Jan 03, 2025

Public workspaceBleach life-stage synchronization of C. elegans 

DOI
dx.doi.org/10.17504/protocols.io.rm7vzj91xlx1/v1
  • 1Arcadia Science
  • Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzj91xlx1/v1
Protocol Citation: Justin Donnelly 2025. Bleach life-stage synchronization of C. elegans . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj91xlx1/v1
Manuscript citation:
Avasthi P, Borges AL, Cheveralls K, Donnelly J, Mets DG, Reiter T. (2025). An experimental and computational workflow to characterize nematode motility behavior. https://doi.org/10.57844/arcadia-b89a-7e76
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 23, 2024
Last Modified: March 14, 2025
Protocol Integer ID: 106302
Keywords: C. elegans, bleach, synchronization, life-stage, L1, nematode
Abstract
This protocol describes the procedure for synchronizing the life stage of C. elegans worms by bleaching gravid adults so that they release their eggs, which have greater resistance to bleach than hatched worms. This procedure can be useful for eliminating the variable of life stage from downstream experiments, and is also necessary for cryopreservation of this organism.
Materials
ReagentPotassium hydroxideP212121 ReagentEthanol 200 ProofDecon LabsCatalog #2716
Concentrated bleach
Autoclaved ultrapure water
M9 buffer
15 mL centrifuge tubes
NGM(OP50) plates
Clinical centrifuge
Tube rocker for 15 mL centrifuge tubes
Dissection scope
Protocol materials
ReagentPotassium hydroxideP212121
ReagentPotassium hydroxideP212121
ReagentEthanol 200 ProofDecon LabsCatalog #2716
ReagentEthanol 200 ProofDecon LabsCatalog #2716
Preparation of plates
Preparation of plates
Chunk worms onto at least six NGM (OP50) plates: Dip a spatula in 100% ReagentEthanol 200 ProofDecon LabsCatalog #2716 . Flame-sterilize. Once the flame is extinguished, cut a chunk from the plate with an edge length of ~Amount0.5 cm . Select a chunk with several larvae. Transfer this chunk to a new plate under flame, with the worm side facing down.

Incubate worms for 3–5 days, until there are many gravid adults.
Note
Synchronization works best on young adults, which still have many fertilized eggs. The older adults become, the fewer L1s there will be at the end of the protocol.

Synchronization via alkaline bleach treatment
Synchronization via alkaline bleach treatment
12m
12m
Under a flame, add Amount0.5-1 mL autoclaved water to a plate. Swirl and tilt plate to ensure that water passes over the entirety of the plate, then use a pipette to transfer the water to a sterile 15 mL centrifuge tube. This process should remove the vast majority of worms from the plate.

Repeat step 3 with remaining plates. The total volume should be below Amount8 mL .

Once you've transferred the worms, add autoclaved water to the conical tube to a total volume of Amount8 mL . If synchronizing multiple strains, synchronize one at a time.

Add Amount1.2 mL concentrated bleach and Amount0.6 mL Concentration5 Molarity (M) ReagentPotassium hydroxideP212121 . Mix solution by gently inverting.

Place the tube on a rocker. Monitor under a dissecting microscope to observe lysis of adults and release of fertilized eggs. This process should take Duration00:04:00 –Duration00:08:00 .
Note
It's important that you don't over-bleach the sample. Extended exposure to alkaline-bleach conditions can damage eggs and prevent hatching. In our hands, our most successful bleach treatments were under 5 minutes. For best results, bias to incomplete lysis over excessive lysis.

12m
Fill tube to Amount15 mL with autoclaved water.

Centrifuge in a swinging bucket centrifuge at Centrifigation768.6 x g for Duration00:01:00 –Duration00:02:00 .

3m
Decant supernatant. Perform two additional washes with Amount15 mL autoclaved water.

Decant supernatant. Perform a third wash with Amount15 mL M9 buffer.

Resuspend worms in Amount1-2 mL M9 buffer. Place on rocker DurationOvernight at Temperature20 °C to allow worms to hatch into L1s. Worms can remain on rocker for an additional day without negative effects.

8m
Plating and counting of L1s
Plating and counting of L1s
Optional: To estimate the concentration of L1s, plate several volumes (e.g., Amount5 µL , Amount10 µL , Amount20 µL ) on plates and count the number of L1 larvae.

Under a flame, plate desired quantity of worm larvae on desired number of NGM (OP50) plates by pipetting appropriate volume of buffer onto the surface of the plate. Allow buffer to evaporate before returning to incubator.
Protocol references