1. Harvest cells from several cultures in very late logarithmic to early stationary phase of growth.xa0 Vigorously agitate to suspend the cells.
2. Aseptically transfer the cell suspension to 15 ml plastic centrifuge tubes.
3. Centrifuge at ~800 x g for 5 min.
4. While cells are centrifuging, prepare a 10% solution of DMSO in complete ATCC Medium 1029.xa0 Cool on ice.
5. Remove the supernatant and pool the cell pellets to the final volume desired with fresh growth medium.
6. Combine the cell suspension with an equal volume of 10% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 5% DMSO.
7. Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).
8. Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 At -40°C, plunge ampules into liquid nitrogen.xa0 Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 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.). xa0
9. Store ampules in a liquid nitrogen refrigerator until needed.
10. To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material.xa0 Allow the ampule to thaw completely (2-3 min).
11. Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh complete ATCC medium 1029.
12. Screw the cap on tightly and incubate at 20-25°C.xa0xa0xa0 Observe the culture daily and transfer when numerous trophozoites are observed.xa0xa0xa0xa0xa0xa0xa0xa0xa0

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