Archives of Microbiology

, Volume 107, Issue 1, pp 57–62 | Cite as

The cryopreservation of Chlorella 1. Interactions of rate of cooling, protective additive and warming rate

  • G. J. Morris


The cryoprotective additives glycerol and dimethylsulphoxide were found to be toxic to Chlorella cells at concentrations greater then 2.5% w/v. Polyvinylpyrrolidone, was not damaging up to a concentration of 15% w/v.

Chlorella 211/7a had a recovery rate greater than 95% at all rates of cooling studied. With Chlorella 211/8h the survival was lower than 0.1% at all rates examined. The addition of dimethylsulphoxide (5% w/v) to Chlorella 211/8h increased the recovery, particularly at the faster rates of cooling; with polyvinylpyrrolidone (10% w/v) there was an optimum range of cooling rate.

Cells of Chlorella 211/7a from the exponential phase of growth were found to be damaged both by a temperature reduction from 25°C to 0°C (thermal shock) and by freezing and thawing. In contrast cells from the stationary phase of growth were resistant to these stresses.

Key words

Chlorella Cryopreservation Cooling rate Warming rate Thermal shock 





N-2-hydroxyethylpiperazine-N′-2-ethansulphonic acid




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  1. Bird, C. J., McLachlan, J.: Cold-hardiness of zygotes and embryos of Fucus (Phaeophyceae, Fucales). Phycologia 13, 215–225 (1974)Google Scholar
  2. Cheng, K. H., Grodzinski, B., Colman, B.: Inhibition of photosynthesis in algae by dimethyl sulfoxide. J. Phycol. 8, 399–400 (1972)Google Scholar
  3. Farrant, J., Morris, G. J.: Thermal shock and dilution shock as the causes of freezing injury. Cryobiology. 10, 134–140 (1973)Google Scholar
  4. Forrest, H. S., van Baalen, C., Myers, J.: Occurrence of pteridines in a blue-green alga. Science. 125, 699–700 (1957)Google Scholar
  5. Gorrill, R. H., McNeil, E. M.: The effect of cold diluent on the viable count of Pseudomonas pyocyanea. J. gen. Microbiol. 22, 437–442 (1960)Google Scholar
  6. Heber, U., Tyankova, L., Santarius, K. A.: Effects of freezing on biological membranes in vivo and in vitro. Biochim. biophys. Acta (Amst.) 291, 23–37 (1973)Google Scholar
  7. Hegarty, C. P., Weeks, O. B.: Sensitivity of Escherichia coli to cold shock during the logarithmic growth phase. J. Bact. 39, 475–484 (1940)Google Scholar
  8. Holm-Hansen, O.: Viability of blue-green and green algae after freezing. Physiol. Plant. 16, 530–540 (1963)Google Scholar
  9. Holm-Hansen, O.: Preservation by freezing and freeze-drying. In: Phycological methods, J. R. Stein, ed., pp. 195–205. Cambridge: Cambridge University Press 1971Google Scholar
  10. Hwang, S., Horneland, W.: Survival of algal cultures after freezing by controlled and uncontrolled cooling. Cryobiology 1, 305–311 (1965)Google Scholar
  11. Jansz, E. R., MacLean, F. I.: Photosynthetic properties of extracts of Anacystis nidulans prepared by lysozyme digestion. Canad. J. Microbiol. 18, 1727–1731 (1972)Google Scholar
  12. Jansz, E. R., MacLean, F. I.: The effect of cold shock on the blue-green alga Anacystis nidulans. Canad. J. Microbiol. 9, 381–387 (1973)Google Scholar
  13. Kessler, E.: Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella. 1. Säureresistenz als taxonomisches Merkmal. Arch. Mikrobiol. 52, 291–296 (1963)Google Scholar
  14. Kessler, E.: Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella. 9. Salzresistenz als taxonomisches Merkmal. Arch. Mikrobiol. 100, 51–56 (1974)Google Scholar
  15. Leibo, S. P., Farrant, J., Mazur, P., Hanna, M. G., Smith, L. H.: Effects of freezing on marrow stem cell populations; interactions of cooling and warming rates in the presence of PVP, sucrose or glycerol. Cryobiology, 6, 315–332 (1970)Google Scholar
  16. Levitt, J.: Responses of plants to environmental stresses. New York-London: Academic Press 1972Google Scholar
  17. Lorenzen, H.: Temperatureinflüsse auf Chlorella pyrenoidosa unter besonderer Berücksichtigung der Zellentwicklung. Flora (Jena) 153, 554–592 (1963)Google Scholar
  18. Lovelock, J. E.: Haemolysis by thermal shock. Brit. J. Haemat 1, 117–129 (1955)Google Scholar
  19. Mazur, P.: Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. gen. Physiol. 47, 347–369 (1963)Google Scholar
  20. Mazur, P.: Freezing injury in plants. A. Rev. Plant. Physiol. 20, 419–448 (1969)Google Scholar
  21. Meynell, G. G.: The effect of sudden chilling on Escherichia coli. J. gen. Microbiol. 19, 380–389 (1958)Google Scholar
  22. Morris, G. J., Farrant, J.: Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to-196°C. Cryobiology 9, 173–181 (1972)Google Scholar
  23. Morris, G. J., Farrant, J.: Effects of cooling rate on thermal shock haemolysis. Cryobiology, 10, 119–125 (1973)Google Scholar
  24. Patterson, G. W.: The distribution of sterols in algae. Lipids 6, 120–127 (1971)Google Scholar
  25. Pirson, A., Lorenzen, H., Koepper, A.: A sensitive stage in synchronized cultures of Chlorella. Plant. Physiol. 34, 353–355 (1959)Google Scholar
  26. Plattner, H., Fischer, W. M., Schmitt, W. W., Bachman, L.: Freeze etching of cells without cryoprotectants. J. Cell Biol. 53, 116–126 (1972)Google Scholar
  27. Ring, K.: The effect of low temperatures on permeability in Streptomyces hydrogenans. Biochem. biophys. Res. Commun. 19, 576–581 (1965)Google Scholar
  28. Sakai, A., Yoshida, S.: Survival of plant tissue at super-low temperature. 6. Effects of cooling and rewarming rates on survival. Plant Physiol. 42, 1695–1701 (1967)Google Scholar
  29. Smith, A. U.: Effects of low temperatures on living cells and tissue. In: Biological applications of freezing and drying, R. J. C. Harris, ed., pp. 1–59. New York: Academic Press 1954Google Scholar
  30. Staehelin, A.: Die Ultrastruktur der Zellwand und des Chloroplasten von Chlorella. Z. Zellforsch. 74, 325–350 (1966)Google Scholar
  31. Strange, R. E., Dark, F. A.: Effect of c illing on Aerobacter aerogenes in aqueous suspension. J. gen. Microbiol. 29, 719–730 (1962)Google Scholar
  32. Thorpe, P., Knight, S. C., Farrant, J.: Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques. Cryobiology (in press)Google Scholar
  33. Toyokawa, K., Hollander, D. H.: Variation in sensitivity of Escherichia coli to freezing damage during the growth cycle. Proc. Soc. exp. Biol. (N. Y.) 92, 499–500 (1956)Google Scholar
  34. Tsuru, S.: Preservation of marine and fresh water algae by means of freezing and freeze-drying. Cryobiology 10, 445–452 (1973)Google Scholar

Copyright information

© Springer-Verlag 1976

Authors and Affiliations

  • G. J. Morris
    • 1
  1. 1.Culture Centre of Algae and ProtozoaCambridgeEngland

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