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Levamisole is a synthetic anthelmintic drug extensively employed in veterinary medicine to treat parasitic infections caused by nematodes. As an anthelmintic, it targets the nematode acetylcholine ion-channel receptors (Martin et al). Besides its medical applications, Levamisole also serves as a widely used reagent in various research fields, particularly in C. elegans paralysis assays. Its ability to induce paralysis in these nematodes makes it invaluable for studying neuronal function and muscle physiology. To provide insight into toxicological experimentation, we present an example below utilizing this compound. Researchers can use this example as a framework to conduct their own toxicity studies using different soluble drugs.


  • 6 well plate with lid.
  • NGM.
  • M9 buffer.
  • Levamisole – Stock solution: 100mM in ddH20.
  • Synchronized populations of young adult worms. 
  • wMicrotracker Arena device.

Protocol Brief:

Microplate preparation: 

  • NGM was prepared following standard procedure. 
  • Levamisole is added to the 55°C NGM agar solution immediately after the addition of the salts and cholesterol
  • Levamisole plates were not seeded with OP50 
  • Plates can be used within two weeks after prepared when stored in a container at 4 °C.
  • The assay is performed on plates that had been allowed to template to room temperature prior to the start of the experiment.

Step by Step PROTOCOL: 

1. Grow synchronized populations of adult worms in seeding NGM plates (OP50).

2. Remove worms from plates using M9 buffer and transfer them in a sterile 15 ml tube. 

3. Let the worms settle. Decant the supernatant taking care not to disturb the pellet. 

4. Perform a wash with 5 ml of M9 buffer. Briefly shake or invert the tube

5. Repeat the decantation step. Throw out the supernatant and add 3 ml of M9 buffer. 

6. Count number of worms in 10 µl in triplicate and calculate the average. 

7. Prepare a suspension to get [35 worms/10 µl]

8. Transfer 10 µl of worm solution to 6-well plate. 

10. Register worm activity using wMicroTracker ARENA

Note: Perform at least three technical replicates and at least two biological replicate.


Dose-inhibition response curve of levamisole on N2 locomotor activity. The effect of different concentration of levamisole (0µM (control),100µM and 200µM) was evaluated. Thirty-five young adult N2 were picked per well in a 6-well plate. The locomotor activity was recorded at 23ºC during 4 hours. Data represents the mean of four independent experiments conducted in duplicate.