1.?? Prepare a 10% (v/v) sterile methanol solution in fresh ATCC 1194 medium.
2.?? Harvest cells from a culture which is at or near peak density.? Centrifuge at 800 x g for 5 min.
3. Adjust the concentration of cells to 2 x 106/ml in fresh methanol solution.
4.?? Mix the cell preparation in equal volume with the methanol solution.? The final concentrations will be 1 x 106 cells/ml in 5% methanol.
5.? 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.
6.? 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.) ?
?
7.?? Store ampules 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.? Shelf life must be determined empirically for storage temperatures above
-130°C.
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 add to 5 ml of ATCC medium 1194.
10.????????? Incubate on a 15° horizontal slant at 50-100 μEinsteins/m2/s irradiance at 25°C.? Maintain under a 14/10 h light-dark photoperiod.
