Acanthamoeba castellanii (Douglas) Page (ATCC^® 30010^™)

Harvest and Preservation

1. To achieve the best results, set up cultures with several different inocula (e.g. 0.25 mL, 0.5 mL, 1.0 mL). Harvest cultures and pool when the culture that received the lowest inoculum is at or near peak density.
2. If the cell concentration exceeds the required level, do not centrifuge, but adjust the concentration to between 2 x 10⁶ and 2 x 10⁷ cysts/mL with fresh medium. If the concentration is too low, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
3. 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.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 10⁶ and 10⁷ cells/mL 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 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 are stored in either the vapor or liquid phase of a nitrogen freezer.
8. 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 just sufficiently to cover the frozen material. Do not agitate the vial.
9. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5 mL of fresh ATCC medium 712 in a T-25 tissue culture flask or plastic 16 x 125 mm screw-capped test tube. Incubate at 25°C.

Hall J, Voelz H. Bacterial endosymbionts of Acanthamoeba sp.. J. Parasitol. 71: 89-95, 1985. PubMed: 3981353

Neff RJ. Purification, axenic cultivation, and description of a soil amoeba, Acanthamoeba sp.. J. Protozool. 4: 156-182, 1957.

Brooks SE, Schneider DL. Oxidative metabolism associated with phagocytosis in Acanthamoeba castellanii. J. Protozool. 32: 330-333, 1985. PubMed: 3925134

MacKay RM, Doolittle WF. Nucleotide sequences of Acanthamoeba castellanii 5S and 5.8S ribosomal ribonucleic acids: phylogenetic and comparative structural analyses. Nucleic Acids Res. 9: 3321-3334, 1981. PubMed: 7279665

Lonergan KM, Gray MW. Editing of transfer RNAs in Acanthamoeba castellanii mitochondria. Science 259: 812-816, 1993. PubMed: 8430334

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

Chung DI, et al. Biochemical and molecular characterization of a strain KA/S2 of Acanthamoeba castellanii isolated from Korean soil. Korean J. Parasitol. 34: 79-85, 1996. PubMed: 8820744

Neff RJDetection of synchrony induction in amoebae; problems in cell countingIn: Neff RJSynchrony in cell division and growth1964New YorkInterscience Publishers509-520, 1964

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

Tomlinson G. The glyoxylate pathway in Acanthamoeba sp.. J. Protozool. 14: 114-116, 1967.

Cerroni RE, Neff RJ. Inhibition of the feulgen reaction by ions. Exp. Cell Res. 16: 465-470, 1959. PubMed: 13653017

Weihing RR, Korn ED. Ameba actin: The presence of 3-methylhistidine. Biochem. Biophys. Res. Commun. 35: 906-912, 1969. PubMed: 5791528

Neff RJ. Mechanisms of purifying amoebae by migration on agar surfaces. J. Protozool. 5: 326-231, 1958.

Weisman RA, Korn ED. Phagocytosis of latex beads by Acanthamoeba. I. Biochemical properties. Biochemistry 6: 485-497, 1967. PubMed: 4860149

Lonergan KM, Gray MW. Predicted editing of additional transfer RNAs in Acanthamoeba castellanii mitochondria. Nucleic Acids Res. 21: 4402, 1993. PubMed: 8415006

Daggett PM, et al. Distribution and possible interrelationships of pathogenic and nonpathogenic Acanthamoeba from aquatic environments. Microb. Ecol. 8: 371-386, 1982.

