John G. Day and Mitzi M. DeVille. 1. Introduction .... Then add 0.5 g sodium acetate .... Colony formation in agar: Dilute control cultures (Section 2., item 2).
CHAPTER9
Cryopreservation John G. Day and Mitzi
of Algae M. DeVille
1. Introduction Eukaryotic microalgae and prokaryotic cyanobacteria (blue-green algae) have traditionally been maintained by serial subculture, with the frequency of transfer being largely determined by the growth characteristics of the strain. This technique has the dual disadvantages of being labor intensive and carrying risk of loss in genetic stability of the strain. Compared with other groups of microorganisms, relatively little research has been carried out on the development of long-term preservation methods for microalgae and cyanobacteria. Freeze-drying has not been found to be a successful biostorage method for microalgae, with very low levels of viability (4% of original population) (1,2) and a further reduction in viability on prolonged storage (3,4). The successful lyophilization of the cyanobacterium Nostoc muscorum, using a method similar to that detailed in Chapter 3, has been reported, with no observed reduction in viability after 5 yr storage (5). This technique has been adopted by a small number of researchers to preserve selected cyanobacterial strains. Cryopreservation has been successfully employed to maintain algae (6-8). Using an appropriate protocol, high levels of viability (>90%) may be observed (7,s) and viability levels are apparently unaffected by storage times up to 13 yr (6). No single protocol has been found to be successful for a wide range of organisms. Direct immersion in liquid nitrogen, either with or without the addition of cryoprotectants, has been successfully employed to preserve a small number of unicellular, Chlorococcales and cyanobacteria (9,lO). Most protocols that have been From: Methods m Molecular Bology, Vol. 38: Cryopreservation and Freeze-Drying Profocols Edited by: J. G. Day and M R McLellan Copyright Q 1995 Humana Press Inc., Totowa, NJ
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developed employ a simple two-step system with controlled/semicontrolled cooling from room temperature to an intermediate subzero holding temperature prior to plunging into liquid nitrogen. The majority of protocols in current use have been developed empirically with the prefreezing culture regime, cryoprotectant choice and concentration, cooling rate, and thawing regime being manipulated to minimize the amount of damage to the algal cells and hence maximize postthaw viability levels. Damage may be induced by changes in the composition of intra- and extracellular solutions, mechanical and physical stressescaused by ice formation, as well as direct cold shock and chilling injuries. High levels of viability are required to minimize the possibility of selecting an unrepresentative freeze-tolerant subpopulation. Furthermore, the relatively slow growth rate of algae (generation times in the range of 8-72 h are not uncommon), along with other problems associated with low viable cell density (see Note l), can result in practical difficulties in reestablishing a viable culture. At the Culture Collection of Algae and Protozoa (CCAP), a minimum level of 50% postthaw viability is required before an algal strain is maintained only in a cryopreserved state. Currently, approx 30% of the collection is cryopreserved and another 5%, where viabilities are in the range of 15-50%, are maintained by both serial subculture and cryopreservation. Only a relatively small taxonomic section of the algae currently held in culture collections has been successfully preserved. At present, 380 of the 470 strains cryopreserved at CCAP are members of the Chlorococtales or Cyanophyceae; most of these have been frozen using the protocol outlined below (see Section 3., steps l-8). 2. Materials 1. Laminar flow cabinetand appropriatefacilities for following good microbiological practice. 2. Culture: Use late log or early stationaryphasecultures if available (seeNote 2).
3. Medium: This ISdependenton the nutritional requirementsof the alga. For strains currently cryopreserved,the most commonly employed media are detailed in items 4-6.
4. BG 11 medium (Table l), used for freshwater cyanobacteria and nonaxenic
algae. Add 100 mL, of solution 1, 10 mL of solutions 2-8, and 1 mL of solution 9 to distilled or deionized water to obtam a total vol of 1 L. pH is adjustedto 7.8 prior to sterilization.
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of Algae Table 1 BG 11 Medium Stock solutions NaN03 KzHP04a3H20 MgSO,.7H,O CaC1,.2H,O Citric acid Ferric ammonium citrate EDTA (disodium salt) Na2C03 Trace metal mixture:
15.0a 4.0 7.5 36 0.6 0.6 0.1 2.0
2.86 MnC12+4Hz0 1.81 0.222 ZnS04e7Hz0 0.39 Na2Mo04-2Hz0 0.079 CuS04.5Hz0 0.0494 Co(NO,),.6H,O OMaybe omltted for nitrogen fixmg cyanobactena. Based on ref. II. H3J303
5. Euglena gracilis: Jaworski’s medium (Table 2), used for axenic freshwater
6.
7.
