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.xa0 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.xa0 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.xa0xa0 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.)
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. xa0Immediately 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.

xa0

Alternative Thawing Procedure

1. Aseptically add 0.5 mL of fresh medium to the frozen ampule.xa0 Immediately place in a 35°C water bath until thawed (2-3 min).xa0 Immerse the ampule just sufficient to cover the frozen material.xa0 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. xa0The 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.xa0 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.xa0
4. Once the culture has been established subculture into a T-25 flask and follow the protocol for maintenance of culture

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