C. elegans Freezing Media Calculator

Standard C. elegans Freezedown Protocol

• 5.85 g NaCl
• 50 mL 1 M K-phosphate buffer (pH 6)
• 1 mL cholesterol stock (5 mg/mL in ethanol)
• 300 mL glycerol
• Distilled water to 1 L

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.

Storage & Catalog Logistics

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.