Safety Warning:
Ethyl methanesulfonate (EMS) is a potent mutagen and suspected carcinogen. Always handle EMS with nitrile gloves, use a fume hood, and dispose of all EMS-contaminated materials according to your institution’s hazardous waste guidelines. Use disposable plasticware only, and inactivate all EMS waste prior to disposal (see Step 9).

Materials Needed
• M9 buffer
• 15 mL and 50 mL plastic conical tubes
• EMS
• Clinical centrifuge
• Rocking platform
• E. coli-seeded NGM plates
• Pasteur pipettes
• Sodium thiosulfate & NaOH (for inactivation solution)

Step-by-Step Procedure

1. Prepare Worms
Grow C. elegans on 1–3 NGM plates until most animals reach the late L4 stage. Timing is critical for optimal mutagenesis.
2. Collect Worms
Wash worms off plates using M9 buffer into a 15 mL plastic conical tube.
3. Centrifuge
Spin down worms in a clinical centrifuge at high speed for 30 seconds. Carefully aspirate the supernatant—worm pellets may be loose and easily disturbed.
4. Wash Worms
Add fresh M9 to the worm pellet, invert the tube several times, and centrifuge again. Aspirate the supernatant.
5. Resuspend
Resuspend the washed worm pellet in 2 mL of M9 buffer.
6. Prepare EMS Solution (Fume Hood)
In the fume hood:
• Prepare a 0.1 M EMS stock by adding 20 µL of EMS to 2 mL of M9 in a fresh 15 mL conical tube.
• Mix gently until the oily EMS is fully dissolved.
• Immediately dispose of the EMS pipette tip into a 50 mL EMS waste tube.
7. Mutagenesis
• Add 2 mL of worm suspension to the EMS solution, achieving a final concentration of 0.05 M EMS.
• Seal the tube with Parafilm and place it on a rocker at 20°C for 4 hours.
• Keep all tubes containing EMS inside the fume hood during incubation.
8. Post-Treatment Washing & Recovery
• After incubation, wash the worms twice with fresh M9 buffer to remove residual EMS.
• Place all pipette tips and other disposable items into the 50 mL EMS waste tube.
• Transfer worms using a glass Pasteur pipette (not plastic tips, which worms stick to) to the edge of the E. coli lawn on an NGM plate.
9. Inactivate EMS Waste
Neutralize all EMS waste by mixing with an equal volume of inactivation solution:
0.1 M NaOH + 20% (w/v) Sodium Thiosulfate
Let soak for at least 24 hours before disposal. All tubes and pipettes exposed to EMS must be soaked in this solution.
10. Recovery and Selection of P0s
Allow treated worms to recover for 15–20 minutes (some protocols recommend up to 2 hours).
Pick healthy-looking late L4s as P0 animals for mutagenesis experiments.

Timing matters:
Mutagenizing too early (before germline proliferation) may result in fewer independently mutagenized genomes. Mutagenizing too late can lead to poor efficiency.

Note:
After EMS treatment, L4 identification can be tricky—animals appear starved and may lack the typical white vulval crescent. However, the internal “dot” visible near the end of L4 is still distinguishable.