Legionella pp 45-53 | Cite as

Quantification of Viable but Non-Culturable Cells of Legionella pneumophila

  • Rosalie S. Braun
  • Nilmini Mendis
  • Laam Li
  • Sebastien P. FaucherEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)


Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry.

Key words

Viable but non-culturable state Staining and labeling Flow cytometry Legionella pneumophila 



This work was supported by Discovery Grant 418289-2012 from the National Sciences and Engineering Research Council of Canada (NSERC) and a John R. Evans Leaders Fund—Funding for research infrastructure from the Canadian Foundation for Innovation to SPF. NM was the recipient of a PhD scholarship from Fond de Recherche du Québec—Nature et Technologie.


  1. 1.
    Baum von H, Ewig S, Marre R et al (2008) Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin Infect Dis 46:1356–1364. Scholar
  2. 2.
    Taylor M, Ross K, Bentham R (2009) Legionella, protozoa, and biofilms: interactions within complex microbial systems. Microb Ecol 58:538–547. Scholar
  3. 3.
    World health organization (2003) Emerging issues in water and infectious disease. pp 1–24Google Scholar
  4. 4.
    Lau HY, Ashbolt NJ (2009) The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol 107:368–378. Scholar
  5. 5.
    Li L, Mendis N, Trigui H et al (2014) The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol 5:258. Scholar
  6. 6.
    Steinert M, Emödy L, Amann R, Hacker J (1997) Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl Environ Microbiol 63:2047–2053PubMedPubMedCentralGoogle Scholar
  7. 7.
    Garcia MT, Jones S, Pelaz C et al (2007) Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection. Environ Microbiol 9:1267–1277. Scholar
  8. 8.
    Alleron L, Merlet N, Lacombe C, Frère J (2008) Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment. Curr Microbiol 57:497–502. Scholar
  9. 9.
    Alleron L, Khemiri A, Koubar M et al (2013) VBNC Legionella pneumophila cells are still able to produce virulence proteins. Water Res 47:6606–6617. Scholar
  10. 10.
    Buse HY, Donohue MJ, Ashbolt NJ (2013) Hartmannella vermiformis inhibition of Legionella pneumophila cultivability. Microb Ecol 66:715–726. Scholar
  11. 11.
    Li L, Faucher SP (2016) The membrane protein LasM promotes the culturability of Legionella pneumophila in water. Front Cell Infect Microbiol 6:1613. Scholar
  12. 12.
    Ducret A, Chabalier M, Dukan S (2014) Characterization and resuscitation of “non-culturable” cells of Legionella pneumophila. BMC Microbiol 14:3. Scholar
  13. 13.
    Aurass P, Gerlach T, Becher D et al (2016) Life stage-specific proteomes of Legionella pneumophila reveal a highly differential abundance of virulence-associated Dot/Icm effectors. Mol Cell Proteomics 15:177–200. Scholar
  14. 14.
    Mendis N, McBride P, Faucher SP (2015) Short-term and long-term survival and virulence of Legionella pneumophila in the defined freshwater medium Fraquil. PLoS One 10:e0139277. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Rosalie S. Braun
    • 1
    • 2
  • Nilmini Mendis
    • 2
  • Laam Li
    • 3
    • 2
  • Sebastien P. Faucher
    • 2
    Email author
  1. 1.New York University College of DentistryNew YorkUSA
  2. 2.Faculty of Agricultural and Environmental Sciences, Department of Natural Resource SciencesMcGill UniversitySte-Anne-de-BellevueCanada
  3. 3.Department of ChemistryCity University of Hong KongKowloonHong Kong

Personalised recommendations