8. 9. 10.
Chlorococcales. Add 0.5 mL of solutions l-10 to distilled or deionized water to obtain a total vol of 1 L of medium. Then add 0.5 g sodium acetate (trihydrate), 0.5 g Lab-Lemco powder (Oxoid L29, Unipath Ltd., Basingstoke, UK), 1.0 g tryptone, and 1.0 g yeast extract. Guillard’s (f/2) medium (Table 3), used for marine algae. Add 10 mL of solution 1 and 1 mL of solutions 2-4 to filtered natural seawater to obtain a total vol of 1 L. Adjust pH to 8.0 prior to sterilization. Solution 5 (1 mL) is added to the medium prior to pH adjustment and sterilization if the medium is to be used for diatoms. Cryoprotectant: 10% (v/v) Dimethyl sulfoxide (DMSO) in the appropriate medium. DMSO is cytotoxrc and care should be taken when handling. Alternative cryoprotectants are also regularly employed for some strains (see Note 3). Cryovials: Presterilized plastic screwcap 1.8 mL cryovlals (Costar, Cambridge MA) (see Note 4). Refrigeration system: Refrigerated methanol bath precooled to -30°C. Note: Methanol is toxic and flammable. Alternatively, more controlled rate coolers may also be used (see Note 5). Liquid nitrogen dewar: Small l-2 L wide-neck dewar.
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Day and DeVille Table 2 Euglena gracilis: Jaworski’s Medium
Stock solutions Ca(N0&4H20 KH2P04 MgSO,.7H,O NaHCO, CaClz EDTA FeNa EDTA Naz H3BO3
MnC12.4Hz0 WH,)~o,O,~4H,O Cyanocobalamin (vitamin Bt2) Thiamine HCl (vitamin Bt) Biotin NaN03 N%HPO,- 12H,O
g/200 mL
4.0 2.48 10.0 3.18 2.00 0.45 0.45 0.496 0.278 02 0.008 0.008 0.008 16.0 7.2
Based on ref. 12.
Table 3 Guillard’s (f/2) Medium Stock solutions NaN03
NaH2P04.2H20 NazEDTA FeC1s.6H20 CuS04-5H20 ZnS04.7H20 CoC12*6H20 MnC12.4H20 NazMo04.2Hz0 Cyanocobalamin (vitamin B12) Thiamine HCl (vitamin B,) Biotin NazSi03.9H20
7.5 5.65 4.36 3.15 0.01 0.022 0.01 0.18 0.006 0.5Q 100.0* 0.5a 40.18
@mg/l. Based on ref 12
11. Safety equipment: Long forceps, cryogloves, cryoapron, goggles. 12. Storage system: Cryogenic storage containers, with appropriate storage racks and inventory system suitable for holdmg cryovials (see Note 6).
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13. Fluorescein diacetate (FDA) stain stock solution: 25 mg fluorescein diacetate in 25 mL of acetone.
3. Method All culture manipulations should be carried out following good microbiological practice/aseptic technique, in a laminar flow cabinet if possible. 1. Grow cultures in the appropriate medium (see Section 2., items 3-6) under controlled environmental conditions. Flasks (50 mL) containing 30 mL of medium should be incubated static at 15OC under a photo fluence rate of 25-100 pmol m2/s (see Note 7). Light should be provided on a 1ight:dark cycle, 16:8 h is generally regarded as optimal. Incubate cultures until they reach stationary phase; 30 d is used as a standard culture interval at CCAP. 2. Aseptically transfer sedimented cells with 15 mL of medium into a presterilized beaker. Alternatively, for uniform cell suspensions, centrifuge at 500g for 10 min then decant the supernatant and resuspend the algae in 15 mL of fresh sterile medium. Remove a 5-mL aliquot for use as a control for viability assays (see Section 3., step 11). 3. Add 10 mL of medium containing 10% (v/v) DMSO in culture medium to the remaining 10 mL of dense culture and mix thoroughly to give a final concentration of 5% (v/v) DMSO. 4. Dispense the mixture in I-mL aliquots into sterile prelabeled cryovials. Seal the vials and incubate at room temperature for 5 min (see Note 8). 5. Transfer the filled cryovials to a precooled refrigerated bath (-30°C) and incubate for 15 min (see Note 9). 6. Transfer the vials rapidly, using forceps, to a wide-necked dewar containing liquid nitrogen. Then transport the vials in the dewar to the storage system. 7. Transfer the vials rapidly, using long forceps, to the storage/racking system. Storage is generally in the liquid phase of liquid nitrogen (see Note 6). 8. A full inventory/stocklist, including the locations of the vials within the storage system, should be maintained. This can most easily be retained either on a computer database, as card indices, or any other appropriate systems. 9. Thawing: Transfer stored cryovials (as in Section 3., step 6) to a dewar containing liquid nitrogen for transport and temporary storage, When ready, place the vials in a preheated water bath (40°C) and agitate until the last ice crystal has melted. On completion of the thawing remove immediately and transfer to the larninar flow cabinet (see Note 10).
