Freezing Bacteria Using Glycerol


 Centrifuge tube (15mL)
 Sterile cryovial
 Cryotags
 Autoclaved distilled water
 Vortex mixer
 Freezer
 Sterile pipette tips
 Glycerol (30%, v/v)


1. Prepare a solution of 30% glycerol (v/v) by mixing 30mL of glycerol with 70mL autoclaved
distilled water. Transfer the solution to a screw cap glass bottle by autoclaving at 121˚C at 15
2. Aliquot 500uL of sterile 30% glycerol into sterile 2ml sterile cryovial.
3. Add 500uL of overnight bacterial culture to the cryovial and mix with glycerol using a vortex
4. Label the tube with organism name and date.
5. Place the tube in the freezer and record its location.
6. To revive stored bacteria, only a small volume of culture needs to be removed from the tube.
The tube doesn’t need to be a thawed. Open the tube and scrape the frozen culture with a
sterile pipette tip. Replace the tube into the freezer immediately. Transfer the small volume of
frozen/thawed cells to an agar plate and streak. If the culture thaws, do not re-freeze it as cells
are typically very sensitive to freezing and thawing. Discard the thawed culture appropriately.

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