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Bacteriophage Therapy pp 219–230Cite as

Methods for Bacteriophage Preservation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1693))

Abstract

In a view of growing interest in bacteriophages as the most abundant members of microbial communities and as antibacterial agents, reliable methods for bacteriophage long-term preservation, that warrant the access to original or mutant stocks of unchanged properties, have become of crucial importance. A storage method that retains the infectivity of any kind of bacteriophage virions, either in a cell lysate or in a purified suspension, does not exist, due to the enormous diversity of bacteriophages and hence the differentiation of their sensitivity to various storage conditions. Here, we describe a method of long-term bacteriophage preservation, which is based on freezing of freshly infected susceptible bacteria at early stages of bacteriophage development. The infected bacteria release mature bacteriophages upon melting enabling the recovery of bacteriophage virions with high efficiency. The only limitation of this method is the sensitivity of bacteriophage host to deep-freezing, and thus it can be used for the long-term preservation of the vast majority of bacteriophages.

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References

  1. Ackermann HW, Tremblay D, Moineau S (2004) Long-term bacteriophage preservation. World Fed Cult Collect Newslett 38:35–40

    Google Scholar 

  2. Carlson K (2005) Appendix: working with bacteriophages: common techniques and methodological approaches. In: Kutter E, Sulakvelidze A (eds) Bacteriophages: biology and applications. CRC Press, London

    Google Scholar 

  3. Fortier LC, Moineau S (2009) Phage production and maintenance of stocks, including the expected stock lifetime. Methods Mol Biol 501:203–219

    Article  CAS  PubMed  Google Scholar 

  4. Zierdt CH (1988) Stabilities of lyophilized Staphylococcus aureus typing bacteriophages. Appl Environ Microbiol 54:2590

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Warren JC, Hatch MT (1969) Survival of T3 coliphage in varied extracellular environments. I Viability of the coliphage during storage and in aerosols. Appl Microbiol 17:256–261

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Clark WA (1962) Comparison of several methods for preserving bacteriophages. Appl Microbiol 10:466–471

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Clark WA, Horneland W, Klein AG (1962) Attempts to freeze some bacteriophages to ultralow temperatures. Appl Microbiol 10:463–465

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Clark WA, Geary D (1973) Proceedings: preservation of bacteriophages by freezing and freeze-drying. Cryobiology 10:351–360

    Article  CAS  PubMed  Google Scholar 

  9. Weber-Dąbrowska B, Jończyk-Matysiak E, Żaczek M, Łobocka M, Łusiak-Szelachowska M, Górski A (2016) Bacteriophage procurement for therapeutic purposes. Front Microbiol 7:1177

    PubMed  PubMed Central  Google Scholar 

  10. Merabishvili M, Pirnay JP, Verbeken G, Chanishvili N, Tediashvili M, Lashkhi N, Glonti T, Krylov V, Mast J, Van Parys L, Lavigne R, Volckaert G, Mattheus W, Verween G, De Corte P, Rose T, Jennes S, Zizi M, De Vos D, Vaneechoutte M (2009) Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. PLoS One 4(3):e4944

    Article  PubMed  PubMed Central  Google Scholar 

  11. Adriaenssens EM, Lehman SM, Vandersteegen K, Vandenheuvel D, Philippe DL, Cornelissen A, Clokie MR, García AJ, De Proft M, Maes M, Lavigne R (2012) CIM(®)monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles. Virology 434:265–270

    Google Scholar 

  12. Bourdin G, Schmitt B, Marvin Guy L, Germond JE, Zuber S, Michot L, Reuteler G, Brüssow H (2014) Amplification and purification of T4-like Escherichia coli phages for phage therapy: from laboratory to pilot scale. Appl Environ Microbiol 80:1469–1476

    Article  PubMed  PubMed Central  Google Scholar 

  13. American Type Culture Collection, American Type Culture Collection Bacterial Culture Guide, Tips and Techniques for Culturing Bacteria and Bacteriophages, p 35. 2012. http://www.atcc.org/en/Guides/Bacteriology-Culture-Guide.aspx

  14. Merabishvili M, Vervaet C, Pirnay JP, De Vos D, Verbeken G, Mast J, Chanishvili N, Vaneechoutte M (2013) Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization). PLoS One 8(7):e68797

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cuevas JM, Duffy S, Sanjuan R (2009) Point mutation rate of bacteriophage PhiX174. Genetics 183:747–749

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Drake JW (1991) A constant rate of spontaneous mutation in DNA-based microbes. Proc Natl Acad Sci U S A 88:7160–7164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Santos ME, Drake JW (1994) Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants. Genetics 138:553–564

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Wichman HA, Millstein J, Bull JJ (2005) Adaptive molecular evolution for 13,000 phage generations: a possible arms race. Genetics 170:19–31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Golec P, Dąbrowski K, Hejnowicz MS, Gozdek A, Łoś JM, Węgrzyn G, Łobocka MB, Łoś M (2011) A reliable method for storage of tailed phages. J Microbiol Methods 84:486–489

    Article  PubMed  Google Scholar 

  20. Koenig GL (2003) Viability of and plasmid retention in frozen recombinant Escherichia coli over time: a ten-year prospective study. Appl Environ Microbiol 69:6605–6609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Heckly RJ (1978) Preservation of microorganisms. Adv Appl Microbiol 24:1–53

