Abstract
Microorganisms are valuable and irreplaceable resources for scientific research and biotechnological innovation and should be safeguarded. Therefore, systematic preservation of isolated pure cultures, enriched mixed cultures, or environmental samples should become an integral part of good research practice. Cryopreservation of biological material is a low-tech, widely applicable way of long-term and stable storage. Its success is mostly dependent on the cryoprotective agent, used to protect cells from mechanical injuries due to ice formation, the stability of the freezing temperature, and the correct manipulations before and after storage. Although cryopreservation success can be organism dependent, the protocol described here proved successful for various fastidious pure and mixed cultures when frozen at −80°C using 5% (v/v) dimethyl sulfoxide as cryoprotective agent. Numerous parameters of the protocol can be changed or optimized, and guidelines are given to develop a custom-made cryopreservation protocol.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Emerson D, Wilson W (2009) Giving microbial diversity a home. Nature Rev Microbiol 7:758
Joint I, Mühling M, Querellou J (2010) Culturing marine bacteria – an essential prerequisite for biodiscovery. Microbial Biotechnol 3:564–575
Stackebrandt E (2010) Diversification and focusing: strategies of microbial culture collections. Trends Microbiol 18:283–289
Cary SC, Fierer N (2014) The importance of sample archiving in microbial ecology. Nature Rev Microbiol 12:789–790
Paoli PD (2005) Biobanking in microbiology: from sample collection to epidemiology, diagnosis and research. FEMS Microbiol Rev 29:897–910
Morgan CA, Herman N, White PA, Vesey G (2006) Preservation of micro-organisms by drying: a review. J Microbiol Meth 66:183–193
Smith D (2003) Culture collections over the world. Int Microbiol 6:95–100
Janssens D, Arahal DR, Bizet C, Garay E (2010) The role of public biological resource centers in providing a basic infrastructure for microbial research. Res Microbiol 16:422–429
Vogelsang C, Gollenbiewski K, Ostgaard K (1999) Effect of preservation techniques on the regeneration of gel entrapped nitrifying sludge. Water Res 33:164–168
Laurin V, Labbe N, Juteau P, Parent S, Villemur R (2006) Long-term storage conditions for carriers with denitrifying biomass of the fluidized, methanol-fed denitrification reactor of the Montreal Biodome, and the impact on denitrifying activity and bacterial population. Water Res 40:1836–1840
Rothrock MJ, Vanotti MB, Szögi AA, Gonzalez MCG, Fuji T (2011) Long-term preservation of anammox bacteria. Appl Microbiol Biotechnol 92:147–157
Heylen K, Ettwig K, Hu Z, Jetten M, Kartal B (2012) Rapid and simple cryopreservation of anaerobic ammonium-oxidizing bacteria. Appl Environ Microbiol 78:3010–3013
Kerckhof FM, Courtens EN, Geirnaert A, Hoefman S, Ho A, Vilchez-Vargas R et al (2014) Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity. PLoS One 9:e99517
Smith D, Ryan MJ (2008) The impact of OECD best practices on the validation of cryopreservation techniques for microorganisms. CryoLetters 29:63–72
Vekeman B, Hoefman S, De Vos P, Spieck E, Heylen K (2013) A generally applicable cryopreservation method for nitrite-oxidizing bacteria. Syst Appl Microbiol 36:579–584
Hoefman S, Van Hoorde K, Boon N, Vandamme P, De Vos P, Heylen K (2012) Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. Plos One 7:e34196
Hoefman S, Pommerening-Roser A, Samyn E, De Vos P, Heylen K (2013) Efficient cryopreservation protocol enables accessibility of a broad range of ammonia-oxidizing bacteria for the scientific community. Res Microbiol 164:288–292
Hubàlek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229
Mazur P (1984) Freezing of living cells: mechanisms and implications. Am J Physiol 247:125–142
Tindall BJ (2007) Vacuum-drying and cryopreservation of prokaryotes. In: Day JG, Stacey GN (eds) Cryopreservation and freeze-drying protocols. Humana, Totowa, pp 73–98
Food Safety and Inspection Service (2008) Most probable number procedures and tables. MLG Appendix 2.03. Microbiology Laboratory Guidebook. http://www.fsis.usda.gov/PDF/MLG_Appendix_2_03.pdf
Baati L, Fabre-Gea C, Auriol D, Blanc PJ (2000) Study of the cryotolerance of Lactobacillus acidophilus: effect of culture and freezing conditions on the viability and cellular protein levels. Int J Food Microbiol 59:241–247
Stacey GN, Day JG (2007) Long-term ex situ conservation of biological resources and the role of biological resource centers. In: Day JG, Stacey GN (eds) Cryopreservation and freeze-drying protocols. Humana, Totowa, pp 1–14
Pegg DE (2007) Principles of cryopreservation. In: Day JG, Stacey GN (eds) Cryopreservation and freeze-drying protocols. Humana, Totowa, pp 39–58
Vandamme P, Pot B, Gillis M, Vos PD, Kerstens K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60:407–438
Zeigler DR (2003) Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53:1893–1900
Marzorati M, Wittebolle L, Boon N, Daffonchio D, Verstraete W (2008) How to get more out of molecular fingerprints: practical tools for microbial ecology. Environ Microbiol 10:1571–1581
Baust JM (2002) Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell Preserv Technol 1:17–31
Fuller BJ (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. CryoLetters 25:375–388
Siaterlis A, Deepika G, Charalampopoulos D (2009) Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying. Lett Appl Microbiol 48:295–301
Wang Y, Claeys L, Ha DVD, Verstraete W, Boon N (2010) Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry. Appl Microbiol Biotechnol 87:331–341
Vartoukian SR, Palmer RM, Wade WG (2010) Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiol Lett 309:1–7
Bruns A, Cypionka H, Overmann J (2002) Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the Central Baltic Sea. Appl Environ Microbiol 68:3978–3987
Nichols D, Lewis K, Orjala J, Mo S, Ortenberg R, O’Connor P et al (2008) Short peptide induces an “uncultivable” microorganism to grow in vitro. Appl Environ Microbiol 74:4889–4897
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Vekeman, B., Heylen, K. (2015). Preservation of Microbial Pure Cultures and Mixed Communities. In: McGenity, T., Timmis, K., Nogales , B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_51
Download citation
DOI: https://doi.org/10.1007/8623_2015_51
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-662-45178-6
Online ISBN: 978-3-662-45179-3
eBook Packages: Springer Protocols