4.8. Preservation of bacterial cultures

Cryopreservation is a standard practice for the long-term storage of bacterial cultures.  Although a wide range of preservation methods are available, the protocol described here is sufficient for general strain archival purposes.

  1. Streak out the strain on a plate and incubate for 1 day or until it is dense enough to harvest.  Liquid media may also be used, if the strain is able to grow in it.
  2. Prepare the cryoprotectant:  Sterilize a 30% (v/v) glycerol solution by autoclaving at 121°C for 15-20 min.
  3. Harvest the culture and resuspend in 500 µl of suitable liquid media (ex. for Snodgrassella, use trypticase soy broth).
  4. Add the resuspended culture (500 µl) and 30% glycerol (500 µl) to a 2 ml microfuge tube and mix thoroughly. Cryotubes designed for storage at low temperatures, such as Nunc Cryotubes (Sigma-Aldrich; St. Louis, MO, USA), are recommended.
  5. Store frozen at -80°C.
  6. To reactivate frozen bacteria, remove tube from freezer.  Do not thaw; keep tube on ice or dry ice, or work quickly.  Scrape a small amount of frozen culture out of the tube using a sterile tool (an inoculation loop or toothpick works well), and streak out on a fresh agar plate.  Return tube to -80°C, and incubate the plate in the optimal conditions for your strain.