Harvest and Preservation

1. Harvest cells from cultures which are at or near peak density.  Aseptically transfer cells to 15 mL plastic centrifuge tubes and centrifuge at ~150 x g for 5 min.
2. Adjust the concentration of cells to 2 x 10⁶/mL with fresh medium, then dilute to half this concentration by adding an equal amount of a 15% (v/v) sterile glycerol solution in fresh  medium.
3. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).  The time from mixing of the cell suspension and the glycerol solution, before the cooling cycle begins, should be no less than 15 min and no greater than 30 min.
4. Place vials in a controlled-rate freezing unit. From room temperature, cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.  (The cooling rate in this apparatus is approximately -1°C/min.)   
5. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials can be stored between -80 and -70°C for no longer than one week.
6. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
7. Immediately after thawing, do not leave in the water bath; aseptically remove the contents of the ampule and transfer to a fresh test tube (or T-25 flask) containing 5 mL of ATCC Medium 460. Incubate culture at 20-25°C (test tubes are incubated upright with the cap loosened one-half turn).  Subculture every 10-14d.