Preparing a Glycerol Stock for Long-term Strain Storage

Cryopreservation method for bacterial strains using glycerol as a cryoprotectant, producing indefinitely-storable frozen stocks at −80°C. Critical for preserving verified transformant lineages so that every future reagent-production batch starts from identical genetic material rather than fresh unverified transformants. Written for a novice-to-intermediate audience.

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Version History

Version 0.1.1 Viewing Latest

Effective: 2026-04-20

All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.

Version 0.1.0

Effective: 2026-04-20

Initial release. Atomic technique supporting procedure 59 (OpenTaq Cellular Reagent Production) and any future strain-handling workflow. Procedural content written from standard microbiology practice.

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Procedure Details

Safety & Hazards

PC1 / CL1 containment required for GMO strains.
BSL-1 host (for standard lab E. coli strains). Autoclave all liquid and solid culture waste.
Glycerol is non-toxic at the concentrations used here but is a skin irritant in pure form. Gloves when preparing the 50% stock.
Liquid nitrogen (optional snap-freeze step). If used: insulated gloves, face shield, well-ventilated room. Never in a sealed container.
−80°C freezer. Avoid prolonged skin contact with stored vials. Label everything — frostbitten eyes can't read faded Sharpie.

Preparation Notes

What you need ready before starting:

Saturated overnight culture of the strain you want to preserve, in selective liquid media, grown 14–16 h. Typical starting point: pick a single well-isolated colony from a fresh transformation plate into 5 mL selective media and incubate at 37°C, 225 rpm overnight.
Sterile 50% (v/v) glycerol in water. Prepare by mixing 50 mL glycerol + 50 mL water, autoclaving 121°C × 20 min liquid cycle, and cooling. Store at room temperature; stable for months.
Sterile cryovials (2 mL screw-cap, with internal thread if available — external thread risks contamination during removal). Pre-label with: strain identifier + plasmid + date + your initials + batch lot number. Use a cryo-safe marker (Sharpie industrial, not normal Sharpie — fades at −80°C).
Ice bucket or dry-ice / liquid nitrogen dewar for snap freezing (optional but recommended).
Biosafety cabinet with fresh alcohol spray.

Mental model: glycerol prevents ice crystals from physically destroying cell membranes during freezing. Slow freezing = big crystals = dead cells; fast freezing + cryoprotectant = small crystals = viable cells. You can revive a glycerol stock years later (10+ years documented in the literature) if the freezer held its temperature.

Timing

Overnight (hands-off): grow the overnight culture.
Prep (5 min): label vials, set up workspace.
Mixing (2 min per stock): combine culture and glycerol.
Freezing (2 min): snap-freeze or direct placement at −80°C.
Total hands-on time: ~15 min for a batch of 5–10 stocks.

Equipment (Catalog) 5

Micropipette Liquid handling
Specs: P200, P1000
Filter tips
Vortex mixer Mixing
Specs: Standard
For mixing glycerol/water stock only — NOT for mixing culture/glycerol
Biosafety cabinet Safety
Specs: Class II Type A2 or equivalent
All culture handling
Freezer (−80 °C) Storage
Specs: Ultra-low temperature freezer
Long-term storage
Shaking incubator Thermal regulation
Specs: 37°C, 225 rpm
For overnight culture growth

Materials (Catalog) 3

E. coli BL21(DE3) (NEB) C2527I Bacterial strain
Qty: 500 µL saturated culture per vial
Or any transformant strain you wish to preserve
Ampicillin Antibiotic
Qty: 100 µg/mL final
Match plasmid's resistance marker
Superior Broth (Athena Enzyme Systems) 0105 Growth media
Qty: 5 mL for overnight
Use the same selective media as the plate the colony came from

Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns

Name	Type	Required	Default	Unit	Description
`final_glycerol_concentration_pct`	number	—	`25`	—	Final glycerol concentration in the cryovial. 15–50% all work; 25% is standard. Higher glycerol reduces osmotic damage but increases viscosity.
`freezing_method`	text	—	`snap_freeze_ln2`	—	Method for initial freeze: 'snap_freeze_ln2' (liquid nitrogen, best), 'snap_freeze_dry_ice' (good), or 'direct_minus80' (acceptable).
`storage_temp_c`	number	—	`-80`	degree Celsius (UO:0000027)	Long-term storage temperature. −80°C standard; −20°C reduces viability over weeks-to-months.
`vials_per_strain`	number	—	`5`	—	Redundancy factor. Minimum 5 vials: 1 primary archive + 4+ working stocks.

Procedure Steps (Version 0.1.1)

Verify your overnight culture is saturated and free of contamination. Visual check: uniform cloudiness, no film or flakes on the surface, no pigmented colonies (white E. coli becomes yellow/pink if contaminated with Pseudomonas or Serratia respectively).

In the biosafety cabinet, arrange labelled cryovials in a rack in the order you will fill them.

Verify your 50% glycerol stock is sterile and at room temperature. If previously autoclaved and stored, inspect briefly for cloudiness (should be clear).

For each cryovial: pipette 500 µL of the saturated overnight culture into the vial using a filter tip.

Pipette 500 µL of sterile 50% glycerol into the same vial. This brings the final glycerol concentration to 25% — suitable for long-term −80°C storage.

Cap the vial firmly. Invert gently 5–6 times to mix. Do NOT vortex — shear stress from vortex damages cells before freezing and reduces revival viability.

Optional but recommended: snap-freeze by placing vials into liquid nitrogen for 30 s, or into a dry-ice ethanol/isopropanol bath for 1 min. Faster freezing = smaller ice crystals = higher viability.

Transfer frozen vials to the −80°C freezer. Place in the designated storage box/rack for your strain archive.

Record the Sample in LibreBiotech: one Sample record per vial, with annotations for lot number, strain, plasmid, production date, storage box/rack/position, glycerol concentration, and any QC data.

If possible, perform viability QC: after 24 hours at −80°C, revive one vial by scraping a small quantity onto a selection plate; record colony yield as a Sample annotation.

Prepare redundant stocks. Make at least 5 vials per strain — one 'primary archive' never to be opened except to make secondary working stocks, and 4+ secondary working stocks for daily use.

Completion Notes

Expected outcome. Cryovials of cloudy frozen culture at −80°C. Final glycerol concentration 15–25% in the vial (depending on culture density and glycerol stock concentration). Viability on revival: >99% for routine lab strains, often >10 years shelf life at −80°C.

Storage location. −80°C freezer, in a labelled box. Record the box / rack / position in the LibreBiotech Sample record's storage annotation so anyone on the team can find it.

Reviving a glycerol stock. Do NOT thaw. Scrape a small quantity of frozen culture with a sterile pipette tip or inoculation loop directly into fresh selective liquid media OR streak onto a selective plate. Return the stock to −80°C immediately — repeated freeze-thaw reduces viability over generations.

Batch records. Each glycerol stock lot should have:

Sample record in LibreBiotech for each vial (lot number, contents, storage location, production date).
Process record linking back to the originating colony / transformation.
Viability QC annotation after revival (record how many colonies are seen on revival from 1:1000 dilution).

Troubleshooting.

Symptom	Likely cause	Fix
Vial has no colonies on revival	Stock glycerol not sterile or culture was dead at time of freezing	Restream from original transformation plate; regenerate glycerol stock from a fresh overnight
Contaminant colonies on revival	Non-sterile vials or work surface	Remake with freshly autoclaved vials and stricter biosafety cabinet hygiene
Slow growth on revival	Frost damage from repeated freeze-thaw	Prepare redundant stocks; never thaw the primary archive vial; work from a secondary "working stock"

References

Hubálek Z (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology 46(3):205–29. DOI paper
Standard microbiology practice; see standard molecular biology references (e.g. Green & Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed, CSHL Press 2012). book