Protocol for Freezing Bacteria Using Glycerol

The following protocol provides an alternative to the OPS Diagnostics' Bacterial Freezing Kit. This kit is a ready-to-use product which includes 162 vials (with beads and glycerol), two cryogenic racks and inkjet/ laser labels which are resistant to solvents.

Bacteria can be frozen using a solution of 15% glycerol.  The process is simple and requires screw cap microfuge tubes and sterile glycerol.  The glycerol is diluted to 30% so that it is easy to pipette.  Equal amounts of 30% glycerol and culture broth are mixed, dispensed into tubes and then frozen.  A kit is available (Bacterial Freezing Kit) through OPS Diagnostics that simplifies the preparation of sterile tubes for this process.


Prepare a solution of 30% glycerol (v/v) by mixing 30 ml of glycerol with 70 ml of water.  Transfer the solution to a screw cap glass bottle and sterilize by autoclaving at 121°C for 15 min.  Loosen the cap during autoclaving.

Aliquot 500 µl of sterile 30% glycerol into sterile 2 ml sterile microfuge tubes containing 4 mm glass beads*.

Add 500 µl of bacterial culture to the tube and mix with the glycerol using a vortex mixer.

Label the tube with the organism name, strain, date, etc. 

Place the tube in the freezer and record its location.

To activate bacteria, only a couple of beads need to be removed from the tube.  The tube doesn't need to be thawed.  Open the tube and loosen a couple of beads using a sterile pipette tip.  Pour the loose beads onto an agar plate and roll them around.  Colonies that develop should be re-streaked.  Replace the tube into the freezer immediately.  If the culture thaws, do not re-freeze it as cells are typically very sensitive to freezing and thawing.  Discard the thawed culture appropriately.

*   We prefer tubes that are screw cap and have caps with O-rings.  One option is to fill the tube 1/3 full with 4 mm glass beads (product number BAWG 4000-200-18) before sterilizing.  When retrieving bacteria, remove or chip out one bead onto an agar plate and roll around to disperse the bacteria.  This avoids thawing the entire stock.