Note: Nematodes can be selected and transferred from a suspension by using a fine pipette or suction device to select individual
specimens.
4.1.2 Preparation
of Nematode Mounts
1. Killing and fixing the nematodes.
2. Processing nematodes to glycerin.
3. Mounting the nematodes.
Step 1: Killing and Fixing
of Nematodes
Purpose: For identification to the species level and permanent storage, nematodes must first be heat killed, fixed, and properly
mounted. Nematodes are either killed in hot water or fixed in
cold fixatives or killed and fixed in hot fixatives.
Materials Required
l
Nematodes suspension.
l
26–76 mm glass slide.
l
Hot plate.
l
Water.
Procedure
1. Kill a few specimens by relocating them to a water drop onto a
26–76 mm glass slide.
2. Heat the slide for a few seconds on a flame until the nematodes
abruptly flatten out.
3. Killing by placing the slide on a well-controlled hot plate at
65–70
C is highly successful and prevents injury to specimens
due to overheating.
4. Examine the specimen directly under the microscope.
5. In most cases, it will be transferred to fixative.
Another Method of Killing
Nematodes
Materials Required
l
Nematodes suspension.
l
Ringer’s solution.
l
Water bath.
l
Ringer’s solution (laboratory standard).
– Sodium chloride (NaCl): 9.00 g.
– Potassium chloride (KCl): 0.42 g.
– Calcium chloride dehydrate (CaCl 2 ∙2H 2 O): 0.37 g.
– Sodium bicarbonate (NaHCO3): 0.20 g.
– Distilled water: 1000 ml.
Procedure
1. Add live nematodes to a glass vial.
2. Enable them to set themselves at the bottom.
3. Leave around 2 ml water for them and remove the excess water.
70
Laboratory Techniques for Entomopathogenic Nematodes