1.?? Harvest cells from several cultures in the late logarithmic or early stationary phase of growth.? Vigorously agitate by inverting several times to suspend the cells.? Maintain cells on ice between manipulations.
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 liquid overlay from ATCC medium 2213.? Cool on ice.
5.?? Remove the supernatant and pool the cell pellets to the final volume desired with fresh medium overlay.
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.? From room temperature cool at -1°C/min to -40°C.? 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.). ?
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.? Allow the ampule to thaw completely (2-3 min).
11.????????? Immediately after thawing, aseptically remove the contents and transfer to a fresh test tube of ATCC Medium 2213.
12.Screw the cap on tightly and incubate at 15-20°C on a 15° horizontal slant.? Observe the culture daily and transfer when numerous trophozoites are observed.?????????
