1.?? Harvest cells from a culture which is at or near peak density by adding 3.0-5.0 ml fresh ATCC medium 28 broth to the slant or plate and washing cells into suspension.? It may be helpful to rub the surface of the agar with a spread bar or inoculating loop to detach adhering cells.
2.?? Adjust the concentration of cells to 2 x 107/ml with fresh broth medium, then dilute to half this concentration by adding an equal amount of a 20% (v/v) sterile solution of either DMSO or glycerol in fresh ATCC medium 28 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 cryoprotective solution to the start of the cooling cycle should be no less than 15 min and no greater than 30 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 until thawed (2-3 min). Immerse the ampule enough to cover only the frozen material.? Do not agitate the ampule.
7.?? Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and add to
a fresh slant of ATCC medium 28 or the surface of an agar plate of ATCC medium 28.
8.?? Maintain as described above.
