1. Harvest cells from multiple culture tubes at or near peak density by placing tubes on ice for 5-10 minutes, xa0inverting several times to dislodge attached cells, followed by centrifugation at 800-1000 x g for 8-10 min.
2. While cultures are being centrifuged, prepare a 10% (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.xa0 Allow the DMSO to solidify.xa0 Add the required volume of previously-reduced culture supernatant.xa0 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.
3. Adjust the concentration of cells to at least 2 x 10⁵/mL in culture supernatant.
4. Mix the cell preparation and the cryoprotective solution in equal portions. xa0The final concentration of DMSO will be 5%.
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, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a 16 x 125 mm screw-capped test tube containing 13 mL ATCC Medium 2768.xa0 Tightly seal and then invert the culture tube several times to evenly distribute cells.
9. Incubate the culture on a 15° horizontal slant at 20-25°C with the cap on tight.
10. Follow the protocol for maintenance of culture.

Yubuki N, et al. Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences. J. Eukaryot. Microbiol. 57: 554-561, 2010. PubMed: 20880033

Nucleotide (GenBank) : HM581663 SSU rRNA gene