Cryoprotective Solution
DMSO ?????????????????????????????????????????????????????????????????????????????????? 1.0 ml
Fresh growth medium w/o bacteria???????????????????????????????? 9.0 ml
1.???? Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.? Allow to cool.
2. ??? Harvest cells from a culture in stationary phase (1-2 days after reaching peak density).
3.???? Gently discard most of the supernatant and vigorously agitate the flasks to detach the cells.?
4.???? Determine the cell concentration using a hemocytometer.?? Adjust the concentration to 2 x 105/ml in fresh medium.? If the concentration is too low, centrifuge at 200 x g for 5 minutes and resuspend the pellet with the supernatant to the desired volume.?
5.?? Mix the cell preparation and the cryoprotective solution in equal portions.
6.? ?? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7.? ?? 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.) ?
8.? ?? Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
9. ??? To establish a culture from the frozen place the vial in a 35°C water bath.? Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
10.? Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate the entire contents into a T-25 flask containing 10 ml of bacterized ATCC medium 802.
11.? Incubate at 25°C with the cap on loosely.
12.? Once the culture is established, follow the protocol for maintenance of culture.