Day and DeVille 10. Wipe the cryovial with 70% (v/v) ethanol to sterilize the outer surface of the vial. Aseptically transfer the contents mto 30 mL of appropriate sterile medium and incubate as detailed in step 1 (see Note 11). 11. Viability assays: a. Vital staining: Add 50 pL of FDA stain stock solution to 1 mL of culture, incubate at room temperature for 5 mm, and observe by blue light fluorescence microscopy (see Note 12). Viable cells fluoresce green (FDA positive) and nonviable cells appear red or colorless. It is necessary to dilute out cryoprotectant by transferring the thawed culture into 9 mL of appropriate sterile medium and incubating for up to 24 h prior to staining (seeNote 13). Viability is expressedas a percentage of control (nontreated unfrozen culture; Section 2., item 2) vs FDA positive cells. b. Colony formation in agar: Dilute control cultures (Section 2., item 2) 1:l with sterile medium. Transfer 1-mL aliquots of logarithmic dilutions of control and freeze-thawed cultures into sterile Petri dishes (50mm diameter). Add approx 2.5 mL of 1.O%(w/v) agar in the appropriate medium at 4O”C, agitate gently to mix culture, and agar. When the agar has gelled, seal the Petri dishes with Parafilm and incubate under standard growth conditions as detailed in step 1 (see Note 14). Colonies are counted using a dissecting microscope (magnification x50) and the viability of thawed cultures expressed as a percentage of control culture. Where cultures form clumps or aggregates, colonies may arise from either an individual or clump of viable cells. Cellular multiphcity must be taken into account for both control and frozen and thawed cultures. The frequency of cell aggregation is determined by microscopic observation of at least 500 cells and the percentage postthaw viability determined as detailed: Multiplicity = No. cells/No. clumps Total cell number = No. colonies x multiplicity Percentage viability = Total no. cells before treatment x lOO/ Total no. cells after treatment (see Note 16).
4. Notes 1. At low cell densities, photoinhibition may occur as a result of the lack of self-shading by other algal cells. This in turn may result in the death of cells that have survived cryopreservation. Where cultures are not axemc, low postthaw viabilities may result m the overgrowth of the alga by the competing associated microbial flora. 2. Stationary phase cultures are denser, thus reducmg the time required to reestablish a viable culture. Also, stationary phase cultures may contam
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4.
5. 6.
7. 8. 9. 10.
11.
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mtracellular storage products, including lipids, which may act as additional cryoprotectants. DMSO may also be used at 10% (v/v) final concentration. Alternatively, glycerol at either 5 or 10% (v/v) or methanol at 5 or 10% (v/v) may be used. DMSO and glycerol are sterilized by autoclaving at double the final concentration in culture medium. Methanol is sterilized by filter sterilizing through an alcohol-stable 0.2~pm filter. Choice of cryoprotectant is largely dependent on its effectiveness and its cytotoxicity to the algal strain being frozen. Costar vials have an internal rubber O-ring that appears to reduce the likelihood of liquid nitrogen leaking into the vial. Alternative cryovials, when used at CCAP, tend to have a significantly greater incidence of nitrogen leakage and subsequent potential contamination of algal cultures when stored under liquid nitrogen. Leakage can also lead to the possible rupturing of the vial on thawing, which has obvious safety implications (see also Note 10). Alternatively, Cryoflex (Nunc, Hastrup, Denmark) may be used. This will form a seal and prevent leakage into the vial. Alternative methods of cooling to the holding temperature include the use of controlled-rate coolers. Slow cooling rates (I-S”C/min) are generally used for many larger and more complex microalgae (13-15). Vials must be stored at below -135OC. This is achieved by storage either in the liquid or vapor phase in an insulated cryobin or alternatively in a more sophisticated autofill cryostore. At CCAP, liquid phase storage is preferred as this ensures that stored material is always maintained at below -135OC. Cyanobacteria, particularly when physiologically stressed, should be cultured at relatively low light levels. Light intensity is less critical for postthaw eukaryotic algae. For some slow and nonpenetrating cryoprotectants (e.g., glycerol), cultures are incubated for 30 min in the presence of cryoprotectant prior to cooling. For some algae, including members of the Prasinophyceae incubation for 30 min at -3OOC is used (see also Note 5). Liquid nitrogen leakage into the vial IS potentially dangerous. On thawing the nitrogen will evaporate and this could cause the vial to rupture. Care should be taken when handling vials containing nitrogen and full safety equipment-gloves, apron, goggles, and so on-should be used. Liquid nitrogen contains low levels of viable bacteria and these in turn may contaminate axenic cultures if leakage occurs. Where alternative frozen specimens are available vials containing liquid nitrogen should be discarded. For axenic cultures normally cultured on minimal mineral media, improved recovery rates may be obtained using medium with additional complex organic nitrogen, e.g., proteose peptone (Oxoid) at 1 g/L.
