Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Isolation
Mangrove sediments, Ishigaki Island, Japan, September 2005
Product Format
test tube
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Axenic/Xenic
Xenic
Type Strain
no
Comments
Taxonomic description
Medium
ATCC® Medium 2768: PYNFH, MODIFIED In Seawater
Growth Conditions
Temperature: 20°C to 25°C
Atmosphere: Microaerophilic
Cryopreservation
Reagents
Cryoprotective Solution
DMSO 1.0 mL
Spent culture supernatant 9.0 mL
Harvest and Preservation
Harvest cells from multiple culture tubes at or near peak density by placing tubes on ice for 5-10 minutes, inverting several times to dislodge attached cells, followed by centrifugation at 800-1000 x g for 8-10 min.
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. Allow the DMSO to solidify. Add the required volume of previously-reduced culture supernatant. 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.
Adjust the concentration of cells to at least 2 x 105/mL in culture supernatant.
Mix the cell preparation and the cryoprotective solution in equal portions. The final concentration of DMSO will be 5%.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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. 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.)
Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
To establish a culture from the frozen state place the vial in a 35°C water bath. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial. 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. Tightly seal and then invert the culture tube several times to evenly distribute cells.
Incubate the culture on a 15° horizontal slant at 20-25°C with the cap on tight.
Follow the protocol for maintenance of culture.
Name of Depositor
N Yubuki
Year of Origin
2005
References
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
