Scale the WormBook S-buffer freezing solution recipe for any number of cryovials and get the slow-cool freezedown protocol with recovery test plate recommendations.
Try it out
Load example celegans freezing media calculator data to see the full workflow
Standard freezedown is 4–10 vials per strain for redundancy.
Worms + freezing solution at 1:1 — half the vial is buffer.
| Reagent | Quantity |
|---|---|
| NaCl | 0.097 g |
| 1 M K-phosphate buffer (pH 6) | 0.83 mL |
| Cholesterol stock (5 mg/mL in ethanol) | 0.017 mL |
| Glycerol | 4.95 mL |
| Distilled water (to volume) | 10.71 mL |
When to use
Do not use for
A failed freeze that goes straight into the Dewar wastes a year of liquid nitrogen storage and gives false confidence. Spend the extra day to thaw and score 1 vial.
L1 and L2 larvae tolerate freezing best. Adult worms have higher mortality and produce contaminated thaws. Score the source plate before freezing.
Sharpie ink fades in liquid nitrogen vapor over months. Use a freezer-grade pen or, better, printed cryolabels with barcodes that survive long-term storage.
A single -80 °C failure can wipe out an entire stock collection. Split each freezedown into at least 2 freezers and 1 N₂ Dewar.
Total freezing solution = vials vial volume 0.5 (1:1 worm:buffer ratio) 1.1 overage. Each reagent in the WormBook S-buffer recipe (NaCl, K-phosphate buffer, cholesterol stock, glycerol, water) is scaled proportionally. Recovery test plate count = ceiling(vials / 10), minimum 1. Cholesterol volumes below 10 µL trigger a pipetting-minimum warning.
Last validated 2026-04-06. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience C. elegans Freezing Media Calculator (v0.98.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/celegans-freezing-media-calculator
Stiernagle T. Maintenance of C. elegans. WormBook. 2006. doi/10.1895/wormbook.1.101.1
Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71-94.
Filter-sterilize and store at 4 °C for up to 6 months.
1. Grow worms to a healthy starved L1/L2 stage (the most freeze-tolerant) 2. Wash 3× in M9 to remove debris 3. Mix worm pellet 1:1 with freezing solution in 1.5 mL cryovials 4. Label with strain, date, freezer location, recovery status 5. Place in styrofoam rack at -80 °C for overnight slow cooling 6. Next morning, transfer to liquid N₂ Dewar for long-term storage
Always thaw 1 test vial 24 h after the freeze to verify viability before committing the batch. Plate the test vial to a fresh OP50 plate and score motility within 1 h. Lab convention: ≥30% motile worms = successful freeze.
A C. elegans stock collection is only useful if you can find what you froze.
Each vial label should have: strain name (e.g., N2), genotype, freeze date, freezer + rack + box position, your initials, and recovery test status. A spreadsheet or LIMS that mirrors this info is essential — handwritten labels fade in liquid N₂.
Most labs run 1 active -80 °C box and 1 archival liquid N₂ Dewar per strain. The -80 °C box is for the next 1–2 thaws; the N₂ Dewar is for everything after that. Never store the only copy of anything at -80 °C.
A frozen stock is useless if you can't find the box 10 years later. Catalog every freeze with a unique ID, store the catalog off-site (Zotero, Notion, GitHub), and audit the freezer once a year against the catalog.
Catalogs every C. elegans freezedown with strain ID, freezer location, freeze date, recovery test status, and decade-scale audit reports.
Open app EquipmentSterile cryovials, glycerol, K-phosphate buffer, and cholesterol stock for C. elegans freezedowns
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