Tetraselmis sp. (ATCC® PRA-361)

Organism: Tetraselmis sp.  /  Depositor: N Yubuki

Permits and Restrictions

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Deposited As Tetraselmis sp.
Strain Designations PB25
Biosafety Level 1
Isolation Tide pool, Pachena Beach, Bamfield, BC, Canada, 2010
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
Comments Food source for ATCC® PRA-360™
Medium ATCC® Medium 2846: F/2 Medium
Growth Conditions Temperature: 15-25°C
Atmosphere: Aerobic
Culture system: Xenic, with mixed bacteria
Cryopreservation Reagents
Cryoprotective Solution
DMSO, 1.5 mL
Fresh complete growth medium, 8.5 mL

Harvest and Preservation
  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 the cells from a culture that is at or near peak density by centrifuging at 400 x g for 5 minutes.
  3. Adjust the concentration to between 2 x 105 and 2 x 106 cells/mL with fresh medium.  If the concentration is too low, centrifuge at 400 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield the desired concentration.
  4. Mix the cell preparation and the DMSO in equal portions.  The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no longer than 30 min.
  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.  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.)
  7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
  8. To establish a culture from the frozen state, place the vial in a 35°C water bath until thawed (2-3 min). 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 T-25 containing 10 mL fresh medium.
  9. Incubate the culture at 15-25°C under a 14 hour light (~50 µEinsteins/m2/s irradiance)/10 hour dark cycle with the cap screwed on tightly.

 

Alternative Thawing Procedure

  1. Aseptically add 0.5 mL of fresh medium to the frozen ampule.  Immediately place in a 35°C water bath until thawed (2-3 min).  Immerse the ampule just sufficient to cover the frozen material.  Do not agitate the ampule.
  2. Immediately after thawing, aseptically remove the contents of the ampule and gently add the material to the edge of a 20 x 100 mm petri plate containing ATCC Medium 919 (non-nutrient agar) and position on a 15 degree slant.  The cell suspension will pool at the edge of the plate.
  3. Continue to double the volume of the cell suspension at 10 minute intervals by dropwise addition of fresh medium.  When the volume reaches 16.0 mL place the plate in a horizontal position and incubate at 15-25°C under a 14 hour light (~50 µEinsteins/m2/s irradiance)/10 hour dark cycle. 
  4. Once the culture has been established subculture into a T-25 flask and follow the protocol for maintenance of culture
Name of Depositor N Yubuki
Chain of Custody ATCC <-- N Yubuki
References

Yamaguchi A, et al. Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of Rapaza viridis n. gen. et sp. (Euglenozoa, Euglenida). BMC Evol. Biol. 8: 12-29, 2012. PubMed: 22401606

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation