Temperature:xa025°C

1. Allow the cells to encyst.xa0 To detach cysts from the plate flush the surface with 5 ml fresh ATCC medium 1323 (Pages Balanced Salt Solution).xa0 Rub the surface of the plate with a spread bar to detach adhering amoebae.
2. Transfer the cyst suspension to a sterile centrifuge tube.
3. If the cyst concentration does not exceed 2 x 10⁶ cysts/ml adjust the suspension to that concentration.xa0 To adjust the concentration, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield 2 x 10⁶.
4. While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows:xa0 Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.xa0 Allow the DMSO to solidify.xa0 Add the required volume of refrigerated medium.xa0 Dissolve the DMSO by inverting the tube several times.xa0
   NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
5. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be at least 10⁶ cysts/ml and 7.5% (v/v) DMSO.xa0 The equilibration time (the time between addition of DMSO and the start ofxa0 the cooling cycle) should be no less than 15 min and no longer than 60 min.
6. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. 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.
8. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
9. 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 to a level just above the surface of the frozen material. Do not agitate the vial.
10. Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 997.xa0 Distribute the material evenly over the plate using a spread bar.xa0 Incubate at 25°C.

Adams M, et al. A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis. Int. J. Parasitol. 19: 823-834, 1989. PubMed: 2635158