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1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min. The cells grown in a medium containing agar are concentrated by centrifugation, a solid pellet does not form. The soft pellet is resuspended to desired cell concentration with agar-free supernatant.
2. Adjust the concentration of cells to 2 x 10⁶ - 2 x 10⁷/mL in fresh medium.
3. While cells are centrifuging prepare a 10% (v/v) solution of sterile DMSO in fresh medium.
   1. Add 1.0 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 9.0 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 in equal portions. Thus, the final concentration will be 10⁶ - 10⁷ cells/mL 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 30 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 the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.  At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.  Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.  (The cooling rate in this apparatus is approximately -1°C/min.)  
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 either 9 mL of ATCC medium 361 (completed with serum) or 13 mL ATCC Medium 2154 adjusted to pH 6.0.

10. Incubate the culture at 35ºC with the cap screwed on tightly (tube should be vertical for medium 361 or on a 15º horizontal slant for medium 2154).

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Muller M, Lindmark DG. Uptake of metronidazole and its effect on viability in Trichomonads and Entamoeba invadens under anaerobic and aerobic conditions. Antimicrob. Agents Chemother. 9: 696-700, 1976. PubMed: 1083712

Yarlett N, et al. Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Trtrichomonas foetus. J. Eukaryot. Microbiol. 41: 554-559, 1994. PubMed: 7866382

Yano A, et al. Antigen-specific proliferation responses of peripheral blood lymphocytes to Trichomonas vaginalis antigen in patients with Trichomonas vaginalis. J. Clin. Microbiol. 17: 175-180, 1983. PubMed: 6601112

Lloyd D, et al. Metronidazole-resistant clinical isolates of Trichomonas vaginalis maintain low intracellular metronidazole radical anion levels as a consequence of defective oxygen scavenging. Biologica 30: 521-528, 1988.

Mertens E, et al. Presence of a fructose-2,6-bisphosphate-insensitive pyrophosphate: fructose-6-phosphate phosphotransferase in the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma. Mol. Biochem. Parasitol. 37: 183-190, 1989. PubMed: 2558319

Wang AL, Wang CC. A linear double-stranded RNA in Trichomonas vaginalis. J. Biol. Chem. 260: 3697-3702, 1985. PubMed: 2982874

Smith RF. Viability of Trichomonas vaginalis in vitro at four temperatures. J. Clin. Microbiol. 18: 834-836, 1983. PubMed: 6605364

Turner G, Muller M. Failure to detect extranuclear DNA in Trichomonas vaginalis and Tritrichomonas foetus. J. Parasitol. 69: 234-236, 1983. PubMed: 6600788

Mack SR, Muller M. Effect of oxygen and carbon dioxide on the growth of Trichomonas vaginalis and Tritrichomonas foetus. J. Parasitol. 64: 927-929, 1978. PubMed: 309937

Gelbart SM, et al. Growth of Trichomonas vaginalis in commercial culture media. J. Clin. Microbiol. 28: 962-964, 1990. PubMed: 2351739

Tai JH, et al. The divergence of Trichomonas vaginalis virus RNAs among various isolates of Trichomonas vaginalis. Exp. Parasitol. 76: 278-286, 1993. PubMed: 8500587

Kleiner DE, Johnston M. Purification and properties of a secondary alcohol dehydrogenase from the parasitic protozoan Tritrichomonas foetus. J. Biol. Chem. 260: 8038-8043, 1985. PubMed: 3159722

Goosen NK, et al. Effect of fixation on activity and cytochemistry of hydrogenosomal enzymes in Trichomonas vaginalis. J. Gen. Microbiol. 136: 2189-2193, 1990. PubMed: 1706759

Yarlett N, et al. Ferredoxin-dependent reduction of nitroimidazole derivatives in drug-resistant and susceptible strains of Trichomonas vaginalis. Biochem. Pharmacol. 35: 1703-1708, 1986. PubMed: 3486660

