C. elegans Bleach Synchronization Timing Calculator

Standard Alkaline Hypochlorite Bleach Protocol

Adult C. elegans cuticles dissolve in alkaline hypochlorite within 5–8 minutes, but eggshells (chitin) survive intact. This gives you a clean cohort of eggs that hatch synchronously as L1s — the foundation of any age-matched experiment.

• 4 mL distilled water
• 1.5 mL household bleach (5–6% sodium hypochlorite)
• 0.5 mL 5N NaOH
• = 6 mL total alkaline hypochlorite solution

Always make this fresh — the chlorine activity drops fast once NaOH is added.

1. Wash gravid plates into a 15 mL conical with M9 buffer 2. Spin at 1,500 $\times$ g for 1 min, aspirate supernatant 3. Add 6 mL fresh bleach solution, vortex briefly 4. Watch under a scope — adults dissolve in 5–8 min, leaving eggs intact 5. Spin, aspirate, wash 3× with sterile M9 6. Resuspend in 5 mL M9 in a fresh conical and rotate overnight at 20 °C 7. Eggs hatch as starvation-arrested L1s in 12–16 hours

Picking Synchronized Stages from a Bleach

After hatching as L1 in M9, the cohort develops on OP50-seeded NGM plates with predictable timing. Use the calculator to schedule pickups.

Keep eggs in M9 with no food for 12–16 h at 20 °C. Worms hatch and arrest at L1 — the most synchronized timepoint you can get. Plate to OP50 to release the arrest and start development.

At 20 °C, L4s appear ~42 h after bleach start. The vulval crescent is unmistakable under a dissecting scope. Pick L4s for: lifespan setup, RNAi feeding plates, synchronized aging cohorts, mating crosses.

At 20 °C, gravid young adults appear ~54 h after bleach start. Pick YAs before egg laying begins (no embryos in the gonad) for: brood size assays, fecundity scoring, single-worm sorting.

• 15 °C: 1.6× slower, useful when you need to slow a cohort to fit your schedule
• 20 °C: standard, baseline for all published timing
• 25 °C: 0.65× faster, useful for quick experiments but stresses some mutants