1. Harvest cells from a culture that is at or near peak density by centrifugation at 300 to 400 x g for 5 min.
2. Adjust the concentration of cells to 2 x 10⁵xa0to 2 x 10⁶/mL in fresh medium.
3. While cells are centrifuging prepare a 18% (v/v) solution of sterile DMSO in fresh medium.
   1. Add 1.8 mL of DMSO to an ice cold 20 x 150 mm screw-capped test tube.
   2. Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 8.2 mL of ice cold medium.
   3. Invert several times to dissolve the DMSO.
   4. Allow to warm to room temperature.
4. Mix the cell preparation and the DMSO solution in equal portions. Thus, the final concentration will be 10⁶xa0to 10⁷ cells/mL and 9% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should no less than 15 min and no longer than 30 min.
5. Dispense in 0.5 mL aliquots into 1.0 mL to 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.) xa0
7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of 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 a 16 x 125 mm screw-capped test tube containing 13 mL ATCC Medium 2154.
10. Incubate the culture on a 15° horizontal slant at 35°C with the cap screwed on tightly.

Chavez LA, et al. A light and electron microscopical study of a new, polymorphic free- living amoeba, Phreatamoeba balamuthi n. g., n. sp.. J. Protozool. 33: 397-404, 1986. PubMed: 3746722

Hannaert V, et al. Enolase from Trypanosoma brucei, from the amitochondriate protist Mastigamoeba balamuthi, and from the chloroplast and cytosol of Euglena gracilis: pieces in the evolutionary puzzle of the eukaryotic glycolytic pathway. Mol. Biol. Evol. 17: 989-1000, 2000. PubMed: 10889212

Hinkle G, et al. The unusually long small subunit ribosomal RNA of Phreatamoeba balamuthi. Nucleic Acids Res. 22: 465-469, 1994. PubMed: 8127686

Martin JB, et al. Metabolites of the free-living amoeba Phreatamoeba balamuthi analyzed by 13C- and 31P-NMR spectroscopy: Occurence of phosphoinositol diphosphates. J. Eukaryot. Microbiol. 42: 183-191, 1995.

Muller M, et al. Presence of prokaryotic and eukaryotic species in all subgroups of the pp(i)-dependent group ii phosphofructokinase protein family. J. Bacteriol. 183: 6714-6716, 2001. PubMed: 11673446

Sanchez LB, et al. Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships. Gene 295: 51-59, 2002. PubMed: 12242011

Simpson AG, et al. The organization of Mastigamoeba schizophrenia n. sp.: more evidence of ultrastructural idiosyncrasy and simplicity in pelobiont protists. Eur. J. Protistol. 33: 87-98, 1997.

Nucleotide (GenBank) : L23799 ribosomal RNA small subunit gene

Nucleotide (GenBank) : AF205070 enolase mRNA, complete coding sequence

Nucleotide (GenBank) : AF140033 histone H3 mRNA, complete coding sequence

Nucleotide (GenBank) : AF140034 histone H4 mRNA, complete coding sequence