Cryoprotective SolutionDMSO ?????????????????????????????????????????????????????????????????????????????????? 1.5 ml
Fresh growth medium w/o bacteria???????????????????????????????? 8.5 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 cells from a culture which is at or near peak density by adding 5 ml ATCC medium 5080 (Dryl's solution) and washing cells into suspension.? Rub the surface of the plate with a spread bar to detach adhering trophozoites.
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.
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, aseptically transfer the contents of the ampule to the center of a fresh plate of ATCC medium 2432.? Distribute the material evenly over the plate using a spread bar.
9. Wrap the entire edge of the plate with parafilm and incubate upright at 24°C.
10. Follow the protocol for maintenance of culture.
