C. elegans bleach synchronization.

Materials

• M9
• 15 mL conical tubes
• Bleaching solution — see C. elegans Recipes
• K medium or S medium, depending on application — see C. elegans Recipes
• Glass slide, if titering

Protocol

1. Grow up the desired number of 10 cm plates for bleaching. You want the maximal number of gravid adults. Each 10 cm plate yields approximately 5000 embryos, though strains vary significantly. It is easier to generate more 10 cm plates than you think you will need.

   • Option 1: chunk to 10 cm plates and monitor for ~2–3 days until you see mostly gravid adults.
   • Option 2: pick 30–50 L4s to 10 cm plates and monitor for ~4–5 days until you see mostly gravid adults.

2. Wash worms off plates using a sterile glass pipette into a 15 mL conical tube. You can squirt M9 onto one plate of strain A, transfer the liquid to another plate of strain A, and so on. Wash all plates from a single strain into one tube. With a little practice it is easy to pour from the final 10 cm plate directly into the conical.

3. Spin down worms at 1100 rpm for 1 minute using the table-top clinical centrifuge.

4. Aspirate off the M9, being careful not to suck up any worms.

5. Add 6 mL of bleaching solution to each 15 mL conical. Shake the tubes manually, use a nutator, or use the plate vortex if you have many strains.

   • After four minutes of shaking, check every minute to see if the adult worms are dissolved.
   • Be very careful not to over-bleach. Once the carcasses are nearly dissolved, move to the next step. If only embryos are left, you have gone too long.

   <aside> ⏱️

   The freshness of the bleach solution matters. Ineffective bleaching solution — kept at 4°C longer than one month, or at room temperature longer than eight hours — will require more time and will not dissolve the worms uniformly.

   </aside>

6. Once there are just a few adults remaining, move quickly to spin down the embryos at 1100 rpm for 30 seconds. It is best to have the reagents next to the sink and prepared for the next few steps.

7. Immediately decant off the bleach into the sink.

8. Add 10 mL of M9 using a sterile glass pipette to each 15 mL conical tube.

9. Invert three times as you walk back to the clinical centrifuge.

10. Spin down the embryos at 1100 rpm for 30 seconds.

11. Aspirate off the M9.

12. Repeat steps 8–11 two more times.