Cryoprotective SolutionDMSO ?????????????????????????????????????????????????????????????????????????????????? 2.0 ml
Fresh growth medium w/o bacteria???????????????????????????????? 8.0 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.
2. ?? Harvest Tokophrya cells from a culture that has recently passed peak density by centrifugation at 250-300 x g for 5 min.
3.???? Adjust the concentration of cells to at least 2 x 104/ml in fresh medium.
4.? ?? Mix the cell preparation and the cryoprotective solution in equal portions by adding the cryoprotective solution to the cell suspension in 3 equal aliquots at 2 min. intervals.
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 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.? ?? Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8.? ?? To establish a culture from the frozen state 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. Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and transfer to a petri plate or T-25 tissue culture flask containing a bed of non-nutrient agar (ATCC medium 919) and 10 ml ATCC medium 1323.
9. Aseptically transfer 0.5-2.0 ml of washed Paramecium to the petri plate or T-25 flask (see section on MAINTENANCE OF CULTURE).? Incubate the culture at 20-25°C.
Once the culture is established, follow the protocol for maintenance of culture.
