Cryopreserved holdings save time and money because serially transferring the cultures is not required. This creates less opportunity for mixing up strains. Also, culture tubes and pipettes are not utilized, media does not need to be made, and there is no glassware to be washed and sterilized. As long as the liquid nitrogen levels are maintained in storage canisters, cryopreserved material remains in the same condition as when it was first cryopreserved - reducing the risk of genetic drift.
Many species of algae can be cryopreserved. In general, smaller cells are better candidates than larger ones. Species with many membranes (such as raphidophytes) have not been successfully cryopreserved, at least, here at the NCMA. Certain groups of algae, broadly speaking, have been cryopreserved at the NCMA better than others. These are listed below in descending order of success.
1. green algae
2. Cyanobacteria and pennate diatoms
3. red algae
4. brown coccoid and flagellate species (pelagophytes, pinguiophytes, haptophytes and phaeophytes)
5. smaller centric diatoms
7. some red and green macrophyte algae
8. small dinoflagellates (Symbiodinium, Amphidinium, P. minimum)
We have had the greatest success with strains cryopreserved in log phase. It is important to remember that every species and every strain of a species is different. Feed cultures as necessary to keep them healthy. Feed by splitting one to one, or by removing overlying medium where possible and replacing with fresh medium. 15mL of culture plus cryoprotective agent (CPA), is usually enough material to cryopreserve 15 cryovials.
As cryopreserved material should never warm above -137ºC, the preparation of the cryopreserved material should occur near the storage tank, or LN2 dewers. Heavy duty gloves should be used to bring cryopreserved material to the storage tank. Storage tanks should be on floors that can bear their weight (they are heavy), and should be easily accessible to LN2 deliveries( LN2 feeder tanks are also heavy). Carts should be used to move the heavy feeder tanks.
The thawing technique is as important as the cryopreservation technique. Equipment for thawing cryopreserved material should be near the storage tanks. A clean bench, a water bath and a small centrifuge are also desirable.
Awareness of the hazards associated with LN2 and any other chemicals used in the cryopreservation process is very important. Safety equipment (skin protection, safety glasses or a shield ) is essential as contact with LN2 or LN2 vapor can do extensive damage to skin and mucus membranes. Storage and filler tanks are very heavy and consideration must be given to their placement and handling. Safe access to LN2 tank delivery is important for the same reasons .
Good ventilation is essential. A N2 detector is desirable to monitor air quality and prevent asphyxiation. No open flames should be in use or nearby when working with solvents.