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1. Allow the cells to encyst.  To detach cysts from the plate flush the surface with 5 ml fresh ATCC medium 1323 (Page's Balanced Salt Solution).  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.  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:  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.
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.  The equilibration time (the time between addition of DMSO and the start of  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.  Distribute the material evenly over the plate using a spread bar.  Incubate at 25°C.

Napolitano JJ, et al. Adelphamoeba galeacystis n. g., n. sp., an amoeboflagellate isolated from soil. J. Protozool. 17: 158-161, 1970.

Daggett PM, Nerad TA. The biochemical identification of vahlkampfiid amoebae. J. Protozool. 30: 126-128, 1983. PubMed: 6864593

Clark CG, Cross GA. Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae. J. Protozool. 35: 326-329, 1988. PubMed: 2840492

De Jonckheere JF, et al. On the identity of the amoeboflagellates Didascalus thorntoni and Adelphamoeba galeacystis. J. Eukaryot. Microbiol. 44: 52-54, 1997. PubMed: 9172832

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

De Jonckheere JF. Comparison of partial SSU rRNA sequences suggests revisions of species names in the genus Naegleria. Eur. J. Protistol. 30: 333-341, 1994.