1. Harvest cells from a culture which 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 10% (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 5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should 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 5.0 ml of fresh ATCC medium 1034.
10. Incubate the tube on a 15° horizontal at 35°C with the cap screwed on tightly.

De Jonckheere JF, et al. A comparative study of 14 strains of Naegleria australiensis demonstrates the existence of a highly virulent subspecies: N. australiensis italica n. spp. J. Protozool. 31: 324-331, 1984. PubMed: 6470990

De Jonckheere. Naegleria australiensis sp. nov., another pathogenic Naegleria from water. Protistologica 17: 423-429, 1981.

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

McLaughlin GL, et al. Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae. J. Clin. Microbiol. 26: 1655-1658, 1988. PubMed: 2903176

Carosi G, et al. Ultrastructure of Naegleria australiensis (pp-397 strain) with particular regard to pleomorphic cytoplasmic RER-associated inclusions. Protistologica 22: 169-174, 1986.

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

De Jonckheere JF. Variation of electrophoretic karyotypes among Naegleria spp.. Parasitol. Res. 76: 55-62, 1989. PubMed: 2622896

Huizinga HW, McLaughlin GL. Thermal ecology of Naegleria fowleri from a power plant cooling reservoir. Appl. Environ. Microbiol. 56: 2200-2205, 1990. PubMed: 1975164

Hadas E, Kasprzak W. Comparative biochemical studies on the pathogenic and non-pathogenic amebae of the genus Naegleria. Wiad. Parazytol. 33: 25-38, 1987. PubMed: 3307159

Moss DM, et al. High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species. J. Protozool. 35: 26-31, 1988. PubMed: 2966859

Visvesvara GS, et al. Production of monoclonal antibodies to Naegleria fowleri, agent of primary amebic meningoencephalitis. J. Clin. Microbiol. 25: 1629-1634, 1987. PubMed: 3308948

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

Lares-Villa F, et al. Five cases of primary amebic meningoencephalitis in Mexicali, Mexico: study of the isolates. J. Clin. Microbiol. 31: 685-688, 1993. PubMed: 8458963

Rivera F, et al. Contaminacion del liquido cefolorraquideo de un infante con sindrome de Arnold-Chiari tipo II, hidrocefalia y mielomeningocele, por Naegleria lovaniensis. Rev. Enferm. Infecc. Pediatr. 2: 91-94, 1989.

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

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

Reveiller FL, et al. Species specificity of a monoclonal antibody produced to Naegleria fowleri and partial characterization of its antigenic determinant. Parasitol. Res. 86: 634-641, 2000. PubMed: 10952262

Reveiller FL, et al. Isolation of a unique membrane protein from Naegleria fowleri. J. Eukaryot. Microbiol. 48: 676-682, 2001. PubMed: 11831777