Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

1.xa0 Harvest cells from a culture that is at or near peak density.xa0 Add 2-3 ml fresh ATCC medium 28 broth to each plate and wash cells into suspension.

2.xa0 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.xa0 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.xa0 Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

6.xa0xa0 Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate atxa0xa0xa0xa0xa0xa0xa0 -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately

xa0xa0xa0xa0xa0 -1°C/min.) xa0

7.xa0 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.xa0xa0 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.xa0xa0 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.xa0xa0xa0xa0xa0xa0xa0xa0xa0 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.