1.?? Harvest cells from a culture which is at or near peak density by adding 3.0 ml fresh ATCC medium 5 broth to the slant and washing cells into suspension.
2. Adjust the concentration of cells to 4 x 106/ml with fresh broth medium, then dilute to half this concentration by adding an equal amount of a 10% (v/v) sterile methanol solution in fresh ATCC medium 5 broth.
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 preparation and the methanol solution, before the cooling cycle begins, should be no greater than 15 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 add to a centrifuge tube containing 5 ml of ATCC medium 5 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 fresh tube of ATCC medium 5 and incubate on a 15° horizontal slant at 50-100 μEinsteins/m2/s irradiance at 25°C with the cap loosened one half turn.? Maintain under a 14/10 h light-dark photoperiod.
