Crypthecodinium cohnii (Seligo) Javornicky (ATCC® 30021)

Organism: Crypthecodinium cohnii (Seligo) Javornicky  /  Depositor: K Gold

Strain Designations Puerto Rico
Application
Biofuel production
Biosafety Level 1
Isolation
seawater, Puerto Rico, 1963
Product Format frozen
Type Strain no
Comments
Minor Sibling Species G
growth requirement
cryopreservation
Medium Medium 460: A2E6 medium
Growth Conditions
Temperature: 25.0°C
Duration: axenic
Protocol: ATCCNO: 30021 SPEC: This culture is routinely shipped as a frozen stabilate. Thaw the ampule and aseptically transfer the material to 5 ml of ATCC medium 460 in a 16 x 125 mm screw-capped test tube. Do not distribute the thawed material to a larger volume of medium. It is essential to first establish the culture in a small volume. Screw cap on tightly, loosen one half turn, and incubate culture upright at 25C. The culture should be ready to subculture in approximately 7 days. To subculture, screw the cap on tightly, invert the culture 5 times and aseptically transfer a 0.1 ml aliquot to 5 ml of ATCC medium 460 and incubate as above. Prepare two subcultures weekly. Retain all cultures for up to one month to ensure against loss.
Subcultivation
Protocol: ATCCNO: 30021 SPEC: This culture is routinely shipped as a frozen stabilate. Thaw the ampule and aseptically transfer the material to 5 ml of ATCC medium 460 in a 16 x 125 mm screw-capped test tube. Do not distribute the thawed material to a larger volume of medium. It is essential to first establish the culture in a small volume. Screw cap on tightly, loosen one half turn, and incubate culture upright at 25C. The culture should be ready to subculture in approximately 7 days. To subculture, screw the cap on tightly, invert the culture 5 times and aseptically transfer a 0.1 ml aliquot to 5 ml of ATCC medium 460 and incubate as above. Prepare two subcultures weekly. Retain all cultures for up to one month to ensure against loss.
Cryopreservation

1.   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.

2. 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.

3.  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 greater than 15 min.

4.   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.)   

5.   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.

6.   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.

7.   Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and transfer to a fresh tube containing 5 ml of ATCC Medium 460. Incubate the tube vertically at 20-25°C with the cap loosened one half turn.  Subculture every 10-14d.

Name of Depositor K Gold
Year of Origin 1963
References

Gold K, Baren CF. Growth requirements of Gyrodinium cohnii. J. Protozool. 13: 255-257, 1966.

Provasoli L, Gold K. Nutrition of the American strain of Gyrodinium cohnii. Arch. Mikrobiol. 42: 196-203, 1962. PubMed: 14489006

Beam CA, Himes M. Evidence for sexual fusion and recombination in the dinoflagellate Crypthecodinium (Gyrodinium) cohnii. Nature 250: 435-436, 1974. PubMed: 4851938

Seafood products having good taste containing marine microalgae, particularly Crythecodinium cohnii containing docosahexaenoic acid. Japanese Patent 7,008,221

Iizuka T, et al. Marine micro-algae food material containing docosahexaenoic acid, food containing the same and manufacturing method therefor. US Patent 5,547,699 dated Aug 20 1996

Simione FP Jr., Daggett PM. Recovery of a marine dinoflagellate following controlled and uncontrolled freezing. Cryobiology 14: 362-366, 1977. PubMed: 560941

Sub-culture of a microscopic marine alga. Japanese Patent 7,008,266

. Culture of marine microalgae producing docosahexaenoic acid. Japanese Patent 7,087,959

. Culture of marine microalgae producing docosahexaenoic acid used for e.g. cholesterol lowering action. Japanese Patent 7,087,988

. Handbook of phycological methods. Cambridge: Cambridge Univ. Press; 1975.