NCMA has been cryopreserving algae for nearly 30 years!
We can help you cryopreserve your algae as one of our algae support services, just email us with your interests, or fill out the contact form below.
Here is a general overview of cyropreservation with respect to algae.
1. WHY CRYOPRESERVE?
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.
2. WHICH ALGAL SPECIES ARE GOOD CANDIDATES FOR CRYOPRESERVATION?
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)
3. HOW ARE CULTURES PREPARED FOR CRYOPRESERVATION AND HOW MUCH CULTURE IS NEEDED?
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.
4. WHAT IS NEEDED TO CRYOPRESERVE ALGAE?
- A healthy culture of the strain to be cryopreserved
- Cryovials: These are vials that tolerate ultra-cold temperatures. There are many different types made by a variety manufacturers. We use cryovials with caps that thread internally and have gaskettes. We also use starfoot cryovials, along with special racks made specifically to accommodate starfoot vials. The racks lock the vials in place so that the caps can be unscrewed with one hand, leaving the other free. Cryovials must be labeled before they are exposed to ultra-cold temperatures.
- Cryoprotective Agent (CPA): The CPAs most often used at the NCMA are dimethylsulfoxyide (DMSO) and Methanol (MeOH) at different concentrations.
- A means of controlling the rate of cooling at one degree per minute: A cooling rate of 1°C is a good rate of cooling for successful cryopreservation. We use a controlled rate freezer (CRF) to achieve this rate. A CRF can be programmed to cool at any temperature and compensate for heat released during the freezing process. It can also cryopreserve many samples at once. However, CRFs are expensive. Other devices used to moderate the rate of freezing to ~ 1°C per minute are Mr. Frostie (Nunc/ThermoScientific), CoolCell (Biocision),or a Styrofoam box with one inch walls. All of those cool at approximately 1°C per minute when placed in a -80°C freezer.
- A storage area with temperatures lower than -137ºC: It is essential to store cryopreserved material colder than the temperature at which there is no more ice crystal growth. Appropriate storage temperatures can be achieved by storing in liquid nitrogen (LN2) vapor, LN2 (not recommended) or a mechanical -150°C freezer.
- An inventory system: It is difficult to search for cryopreserved samples once they are at their storage temperature (LN2 vapor is -156 °C). Therefore, a good inventory system is essential.
- Other useful accessories: A grabber made of cold tolerant material is useful for retrieving vials at the bottom of storage tanks. A wet vac ( also known as a shop vac) is useful to remove the “fog”( LN2 vapor) from the top of storage tanks allowing for visibility into the storage tank.
5. SETTING UP A CRYO-LABORATORY
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.
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