Ring-based Aversion Assay
- Unseeded NGM plates
- Cue and diluent (e.g., diacetyl or isoamyl alcohol and ethanol)
- Aversion compounds (water, 4M d-fructose, test compound)
- Copper rings
- Lids of 6 cm plates
- Fast green
- Bleach synchronize worm strains 4 days before the assay.
Wash and prepare worms:
a. Wash worms from 10 cm NGM plates into 1.5 mL eppendorf tubes using 1 mL of M9, maintaining seperate strains per tube.
b. Allow worms to settle for 2 min.
c. Remove supernatant without disturbing the worm pellet.
d. Add 1 mL of M9 and invert tube to wash worms.
e. Repeat b-d two more times, for a total of three M9 washes.
While waiting for worms to settle during washes, prepare the aversion compounds, cue and diluent.
a. All compounds (water, Fructose and test compound) are made with M9 + fast green solution.
b. Spray 3 copper rings with 70% EtOH and flame the copper rings. Allow rings to cool at RT.
Note: Ensure no burnt flakes are on the ring. If there are observable flakes, use a kimwip and 70% EtOH to remove burnt materials. Re-flame after cleaning.
c. Pour 1-2 mL of compound into the lid of a 6 cm plate.
d. Place a cooled copper ring into the compound.
Assemble the first plate:
a. Using forceps pick up the copper ring out of the compound and place it on to the labeled unseeded NGM plate.
Note: You may need to use a pipette tip to pop a bubble in the center of the ring before placing it onto the NGM assay plate.
b. After 30 sec., carefully remove the copper ring and place it back into the compound. There should be a distinct green ring on the plate.
c. Add 1 µl of 1:1000 diluted diacetyl out side of the ring along with 1 µL fast green.
d. Use the plate immediately after making it or the compounds will absorb into the agar and no effect will be observable.
Pipette worms onto a different unseeded 6cm NGM plate.
a. Pick 1 worm without OP50 to the center of the fast green stained compound ring.
b. The plate can only be used for one worm.
Watch the worm under a microscope until it either:
a. Crosses the green ring.
b. Makes 6 attempts but does not cross the ring (exhibits reversal behavior).
c. 10 min. passes without either (a) or (b) occurring.
Record the number of attempts the worm makes to cross the ring.
Repeat steps 3-7 for at least 3 worms per strains for each compound.