    Article  CAS  PubMed  Google Scholar 

  22. Tedeschi R, De Paoli P (2011) Collection and preservation of frozen microorganisms. Methods Mol Biol 675:313–326

    Article  CAS  PubMed  Google Scholar 

  23. Kutter E, Kellenberger E, Carlson K, Eddy S, Neitzel J, Messinger L, North J, Guttma B (1994) Effect of bacterial growth conditions and physiology on T4 infection. In: Karam JD, Drake JW, Kreuzer KN, Mosig G, Hall DH, Eiserling FA, Black LW, Spicer EW, Kutter E, Carlson K, Miller ES (eds) Molecular biology of bacteriophage T4. ASM Press, Washington, DC, pp 406–418

    Google Scholar 

  24. Piuri M, Hatfull GF (2006) A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells. Mol Microbiol 62:1569–1585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bryan D, El-Shibiny A, Hobbs Z, Porter J, Kutter EM (2016) Bacteriophage T4 infection of stationary phase E. coli: life after log from a phage perspective. Front Microbiol 7:1391

    Article  PubMed  PubMed Central  Google Scholar 

  26. Simione FP, Brown EM (eds) (1991) ATCC preservation methods: freezing and freeze-drying. American Type Culture Collection, Rockville, MD

    Google Scholar 

  27. Nei T, Araki T, Matsusaka T (1969) Freezing injury to aerated and non-aerated cultures of Escherichia coli. In: Nei T (ed) Freezing and drying of microorganisms. University of Tokyo Press, Tokyo

    Google Scholar 

  28. Shafia F, Thompson TL (1964) Calcium ion requirement for proliferation of bacteriophage phi Mu-4. J Bacteriol 88:293–296

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Landry EF, Zsigray RM (1980) Effects of calcium on the lytic cycle of Bacillus subtilis phage 41c. J Gen Virol 51:125–135

    Article  CAS  PubMed  Google Scholar 

  30. Wentworth BB, Romig WR (1968) Recombinants of a defective lysogen of staphylococcal strains. Jpn J Microbiol 12:299–307

    Article  CAS  PubMed  Google Scholar 

  31. Kingsbury DT, Ordal EJ (1966) Bacteriophage infecting the myxobacterium Chondrococcus columnaris. J Bacteriol 91:1327–1332

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Welker NE, Campbell LL (1965) Induction and properties of a temperate bacteriophage from Bacillus stearothermophilus. J Bacteriol 89:175–184

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Santos MA, De Lencastre H, Archer LJ (1984) Homology between phages SPP1, 41c, 22a, rho 15 and SF6 of Bacillus subtilis. J Gen Virol 65:2067–2072

    Article  CAS  PubMed  Google Scholar 

  34. Storms ZJ, Arsenault E, Sauvageau D, Cooper DG (2010) Bacteriophage adsorption efficiency and its effect on amplification. Bioprocess Biosyst Eng 33:823–831

    Article  CAS  PubMed  Google Scholar 

  35. Fildes P, Kay D (1959) The function of tryptophan in the adsorption of a bacteriophage. Br J Exp Pathol 40:71–79

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Hyman P, Abedon ST (2009) Practical methods for determining phage growth parameters. Methods Mol Biol 501:175–202

    Article  CAS  PubMed  Google Scholar 

  37. Adams MH (1959) Enumeration of bacteriophage particles. In: Adams MH (ed) The bacteriophages. Interscience Publishers Inc., New York, NY

    Google Scholar 

  38. Rojas-Tapias D, Ortiz-Vera M, Rivera D, Kloepper J, Bonilla R (2013) Evaluation of three methods for preservation of Azotobacter chroococcum and Azotobacter vinelandii. Univ Sci 18:129–139

    Article  CAS  Google Scholar 

  39. Fonseca F, Marin M, Morris GJ (2006) Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: freezing kinetics and storage temperature effects. Appl Environ Microbiol 72:6474–6482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hubálek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by funds from the Operational Program “Innovative Economy, 2007–2013,” project No. POIG 01.03.01-02-003/08, and statutory funds for the Faculty of Agriculture and Biology of the Warsaw University of Life Sciences, SGGW.

Author information

Authors and Affiliations

  1. Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Pawińskiego 5A, Warsaw, 02-106, Poland

    Małgorzata B. Łobocka & Aleksandra Głowacka

  2. Faculty of Agriculture and Biology, Autonomous Department of Microbial Biology, Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, Warsaw, 02-776, Poland

    Małgorzata B. Łobocka

  3. Laboratory of Molecular Biology (affiliated with the University of Gdańsk), Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Pawińskiego 5A, Gdańsk, 02-106, Poland

    Piotr Golec

Authors
  1. Małgorzata B. Łobocka
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  2. Aleksandra Głowacka
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  3. Piotr Golec
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Corresponding author

Correspondence to Małgorzata B. Łobocka .

Editor information

Editors and Affiliations

  1. LIBRO-Laboratory of Research in Biofilms Rosário Oliveira, CEB – Centre of Biological Engineering, University of Minho, Braga, Portugal

    Joana Azeredo

  2. CEB – Centre of Biological Engineering, University of Minho, Braga, Portugal

    Sanna Sillankorva

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Łobocka, M.B., Głowacka, A., Golec, P. (2018). Methods for Bacteriophage Preservation. In: Azeredo, J., Sillankorva, S. (eds) Bacteriophage Therapy. Methods in Molecular Biology, vol 1693. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7395-8_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7395-8_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7394-1

  • Online ISBN: 978-1-4939-7395-8

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