1.? Harvest cells from a culture that is at or near peak density.? Add 2-3 ml fresh ATCC medium 28 broth to each plate and wash cells into suspension.
2.? Collect cells by centrifugation at 800 x g for 5 min. Adjust the concentration of cells to 2 x 106 - 2 x 107/ml in fresh medium.
3.? While cells are centrifuging prepare a 10% (v/v) solution of sterile methanol in fresh broth medium.
4. Mix the cell preparation and the 10% methanol solution in equal portions. Thus, the final concentration will be 106 - 107 cells/ml and 5% (v/v) Methanol. The time from mixing of the cell preparation and methanol stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 min.
5.? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6.?? Place the vials in a controlled rate freezing unit.? From room temperature cool at -1°C/min to -40°C.? If the freezing unit can compensate for the heat of fusion, maintain rate at??????? -1°C/min through the heat of fusion.? At -40°C plunge 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.) ?
7.? 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.
8.?? 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.
9.?? Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and transfer to a test tube containing 5 ml of ATCC medium 28 broth or to the surface of an ATCC medium 28 agar plate.
10.????????? Incubate a test tube culture upright at 25°C with the cap screwed on loosely (loosened one-half turn); incubate a plate upright at 25°C. Subculture every 4-6 weeks when incubated at 25C, or every 6-12 months when incubated at 18C.
