Cryoprotective Solution
DMSO, 1.5 ml
Dryls solution, 8.5 ml

1. Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
2. Harvest cells from a culture which is at or near peak density by adding 5 ml ATCC medium 5080 (Dryls solution) and washing cells into suspension.xa0 Rub the surface of the plate with a spread bar to detach adhering trophozoites.
3. Adjust the concentration of cells to at least 2 x 10⁶/ml in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
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. 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. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place the vial in a 35°C water bath.xa0 Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.xa0xa0 Immediately after thawing, aseptically transfer the contents of the ampule to the center of a fresh plate of ATCC medium 997.xa0 Distribute the material evenly over the plate using a spread bar.
9. Wrap the entire edge of the plate with parafilm and incubate upright at 25°C.

Follow the protocol for maintenance of culture.

Silvany RE, et al. The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga. Ophthalmology 97: 286-290, 1990. PubMed: 2336265

Naginton J, et al. Amoebic infection of the eye. Lancet 2: 1537-1540, 1974. PubMed: 4140981

Silvany RE, et al. Effect of contact lens preservatives on Acanthamoeba. Ophthalmology 98: 854-857, 1991. PubMed: 1866136

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

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

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

Stothard DR, et al. The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types. J. Eukaryot. Microbiol. 45: 45-54, 1998. PubMed: 9495032

Alizadeh H, et al. In vitro amoebicidal activity of propamidine and pentamidine isethionate against Acanthamoeba species and toxicity to corneal tissues. Cornea 16: 94-100, 1997. PubMed: 8985640

Kong HH, et al. Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga. Korean J. Parasitol. 33: 331-340, 1995. PubMed: 8591011

Niszl IA, et al. Cytopathogenicity of clinical and environmental Acanthamoeba isolates for two mammalian cell lines. J. Parasitol. 84: 961-967, 1998. PubMed: 9794638

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

Alves JMP, et al. Random amplified polymorphic DNA probes as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba. Braz. J. Med. Biol. Res. 33: 19-26, 2000.

Ledee DR, et al. Advantages of using mitochondrial 16S rDNA sequences to classify clinical isolates of Acanthamoeba. Invest. Ophthalmol. Vis. Sci. 44: 1142-1149, 2003. PubMed: 12601042

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

Nucleotide (GenBank) : AF019062 Acanthamoeba polyphaga Nagington 18S ribosomal RNA gene, partial sequence.