Cryopreservation of Cyanobacteria

By Jerry J. Brand, Botany Dept., Univ. of Texas at Austin

Cryopreservation refers to the storage of a living organism at ultra- low-temperature such that it can be revived and restored to the same living state as before it was stored. Indefinitely long storage times require that the organism be maintained below the glass transformation temperature of aqueous solutions, approximately -130degC, the temperature at which frozen water no longer sublimes and recrystallizes. Although ultra-cold freezers may stabilize some living cells for weeks or even years, liquid nitrogen is required for longer storage times. Cyanobacteria, as is the case for all living cells, suffer severe osmotic stress and/or ice crystal damage during the freezing and thawing processes. The most effective known ways to minimize these potentially lethal effects are to add a cryoprotective compound to the culture prior to its freezing for storage, and to control the transient cooling and warming rates during preservation.

We have been able to cryopreserve virtually all of the approximately 200 strains of cyanobacteria in the UTEX collection of algae [R.C. Starr and J.A. Zeikus (1993) J Phycol 29 (supp)] located in the Department of Botany at the University of Texas at Austin. This includes unicells, branching and unbranching filamentous species, marine and freshwater species, and those with heterocysts and akinetes. We also have successfully stored several photosynthetic mutants of cyanobacterial species provided by Robert Tabita of Ohio State University and John Golbeck of the University of Nebraska. The required procedures are straightforward and inexpensive, but require attention to a few details. One-hundred percent viability is never expected but viabilities over 50% are typical. High viabilities (i.e. >10%) are especially desirable for mutant strains. Here I will describe procedures that cryopreserve nearly all of cyanobacterial strains we have examined.

SAMPLE PREPARATION: Transfer cyanobacterial liquid culture into a cryovial[1] in preparation for cryopreservation. Alternatively the culture can be grown directly as a lawn on a tiny agar slant prepared within the cryovial [K. Bodas, K.R. Diller, and J.J. Brand (1995) Cryo-Letters 16:267-274]. If the cryovial contains liquid culture, pellet the cells by centrifugation in a clinical centrifuge and discard the supernatant [2]. Add cryoprotective solution containing 1.0 ml of half-strength growth medium (BG-11 works well for virtually all fresh-water cyanobacteria) containing 5% methanol or 8% DMSO to the pelleted cells [3]. Alternatively, if the culture is growing on an agar slant, transfer 1 ml of cryoprotective solution above the slant [4,5].

[1] Two-ml or 1.8-ml polyethylene or polypropylene cryovials are especially convenient for handling and storage efficiency, although 1-ml and 5-ml cryovials also work well.

[2] We have constructed acrylic sleeves that fit into the tube holders of the rotor, positioning the cryovials securely in place within a clinical centrifuge rotor, flush with the top of the tube holders. The cryovials can also be inserted into unmodified tube holders for centrifugation in a clinical centrifuge.

[3] Although glycerol is an effective cryoprotective agent for many bacteria, it is not effective for most cyanobacteria. Methanol at approximately 5% (v/v) is suitable for most strains. However, we have been successful with concentrations of methanol ranging from 2% to 12.5%, and DMSO ranging from 4 to 15 %, depending on the culture. A small fraction of some cultures survive with no added cryoprotective agent.

[4] When the cryoprotective agent is added directly above the culture on an agar slant, the tube is shaken gently prior to freezing, to dislodge some of the cells and ensure that the liquid penetrates through the culture. Cells pelleted from liquid suspension are fully suspended in the cryoprotective solution.

[5] Cells are killed by exposure to bright light when in cryoprotective solution. Keep the culture in subdued room light while handling, and in complete darkness at other times.

FREEZING: The cryovial containing the culture in cryoprotective agent at room temperature is inserted into a special "freezing container"[6] which has been pre-chilled to refrigerator temperature. The freezing container is then placed into a -70degC freezer for 2 hours. Then the cryovial is quickly removed from the freezing container, placed into a storage container, and plunged into liquid nitrogen for indefinite storage.[7]

[6] The "Mr. Frosty" freezing container (Nalgene) is satisfactory for nearly all cyanobacteria. It is inexpensive to purchase and holds eighteen 2-ml cryovials simultaneously. Its contents cool at slightly less than 1degC per minute when it is placed into a -70degC freezer.

[7] Sterility is a problem when storing plastic cryovials in liquid nitrogen. Vials equipped with gaskets and those with inside threads seal most tightly, but liquid nitrogen always creeps into some cryovials. This provides a conduit for entry of bacteria, some of which remain viable in bulk liquid nitrogen. Several manufacturers sell heat-shrink tubing that serves as a tight-fitting sleeve around the entire cryovial and lid, thereby eliminating liquid nitrogen leakage. Bacterial contamination can be eliminated also by storage in sealed glass ampoules or by storing plastic cryovials in the nitrogen vapor just above the liquid, although these procedures introduce additional safety and convenience considerations.

THAWING AND RECOVERY: Cultures to be revived are removed from liquid nitrogen storage and warmed rapidly to room temperature[8]. Cells are immediately pelleted by centrifugation of the cryovial[9,10], and the supernatant is discarded. One ml of fresh growth medium is placed into the vial to suspend the pellet. The cryovial lid is slightly loosened to allow gas exchange, and the contents of the vial are kept in complete darkness for 1-2 days. The culture can then be placed on agar or in liquid growth media under normal growth conditions. The viable cells should begin normal growth within 1 - 2 days in light, although they are especially susceptible to damage by excessive light intensity for the first day or two of illumination.

[8] Warm rapidly by plunging the tightly sealed, still-frozen cryovials into a dish of water at 35degC. An appropriately selected volume of water will cool to approximately 25degC as the cryovial contents are warmed to that same temperature.

[9] Centrifugation of a thawed culture in a cryovial containing an agar slant is best done in an angle rotor that pellets the cells on the agar surface without appreciably altering the position of the agar in the tube.

[10] Cultures of eukaryotic algae especially, and cyanobacteria to some extent, are susceptible to mechanical damage during recovery from storage at low temperature. Cells should be pelleted at the minimum R.C.F. that facilitates pelleting. Excessive agitation should be avoided when suspending the pellet.

Comments welcome!

Jerry J. Brand, Botany Dept., Univ. of Texas at Austin

E-MAIL: JBrand@UTxsvs.Cc.UTexas.Edu