Viscogliosi E, et al. Phylogenetic implication of iron-containing superoxide dismutase genes from trichomonad species. Mol. Biochem. Parasitol. 80: 209-214, 1996. PubMed: 8892298

Roger AJ, et al. A possible mitochondrial gene in the early-branching amitochondriate protist Trichomonas vaginalis. Proc. Natl. Acad. Sci. USA 93: 14618-14622, 1996. PubMed: 8962102

Lloyd D, Kristensen B. Metronidazole ingibition of hydrogen production in vivo in drug-sensitive and resistant strains of Trichomonas vaginalis. J. Gen. Microbiol. 131: 849-853, 1985.

Vanacova S, et al. Characterization of Trichomonad species and strains by PCR fingerprinting. J. Eukaryot. Microbiol. 44: 545-552, 1997. PubMed: 9435127

Harmych SE, et al. Lactate dehydrogenase from the protozoan parasite, Trichomonas vaginalis. Comp. Biochem. Physiol. 115B: 405-409, 1996.

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Ter Kuile BH, Muller M. Interaction between facilitated diffusion of glucose across the plasma membrane and its metabolism in Trichomonas vaginalis. FEMS Microbiol. Lett. 110: 27-32, 1993. PubMed: 8319891

Madico G, et al. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples. J. Clin. Microbiol. 36: 3205-3210, 1998. PubMed: 9774566

van Leeuwen F, et al. beta-D-glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres. Proc. Natl. Acad. Sci. USA 95: 2366-2371, 1998. PubMed: 9482891

Keeling PJ, et al. Linked genes for calmodulin and E2 ubiquitin-conjugating enzyme in Trichomonas vaginalis. J. Eukaryot. Microbiol. 43: 468-474, 1996. PubMed: 8976604

Marinets A, et al. The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protist Trichomonas vaginalis. J. Mol. Evol. 43: 563-571, 1996. PubMed: 8995053

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Nucleotide (GenBank) : Z70670 T.vaginalis sod1 gene.

Nucleotide (GenBank) : Z70671 T.vaginalis sod2 gene.

Nucleotide (GenBank) : Z70672 T.vaginalis sod3 gene.

Nucleotide (GenBank) : Z70673 T.vaginalis sod4 gene.

Nucleotide (GenBank) : Z70674 T.vaginalis sod5 gene.

Nucleotide (GenBank) : M33717 T.vaginalis ferredoxin gene, complete cds.

Nucleotide (GenBank) : U57000 chaperonin 60 (cpn60) mRNA, partial coding sequence

Nucleotide (GenBank) : AF067404 DNA polymerase epsilon gene, partial coding sequence

Nucleotide (GenBank) : X98016 histone H4-2 and histone H3-2, partial coding sequence

Nucleotide (GenBank) : U27577 Trichomonas vaginalis polyubiquitin (UbA) mRNA, partial cds.

Nucleotide (GenBank) : U28008 Trichomonas vaginalis ubiquitin 1A (Ub1A) gene, partial cds.

Nucleotide (GenBank) : U28009 Trichomonas vaginalis ubiquitin 1C (Ub1C) gene, partial cds.

Nucleotide (GenBank) : U28010 Trichomonas vaginalis ubiquitin 1D (Ub1D) gene, partial cds.

Nucleotide (GenBank) : U28011 Trichomonas vaginalis ubiquitin 1E (Ub1E) gene, partial cds.

Nucleotide (GenBank) : U28012 Trichomonas vaginalis ubiquitin dimer 2B (Ub2B) gene, partial cds.

Nucleotide (GenBank) : U70308 Trichomonas vaginalis mitochondrial-type HSP70 mRNA, complete cds.

Nucleotide (GenBank) : U38692 Trichomonas vaginalis cytosolic malate dehydrogenase gene, complete cds.

Nucleotide (GenBank) : U28013 Trichomonas vaginalis polyubiquitin junction JC (UbJC) gene, partial cds.

