1               cryopreservation

1. Cultivate N. oceanica or N. gaditana on a shaker (150 rpm, 23°C, 100 yE) to a cell concentration of 1*10⁷ cells/ml.
2. Place culture for 20 hours in the dark before cryopreservation.
3. Equilibrate the CoolCell freezing container (check if black core is inside) at 4°C.
4. Prepare a sterile filtered 20% solution of DMSO in the culture medium used to grow the algae.
5. harvest the dark treated cells by centrifugation in sterile 50 ml falcon tubes  (4000g, 10 min). resuspend the culture in appropriate amount of sterile medium to reach a end concentration of 1*10^7 cells/ml.
6. Dispense 20% DMSO solution into the ice-cold, prelabeled 2 ml cryogenic vials to reach the appropriate 10% concentration in a culture. In subdued/dark light, transfer the harvested, concentrated algal culture to the cryogenic vial (e.g.  1 volume of culture to 1 volume of DMSO)–> Concentration of the algae in the cryo-vial is around 6*10^7 cells/ml.
7. Close the vials and gently invert several times. Keep the vials from bright room light.
8. equilibrate the DMSO and algae 30 minutes on ice (4°C) in darkness.
9. Transfer the cryogenic vials to a prechilled freezing container and immediately transfer the container to a -80°C freezer. Leave the container for  3 hours at -80°C to reach the temperature less than -50°C. (CoolCell freezing containers ensure standardized controlled-rate -1°C /minute cell freezing in a -80°C freezer. Place sample vials containing 1 ml of cell suspension in each well. Each well should contain a filled vial. If freezing batch is fewer than 12 vials, fill each empty well with a BioCision CoolCell Filler Vial. Fully seat the lid on CoolCell, Check that the tubes slide in and out freely. Place CoolCell upright into a -80°C freezer or dry ice locker. Ensure that there is at least one inch of free space clearance around CoolCell module.)
10. Quickly transfer the cryogenic vials kept on ice in the CoolCell to the storage box and place the storage box into the liquid nitrogen storage dewar.

2               Thawing process

1. Prepare a water bath at 27°C (magnetic stirrer and milliQ water), use the floating vial-holder for thawing the vials.
2. Prepare erlenmeyer colb with at least 50 ml fresh medium.
3. Quickly transfer the cryogenic vials kept in liquid nitrogen to the water bath. Remove the cryogenic vials from the water bath (clean them with EtOH) as soon as all ice in the cryogenic vial has melted. Take care that the thawing process and transfer to fresh medium is less then 3 min.
4. Transfer the algae to fresh culture medium (1 ml algae from cryovial to 50 ml culture medium). The concentration of the DMSO must be reduced to less than 0.2%.
5. Allow the recovered culture to remain in darkness or in subdued light for several    hours, preferably overnight. Then place the culture under normal culture conditions and expect growth of viable cells to resume within 4-5 days.