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Microbiology

Bacterial glycerol stocks

Store bacterial strains long term at −80 °C in glycerol and revive them without thawing the stock.

Total time10 min
Hands-on10 min
LevelBasic
Steps4
0 of 4 steps done

Before you start

  • Use a fresh overnight culture grown from a single colony with selection.

Reagents and materials

Totals are for 4 stocks (+10% extra). Per-stock amounts are in the middle column.

ReagentPer stockTotal
Overnight culture500 µL500 µL
Sterile 50% glycerol25% final500 µL500 µL
Cryovial (screw cap)1 each1 each

Buffers and recipes

50% glycerol per 100 mL

Glycerol50 mL
Ultrapure water50 mL

Autoclave or filter-sterilise.

Equipment

  • Cryovials
  • −80 °C freezer
  • Freezer box

Procedure

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

  1. Mix

    Combine 500 µL culture with 500 µL sterile 50% glycerol in a cryovial and mix by inversion (15–25% final glycerol is common).

  2. Label

    Label with strain, plasmid, resistance, date and initials.

  3. Freeze

    Store at −80 °C (flash-freezing is optional).

  4. Revive

    Scrape a little frozen material with a sterile tip or loop and streak on a plate — do not thaw the stock.

Critical points

  • Keep a record (database) of all stocks and their locations; make duplicates of important strains.

Troubleshooting

ProblemWhat to check or change
No growth from stockUse more material; check that glycerol was added; avoid freeze–thaw cycles.

Safety

Work at the organism’s biosafety level.

Further reading

  • Addgene protocol: creating bacterial glycerol stocks.

This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.