Qureshi MN, et al. Inhibition of Acanthamoeba species by Pseudomonas aeruginosa rationale for their selective exclusion in corneal ulcers and contact lens care systems. J. Clin. Microbiol. 31: 1908-1910, 1993. PubMed: 8349772

Korn ED, Weisman RA. Phagocytosis of latex beads by Acanthamoeba. II. Electron microscopic study of the initial events. J. Cell Biol. 34: 219-227, 1967. PubMed: 6033533

Smith FR, Korn ED. 7-Dehydrostigmasterol and ergosterol: the major sterols of an amoeba. J. Lipid Res. 9: 405-408, 1968. PubMed: 5725873

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

Anger C, et al. A quantitative bacterial plaque assay for the enumeration of viable Acanthamoeba cells. Rev. Infect. Dis. 13 suppl.5: S396, 1991. PubMed: 2047673

Fischer-Stenger K, et al. Separation of soluble amoebicidal and tumoricidal activity of activated macrophages. J. Protozool. 39: 235-241, 1992. PubMed: 1560419

Rutherford I, et al. Efficacy of a chlorhexidine tablet system for disinfection of soft contact lenses against Acanthamoeba species. Rev. Infect. Dis. 13 suppl.5: S416-S417, 1991. PubMed: 2047679

Bozue JA, Johnson W. Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion. Infect. Immun. 64: 668-673, 1996. PubMed: 8550225

Bottone EJ, et al. Differential binding capacity and internalisation of bacterial substrates as factors in growth rate of Acanthamoeba spp.. J. Med. Microbiol. 40: 148-154, 1994. PubMed: 8107064

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

Howe D, et al. Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.. Parasitol. Res. 83: 435-348, 1997. PubMed: 9197389

Pettit DA, et al. In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study. J. Parasitol. 82: 769-777, 1996. PubMed: 8885887

Marciano-Cabral F, Toney DM. The interacion of Acanthamoeba spp. with activated macrophages and with macrophage cell lines. J. Eukaryot. Microbiol. 45: 452-458, 1998. PubMed: 9703682

Toney DM, Marciano-Cabral F. Resistance of Acanthamoeba species to complement lysis. J. Parasitol. 84: 338-344, 1998. PubMed: 9576508

Berk SG, et al. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp.. Appl. Environ. Microbiol. 64: 279-286, 1998. PubMed: 9435080

Kong HH, et al. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea. J. Clin. Microbiol. 40: 1199-1206, 2002. PubMed: 11923331

Price DH, Gray MW. Confirmation of predicted edits and demonstration of unpredicted edits in Acanthamoeba castellanii mitochondrial tRNAs. Curr. Genet. 35: 23-29, 1999. PubMed: 10022945

Susa M, et al. De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells. Infect. Immun. 64: 1679-1684, 1996. PubMed: 8613378

Fettes PS, et al. Characterization of the Legionella pneumophila gene ligA. Int. J. Med. Microbiol. 290: 239-250, 2000. PubMed: 10959726

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

Bullerwell CF, et al. Discovery and characterization of Acanthamoeba castellanii mitochondrial 5S rRNA. RNA 9: 287-292, 2003. PubMed: 12592002

Lynch D, et al. The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii. FEMS Microbiol. Lett. 219: 241-248, 2003. PubMed: 12620627

Yagita K, et al. Clustering of Acanthamoeba isolates from human eye infections by means of mitochondrial DNA digestion patterns. Parasitol. Res. 85: 284-289, 1999. PubMed: 10099009

Procedures for the Recovery and Identification of Parasites from the Intestinal Tract: Approved Guideline - 2nd Edition. Wayne, PA. Clinical and Laboratory Standards Institute; CLSI M28-A2.

Nucleotide (GenBank) : K00471 A.castellanii (amoeba) 5.8S ribosomal RNA.

Nucleotide (GenBank) : U12386 Acanthamoeba castellanii mitochondrion, complete genome.

Nucleotide (GenBank) : U03732 Acanthamoeba castellanii Neff mitochondrion rRNA large subunit gene.

Nucleotide (GenBank) : M97651 Acanthamoeba castellanii mitochondrial Met-tRNA, Ala-tRNA, Pro-tRNA, and Asp-tRNA genes.

Nucleotide (GenBank) : L20506 Acanthamoeba castellanii transfer RNA-Gln, complete tRNA; transfer RNA-Lys, complete tRNA; transfer RNA-Glu, complete tRNA; transfer RNA-Ile, complete tRNA; transfer RNA-Leu, complete tRNA.