Cryoprotective Solution
DMSO, 1.5 ml
Dryls solution (or similar), 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.)xa0xa0
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.

Nerad TA, et al. Acanthamoeba pearcei n. sp. (Protozoa: Amoebida) from sewage contaminated sediments. J. Eukaryot. Microbiol. 42: 702-705, 1995. PubMed: 8520585

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

type strain

Gast RJ. Development of an Acanthamoeba-specific reverse dot-blot and the discovery of a new ribotype. J. Eukaryot. Microbiol. 48: 609-615, 2001. PubMed: 11831768

Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16: 273-307, 2003. PubMed: 12692099

Nucleotide (GenBank) : AF019053 Acanthamoeba pearcei 18S ribosomal RNA gene, partial sequence.