14. Chemotaxis index can alter from 1.0 (perfect attraction) to 1.0
(perfect repulsion).
5.2 Chemotaxis to
Host Volatiles [141,
154, 155]
5.2.1 Materials Required
l
Chemotaxis media plates.
l
KD scientific pump.
l
PVC (polyvinyl chloride) tube.
l
Nematodes (IJs).
l
Hamilton gastight syringes (50 ml).
l
Petri dish (9 cm diameter).
l
Media: 2% agar, 5 mM potassium phosphate (pH 6.0), 1 mM
CaCl 2 , and 1 mM MgSO 4 .
5.2.2 Procedure
1. Prepare chemotaxis media plates at room temperature at least
12 h before experiments start.
2. Fill test syringes with five alive, non-infected G. mellonella
larvae, and leave the control syringes blank.
3. Load the syringes inside a KD scientific pump.
4. Drill two 10-mm holes on each side of the lid roughly 10 mm
apart from the edges.
5. Attach PVC tube into holes and onto the syringes in order to
join the two.
6. Air in the syringes travels onto the scoring circles through these
tubes that are joined to the bottom of the chemotaxis plate.
7. Place 250 μl IJs (pellet) onto the midpoint of the chemotaxis
plate.
Note: Place an altered lid on top of the plates and position
the tubing placed over its equivalent scoring circle.
8. Set the plates on a vibration-reducing stand for 1 h.
9. Score the assay with the scoring template fastened to the bottom of the chemotaxis plates.
10. Run at least three plates for every experiment at every time
point, and individual experiment consists of nine technical
replicates.
11. Run one complete experiment with unexposed IJs to the host
cuticle, while another complete experiment is conducted with
exposed IJs.
12. Determine the chemotaxis index (CI) values by counting the
number of IJs within each scoring circle, which are being
exposed to the host odors and control.
CI ¼ No. in host circle À No. in control circle/Sum of all
individuals in both circles (Fig. 2).
Chemotaxis Assay
41
(perfect repulsion).
5.2 Chemotaxis to
Host Volatiles [141,
154, 155]
5.2.1 Materials Required
l
Chemotaxis media plates.
l
KD scientific pump.
l
PVC (polyvinyl chloride) tube.
l
Nematodes (IJs).
l
Hamilton gastight syringes (50 ml).
l
Petri dish (9 cm diameter).
l
Media: 2% agar, 5 mM potassium phosphate (pH 6.0), 1 mM
CaCl 2 , and 1 mM MgSO 4 .
5.2.2 Procedure
1. Prepare chemotaxis media plates at room temperature at least
12 h before experiments start.
2. Fill test syringes with five alive, non-infected G. mellonella
larvae, and leave the control syringes blank.
3. Load the syringes inside a KD scientific pump.
4. Drill two 10-mm holes on each side of the lid roughly 10 mm
apart from the edges.
5. Attach PVC tube into holes and onto the syringes in order to
join the two.
6. Air in the syringes travels onto the scoring circles through these
tubes that are joined to the bottom of the chemotaxis plate.
7. Place 250 μl IJs (pellet) onto the midpoint of the chemotaxis
plate.
Note: Place an altered lid on top of the plates and position
the tubing placed over its equivalent scoring circle.
8. Set the plates on a vibration-reducing stand for 1 h.
9. Score the assay with the scoring template fastened to the bottom of the chemotaxis plates.
10. Run at least three plates for every experiment at every time
point, and individual experiment consists of nine technical
replicates.
11. Run one complete experiment with unexposed IJs to the host
cuticle, while another complete experiment is conducted with
exposed IJs.
12. Determine the chemotaxis index (CI) values by counting the
number of IJs within each scoring circle, which are being
exposed to the host odors and control.
CI ¼ No. in host circle À No. in control circle/Sum of all
individuals in both circles (Fig. 2).
Chemotaxis Assay
41