3.5.3. Preservation via lyophilization

(J. Swezey, USDA-ARS Culture Collection (NRRL), Peoria , IL; personal communication)

To prepare lyophilization vials:

  1. Cut Pyrex glass tubing (6 mm diameter glass with 1 mm wall thickness) 15 cm long and seal one end by melting.
  2. Plug the open ends of the vials with cotton.
  3. Autoclave.
    Note: Ampules using rubber septa and aluminium sealing rings may be used as an alternative to the above three steps. However, use of these ampules increase costs and the vacuum may be lost over time.
  4. After 2-3 days incubation of the bacterium from an agar slant/plate, wash with sterile bovine serum (e.g. Colorado Serum Co., Denver, CO).
  5. Place 0.1 ml aliquots of the cell/serum suspension into the sterilized vials, label, date.
  6. Attach the vials to a lyophilizer apparatus.
  7. Lower the vials into a solution of 50 % ethylene glycol and 50 % water (chilled to -50oC with dry ice) to freeze the cell suspensions.
  8. Turn on the lyophilizer vacuum pump.
  9. Evacuate for 3 hours while letting the temperature of the glycol/water bath gradually warm to -4oC.
  10. Lift the vials from the glycol/water bath.
  11. Allow the outer surfaces to dry for 1 hour at RT.
  12. Carefully cut the vials from the lyophilizer apparatus using a dual tip burner, running on natural gas and oxygen, to melt the upper portion of the glass vial while sealing the contents under vacuum.
  13. Store sealed vials refrigerated in the dark.