an incubator or desiccator and results in completely fixed
nematodes.
Step 3: Mounting
Nematodes
Materials Required
l
Concave glass slide.
l
Nematode pick.
l
Glass wool (5 mm in length).
l
Cover glass (18 mm wide).
l
Ethanol.
l
Glycerol.
l
Distilled water.
l
Forceps/needle.
l
Filter paper.
l
Nail polish.
l
Small brush.
l
Dissecting microscope.
l
Phase contrast microscope.
Procedure
1. Dispense a little of the fixative in the midpoint of a clean glass
slide.
72
Laboratory Techniques for Entomopathogenic Nematodes