2. When the culture is at or near peak density, rub the surface of the tube with a sterile cotton swab and vigorously shake the swab to dislodge the cells. Screw the cap on tightly and invert gently 10 times to distribute cells evenly.
3. Transfer approximately 0.25 ml to a T-25 tissue culture flask containing 10 ml of fresh ATCC medium 802.
4. Screw the caps on tightly and incubate at 25°C.
1. Harvest the cells from a culture that is at or near peak density by centrifuging at 850 x g for 5 minutes.
2. If the cell concentration exceeds the required level do not centrifuge, but adjust the concentration to between 2 x 106 and 2 x 107cysts/ml with fresh medium. If the concentration is too low, centrifuge at 850 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
3. While cells are centrifuging prepare a 20% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath. Allow the DMSO to solidify. Add the required volume of refrigerated medium. Dissolve the DMSO by inverting the tube several times.
*NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 106 107 cells/ml and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
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 controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge 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. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
9. Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 12 ml of bacterized ATCC Medium 1773. Incubate the culture at 25°C.
Thomas A Nerad, personal communication