1.?? To harvest the Balamuthia culture, detach cysts by scraping the inside bottom surface of the flask with a sterile cell scraper.
2.?? Transfer the cyst suspension to 15 ml plastic centrifuge tubes. Centrifuge at approximately 800 x g for 5 min.
3.?? Remove all but 0.5 ml of the supernatant from each tube, resuspend the cyst pellets, and pool them to a single tube.
4.?? Adjust the concentration of cysts to 2.0 - 4.0 x 105 cysts/ml with fresh medium or PBS.
????? NOTE: If the concentration of cysts is too low, centrifuge at 800 x g for 5 min and resuspend in the volume of fresh medium or PBS required to yield the desired concentration.
5.?? Prepare a cryoprotective solution containing 15% (v/v) DMSO and 6% (v/v) HIFBS in fresh medium or PBS.
6.?? Mix the cyst preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 105 cysts/ml, 7.5% DMSO, and 3% HIFBS. The time from the mixing of the cyst preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.
7.?? Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
8.?? 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.)
9.?? Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.
10.????????? To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.? Do not agitate the ampule.? Do not leave ampule in water bath after thawed.
11.????????? Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of cells (ATCC CRL-1586 or CCL-81) and 10 ml ATCC 30-2003 with 3% (v/v) HIFBS.
12.????????? Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
13.????????? Incubate in a 35°-37°C CO2 incubator with the cap screwed on tightly.