Day and DeVille 12. Temperature levels of the specimen increase rapidly during fluorescence microscopy and cells will die within l-2 min. Observations and counts must be performed rapidly to prevent an underestimate of the viability level. 13. An incubation period that allows repair of sublethal damage but that is too short for cell division to occur, will give a more accurate index of viability when using vital staining rather than staining immediately after thawing. 14. Exposure time to the hot agar should be minimized; 40°C for more than a few minutes will be lethal for many nonthermophilic algae. 15. For most algae incubation at 25°C will reduce the period required to obtain discrete countable colonies. 16. For many algae, spreadplates of logarithmic dilutions may be used instead of pour-plates. Spread 0.1 mL of logarithmic dilutions of control and frozen and thawed culture onto the surface of Petri dishes (90 mm) containing an appropriate agar (1.5% w/v) solidified medium. Incubate and enumerate as detailed for pour-plates.
References 1. Day, J. G. (in press) Cryopreservationof microalgae and cyanobacteria, in Proceedings of the Seventh International Congress of Culture Collections: Biodiversity and the Role of Culture Collections (Fritz, D., ed.), WFCC, Braunshweig,
Germany. 2. McGrath, M. S., Daggett, P., and Dilworth, S. (1978) Freeze-drying of algae: Chlorophyta and Chrysophyta. J. Phycol. 14,521-525. 3. Day, J. G., Priestley, I. M., and Codd, G. A. (1987) Storage, recovery and photosynthetic activities of immobilised algae, in Plant and Animal Cells, Process Possibilities (Webb, C. and Mavituna, F., eds.), Ellis Horwood, Chichester, UK, pp. 257-261. 4. Holm-Hansen, 0. (1967) Factors affecting the viability of lyophilized algae. Cryobiology 4, 17-23. 5. Holm-Hansen, 0. (1973) Preservation by freezing and freeze-drying, in Handbook of Phycological Methods: Culture Methods and Growth Measurements (Stein, J., ed.), Cambridge University Press, Cambridge, UK, pp. 173-205. 6. McLellan, M. R. (1989) Cryopreservation of diatoms. Diatom Res. 4,301-318. 7. Morris, G. J. (1976) The cryopreservation of Chlorella 1. Interactions of rate of cooling, protective additive and warming rate. Arch. Microbial. 107,57-62. 8. Morris, G. J. (1978) Cryopreservation of 250 strains of Chlorococcales by the method of two-step cooling. Br, Phycol. J. 13,15-24. 9. Holm-Hansen, 0. (1963) Viability of blue-green algae after freezing. Physiologia Planta. 16,530-539.
10. Box, J. G. (1988) Cryopreservationof the blue-greenalga Microcystis
aeruginosa.
Br. Phycol. J. 23,385,386.
11. Rippka, R., Dereuelles, J. B., Waterbury, J. B., and Herdman, M. (1979) Generic
assignments,strain histories and properties of pure cultures of cyanobacteria.J. Gen. Microbial.
111, l-61.
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12. Thompson, A. S., Rhodes, J. C., and Pettman, I. (1988) Culture Collection of Algae and Protozoa Catalogue ofStrains. Culture Collection of Algae and Protozoa, Ambleside, UK. 13. Morris, G. J., Coulson, G. E., and Engels, M. (1986) A cryomicroscopic study of Cylindrocystis brebissonii De Bary and two species of Micrasterias Ralfs (Conjugatophyceae, Chlorophyta) during freezing and thawing. J. Expt. Bat. 37,842-856. 14. Fenwick, C. and Day, J. G. (1992) Cryopreservation of Tetraselmis suecica cultured under different nutrients regimes. J. A&. Phycol. 4,105-109. 15. Watanabe, M. M., Shimizu, A., and Satake, K. N. (1992) NIBS-Microbial culture collection at The National Institute for Environmental Studies: cryopreservation and database of culture strains of algae, in Proceedings of the Symposium on Culture Collection of Algae (Watanabe, M. M., ed.), NIES, Tusukba, Japan, pp. 33-42.
