Jan 02, 2025

Public workspaceC. elegans culture and maintenance

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8y6wl5r/v1
  • 1Arcadia Science
  • Arcadia Science
Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8y6wl5r/v1
Protocol Citation: Justin Donnelly 2025. C. elegans culture and maintenance. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8y6wl5r/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 22, 2024
Last Modified: March 13, 2025
Protocol Integer ID: 106301
Keywords: C. elegans, cryopreservation
Abstract
This protocol describes the maintenance of C. elegans WT and homozygous strains, as well as a procedure for cryopreservation and revival of this organism.
Materials
6 cm Petri dishes with NGM(OP50)
Spatula
Platinum wire
1X trehalose freezing buffer
Cryo-tubes
ReagentEthanol 200 ProofDecon LabsCatalog #2716

Protocol materials
ReagentEthanol 200 ProofDecon LabsCatalog #2716
ReagentEthanol 200 ProofDecon LabsCatalog #2716
C. elegans culture protocol (WT and homozygous mutants)
C. elegans culture protocol (WT and homozygous mutants)
Grow worms in Petri dishes on nematode growth medium (NGM) with OP50 E. coli at Temperature20 °C .

For optimal health, subcultivate worms every 2–4 days.
Subcultivate via chunking
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.
Note
If worms are dauer, you can use this same procedure to revive them from the dauer plate.

Subcultivate via picking
Flame-sterilize a platinum wire. Pick up one or more adult worms from plate, taking care to prevent puncturing the NGM. Transfer worms by gently touching the platinum wire to the new plate under flame. Hold the wire in this position for several seconds to allow worms to crawl off. Flame-sterilize the wire before picking additional worms. Repeat 1–4 times, depending on the desired density.

Return worms to incubator.
Freezing & thawing C. elegans
Freezing & thawing C. elegans
3m
3m
Prepare synchronized L1s via bleach synchronization. See:
Protocol
Bleach life-stage synchronization of C. elegans 
NAME

Bleach life-stage synchronization of C. elegans 

CREATED BY
Arcadia Science

Centrifuge on clinical centrifuge at speed 5 for Duration00:01:00 –Duration00:02:00 .
3m
Resuspend worms in Amount15 mL 1× trehalose freezing buffer. Centrifuge again on clinical centrifuge at speed 5 for Duration00:01:00 –Duration00:02:00 .

3m
Resuspend worms in Amount3.6 mL 1× trehalose freezing buffer. Mix to ensure worms are dispersed.

Quickly after resuspension, pipette Amount1.2 mL into each of three pre-labeled cryotubes.

Freeze the worms in a Mr. Frosty (or similar) at Temperature-80 °C . Keep the container on its side so that worms don't all sink to the bottom before freezing.

After several days, retrieve the worms and run a test thaw by scraping some amount of frozen worm and buffer, and transfer to a fresh NGM (OP50) plate.
Over the following several days, look to see if any worms have revived. If so, you may remove cryotubes from the Mr. Frosty and transfer to long-term storage at Temperature-80 °C .

Once you've confirmed successful revival, you can thaw worms as in step 10.
Protocol references