1. ? Allow the culture to encyst.
2. ? Place the flasks on ice and allow them to equilibrate for at least 30 minutes.? Gently scrap the bottom of each flask with a cell scrapper to detach adherent cysts.?
3.?? Transfer the cyst suspensions to 15 ml plastic centrifuge tubes and centrifuge at 800 x g for 5 minutes.? Place the concentrated cyst suspension on ice. Adjust the concentration of cells to 2 x 105/ml in fresh cold medium.
4.? Mix the cell preparation and the cryoprotective solution in equal portions. ??Keep the suspension on ice.
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 the vials in a Nalgene 1°C freezing apparatus.? Place the apparatus at -80°C for 2.0 to 2.5 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 refer to the
????? instructions above.
