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1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
2. 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 medium.
4. Mix the cell preparation and the 10% methanol in equal portions. Thus, the final concentration will be 10⁶ - 10⁷ cells/mL and 5% (v/v) Methanol. The time from the 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 should not be stored above -55°C.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just 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 add to a centrifuge tube containing 5 mL of ATCC medium 5 (or ATCC medium 847) without agar. Centrifuge at 300 x g for 5 min.
10. Remove most of the supernatant (=methanol, which can inhibit growth) and then resuspend the pellet. Transfer the culture to a 16 x 125 mm screw-capped test tube containing 5 mL of ATCC medium 5 broth (or ATCC medium 847 broth), or to the surface of an ATCC medium 5 agar plate (20 x 100 mm Petri plate containing 20 mL of ATCC medium 5 agar). (Alternately, an ATCC medium 847 agar plate can be used; this is a 20 x 100 mm Petri plate containing 20 mL of ATCC medium 847 agar.)
11. Incubate the culture at 50-100 µEinsteins/m²/s irradiance at 25°C. Maintain under a 14/10h light-dark photoperiod.

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