Chlorella vulgaris (cells)

(Cryopreservation of microalgae Chlorella vulgaris)

Intro/Description:

For the cryopreservation of microalgae. Protocol has been adapted from Muller, et al. (2007).

Materials:

• Dimethylsulfoxide (DMSO)
• Cryopreservation vials
• Controlled-rate cooler
• Cryostorage vessel with liquid nitrogen
• Waterbath at 45°C
• ice

Protocol:

1. Resuspend algal cultures in appropriate medium and adjust density to 1-5x107 cells/ml.

2. In a 2ml cryopreservation vial add 0.9ml cell culture with 10% DMSO (for a final concentration of 5% v/v DMSO)

3. Allow culture to rest for 10mins on ice, then transfer vials to a controlled-rate cooler, pre-chilled to 4°C.

4. Chill cultures at a rate of -1°C/min to a holding temperature of -35°C. Hold at -35°C for 40mins.

5. Again using a controlled cooling rate of -1°C/min, chill cultures to -45°C.

6. Transfer to a cryostorage vessel containing liquid nitrogen.

7. To thaw, submerge the vials in a 45°C water bath until culture is completely thawed (approximately 2-3mins).

8. Immediately dilute cultures 20-fold in fresh medium to reduce concentration of cryoprotective agent.

9. Culture algae following standard culturing procedures.

References:

Muller, J., J.G. Day, K. Harding, D. Hepperle, M. Lorenz, and T. Friedl. 2007. Assessing genetic stability of a range of terrestrial microalgae after cryopreservation using amplified fragment length polymorphism (AFLP). Am J Bot. 94:799-808.

Added by: Carlton Hoyt on July 25, 2012

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