1. Harvest cells from a culture that is at or near peak density by centrifugation at 600 x g for 5 min. Pool the cell pellets into a single tube.
2. Adjust the concentration of cells to 2.0 x 10⁶/ml.xa0 If the concentration is too low, centrifuge at 600 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. Prepare a 15% (v/v) sterile DMSO solution in ATCC medium 1034 as follows:xa0 Add the required volume of DMSO to a glass screw-capped test tube and place on ice.xa0 Allow the DMSO to solidify.xa0 Add the required volume of refrigerated ATCC medium 1034.xa0 Dissolve the DMSO by inverting several times.xa0 If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁶ and 7.5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 60 min.
5. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. 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.
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial enough to cover only the frozen material. Do not agitate the vial.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate into a fresh tube or flask of ATCC medium 1034.
10. Incubate at 25°C with the cap screwed on tightly (incubate a test tube on a 15° horizontal slant).

Carosi G, et al. An electron microscope study of Naegleria jadini nov. sp. (Willaert-le Ray, 1973) in axenic medium, the ameboid stage. Protistologica 12: 31-36, 1976.

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

Environ. Res. 59: 223-226, 1992.

Willaert E, Le Ray D. Caracteres morphologiques, biologiques et immunochimiques de Naegleria jadini sp. nov. (Amoebida, Vahlhamphfiidae). Protistologica 9: 417-426, 1973.

John DTOpportunistically pathogenic free-living amebaeIn: John DTParasitic protozoa2nd ed.3San DiegoAcademic Presspp. 143-246, 1993

Das SR, et al. Studies on mitotic division in Naegleria jadini. Ann. Soc. Belge Med. Trop. 54: 141-146, 1974.

van Belkum A, et al. Genotyping Naegleria spp. and Naegleria Fowleri isolates by Interepeat Polymerase Chain reaction. J. Clin. Microbiol. 30: 2595-2598, 1992. PubMed: 1400959

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

Daggett PM, et al. A molecular approach to the phylogeny of Acanthamoeba. Biosystems 18: 399-405, 1985. PubMed: 4084681

Rivera F, et al. Pathogenic amoebae in natural thermal waters of three resorts of Hidalgo, Mexico. Environ. Res. 50: 289-295, 1989. PubMed: 2583075

Robinson BS, et al. Discontinuous genetic variation among mesophilic Naegleria isolates: Further evidence that N. gruberi is not a single species. J. Protozool. 39: 702-712, 1992. PubMed: 1453360

type strain

De Jonckheere JF. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41: 309-342, 2002.