Nucleotide (GenBank) : AF060233 Trichomonas vaginalis L-lactate dehydrogenase (LDH1) gene, complete cds.

Nucleotide (GenBank) : AF058282 Trichomonas vaginalis elongation factor 1 alpha (tef1) mRNA, partial cds.

Nucleotide (GenBank) : L11394 Trichomonas vaginalis glyceraldehyde 3-phosphate dehydrogenase mRNA, 3' end.

Nucleotide (GenBank) : U07784 Trichomonas vaginalis ATCC 30001 ferredoxin (FD) gene, Inr promoter element.

Nucleotide (GenBank) : M97553 Trichomonas vaginalis succinyl-CoA synthetase beta-subunit gene, complete cds.

Nucleotide (GenBank) : U07785 Trichomonas vaginalis ATCC 30001 P-glycoprotein (Pgp1) gene, promoter element.

Nucleotide (GenBank) : AF022421 glyceraldehyde-3-phosphate dehydrogenase (gap3) gene, partial coding sequence

Nucleotide (GenBank) : U07782 Trichomonas vaginalis ATCC 30001 beta-tubulin (beta-Tub) gene, promoter element.

Nucleotide (GenBank) : U07780 Trichomonas vaginalis ATCC 30001 alpha-tubulin (alpha-Tub) gene, promoter element.

Nucleotide (GenBank) : U07203 Trichomonas vaginalis hydrogenosomal adenylate kinase proprotein gene, complete cds.

Nucleotide (GenBank) : U16836 Trichomonas vaginalis hydrogenosomal malic enzyme subunit A (maeA) gene, complete cds.

Nucleotide (GenBank) : AF022414 Trichomonas vaginalis glyceraldehyde-3-phosphate dehydrogenase (gap2) gene, partial cds.

Nucleotide (GenBank) : AF005075 translation initiation factor 2 gamma subunit (eIF-2 gamma) gene, partial coding sequence

Nucleotide (GenBank) : U38786 calmodulin (CAM) and E2 ubiquitin-conjugating enzyme (TvUBC) genes, partial coding sequence

Nucleotide (GenBank) : AF053370 PPi-dependent fructose 6-phosphate 1-phosphotransferase (pfk3) gene, partial coding sequence

Nucleotide (GenBank) : U16822 Trichomonas vaginalis pyruvate:ferredoxin oxidoreductase proprotein (pfoA) gene, complete cds.

Nucleotide (GenBank) : U16823 Trichomonas vaginalis pyruvate:ferredoxin oxidoreductase proprotein (pfoB) gene, complete cds.

Nucleotide (GenBank) : U16838 Trichomonas vaginalis hydrogenosomal malic enzyme subunit C proprotein (maeC) gene, partial cds.

Nucleotide (GenBank) : U16839 Trichomonas vaginalis hydrogenosomal malic enzyme subunit D proprotein (maeD) gene, partial cds.

Nucleotide (GenBank) : U16837 Trichomonas vaginalis hydrogenosomal malic enzyme subunit B proprotein (maeB) gene, complete cds.

Nucleotide (GenBank) : U07783 Trichomonas vaginalis ATCC 30001 70kDa cystolic heat shock protein (cHSP70 gene), promoter element.

Nucleotide (GenBank) : AF044973 pyrophosphate-dependent fructose 6-phosphate 1-phosphotransferase (Pfk1) gene, complete coding sequence

Nucleotide (GenBank) : AF053371 Trichomonas vaginalis PPi-dependent fructose 6-phosphate 1-phosphotransferase (pfk2) gene, partial cds.

Nucleotide (GenBank) : U07781 Trichomonas vaginalis ATCC 30001 succinyl CoA synthetase beta subunit (beta-SCS) gene, Inr promoter element.

Nucleotide (GenBank) : U07779 Trichomonas vaginalis ATCC 30001 succinyl CoA synthetase alpha subunit (alpha-SCSB) gene, Inr promoter element.