Cryoprotective SolutionDMSO ?????????????????????????????????????????????????????????????????????????????????? 2.0 ml
Filtered artificial seawater (or similar) ??????????????????????????? 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 cells from a culture which is at or near peak density by adding 5 ml sterile filtered artificial seawater 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 106/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 994.? 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 25°C.
10. Follow the protocol for maintenance of culture.
