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.xa0xa0 Prepare a 10% (v/v) sterile methanol solution in fresh ATCC 1194 medium.

2.xa0xa0 Harvest cells from a culture which is at or near peak density.xa0 Centrifuge at 800 x g for 5 min.

3. Adjust the concentration of cells to 2 x 10⁶/ml in fresh methanol solution.

4.xa0xa0 Mix the cell preparation in equal volume with the methanol solution.xa0 The final concentrations will be 1 x 10⁶ cells/ml in 5% methanol.

5.xa0 Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).xa0 The time from mixing of the cell preparation and the methanol solution, before the cooling cycle begins, should be no greater than 15 min.

6.xa0 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.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 -1°C/min.) xa0

xa0

7.xa0xa0 Store ampules in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below

xa0xa0xa0xa0 -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated.xa0 Shelf life must be determined empirically for storage temperatures above

-130°C.

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 add to 5 ml of ATCC medium 1194.

10.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Incubate on a 15° horizontal slant at 50-100 µEinsteins/m²/s irradiance at 25°C.xa0 Maintain under a 14/10 h light-dark photoperiod.