Materials

• Unseeded NGM plates
• Cue and diluent (e.g. diacetyl or isoamyl alcohol, and ethanol)
• Aversion compounds (water, 4 M D-fructose, test compound)
• Copper rings
• Lids of 6 cm plates
• Fast green
• M9
• Forceps

Advance preparation

Bleach synchronize worm strains 4 days before the assay.

Protocol

1. Wash and prepare worms:

   • Wash worms from 10 cm NGM plates into 1.5 mL Eppendorf tubes using 1 mL of M9, keeping strains in separate tubes.
   • Allow worms to settle for 2 minutes.
   • Remove the supernatant without disturbing the worm pellet.
   • Add 1 mL of M9 and invert the tube to wash.
   • Repeat twice more, for a total of three M9 washes.

2. While waiting for worms to settle during washes, prepare the aversion compounds, cue and diluent.

   • All compounds — water, fructose and test compound — are made with M9 + fast green solution.
   • Spray 3 copper rings with 70% EtOH and flame them. Allow the rings to cool at room temperature.
   • Pour 1–2 mL of compound into the lid of a 6 cm plate.
   • Place a cooled copper ring into the compound.

   <aside> ⚠️

   Ensure no burnt flakes are on the ring. If there are visible flakes, use a Kimwipe and 70% EtOH to remove them, then re-flame after cleaning.

   </aside>

3. Assemble the first plate:

   • Using forceps, pick the copper ring out of the compound and place it onto the labeled unseeded NGM plate. You may need to use a pipette tip to pop a bubble in the centre of the ring before placing it.
   • After 30 seconds, carefully remove the copper ring and place it back into the compound. There should be a distinct green ring on the plate.
   • Add 1 μL of 1:1000 diluted diacetyl outside the ring, along with 1 μL fast green.
   • Use the plate immediately — otherwise the compounds will absorb into the agar and no effect will be observable.

4. Pipette worms onto a different unseeded 6 cm NGM plate.

   • Pick 1 worm without OP50 to the centre of the fast green stained compound ring.
   • Each plate can only be used for one worm.

5. Watch the worm under a microscope until it either:

   • Crosses the green ring.
   • Makes 6 attempts but does not cross the ring, exhibiting reversal behavior.
   • 10 minutes pass without either occurring.

6. Record the number of attempts the worm makes to cross the ring.

7. Repeat steps 3–6 for at least 3 worms per strain for each compound.

<aside> ❓

Corrected during migration. The source says to "repeat steps 3-7", which includes the repeat instruction itself and would loop indefinitely. Written here as steps 3–6. Confirm that is the intent.

</aside>