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
Rotting Fucus sp., Trenton, ME, 1974
Product Format
frozen
Storage Conditions
Frozen: -70°C or colder Freeze-Dried: 2°C to 8°C Live Culture: See Protocols Section
Type Strain
no
Comments
Major Sibling Species WH
genetic diversification
cryopreservation
use of plastic ampoules for freeze preservation
Medium
ATCC® Medium 460: A2E6 medium
Growth Conditions
Temperature: 20°C to 25°C
Culture System: Axenic
Cryopreservation
Harvest and Preservation
Harvest cells from cultures which are at or near peak density. Aseptically transfer cells to 15 mL plastic centrifuge tubes and centrifuge at ~150 x g for 5 min.
Adjust the concentration of cells to 2 x 106/mL with fresh medium, then dilute to half this concentration by adding an equal amount of a 15% (v/v) sterile glycerol solution in fresh medium.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation). The time from mixing of the cell suspension and the glycerol solution, before the cooling cycle begins, should be no less than 15 min and no greater than 30 min.
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.)
The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials can be stored between -80 and -70°C for no longer than one week.
To establish a culture from the frozen state place an ampule in a water bath set at 35°C. 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 the water bath; aseptically remove the contents of the ampule and transfer to a fresh test tube (or T-25 flask) containing 5 mL of ATCC Medium 460. Incubate culture at 20-25°C (test tubes are incubated upright with the cap loosened one-half turn). Subculture every 10-14d.
Name of Depositor
CA Beam, M Himes
Year of Origin
1974
References
Salt GW. Changes in the cell volume of Didinium nasutum during population increase. J. Protozool. 22: 112-115, 1975.
Beam CA, Himes M. Sexual isolation and genetic diversification among some strains of Crypthecodinium cohnii-like dinoflagellates evidence of speciation. J. Protozool. 24: 532-539, 1977.
Simione FP Jr., Daggett PM. Recovery of a marine dinoflagellate following controlled and uncontrolled freezing. Cryobiology 14: 362-366, 1977. PubMed: 560941
Simione FP Jr., et al. The use of plastic ampoules for freeze preservation of microorganisms. Cryobiology 14: 500-502, 1977. PubMed: 891238
