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Bacterial Persistence pp 53–66Cite as

A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1333))

Abstract

We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (resazurin assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations.

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References

  1. Gomes LC, Moreira JM, Miranda JM et al (2013) Macroscale versus microscale methods for physiological analysis of biofilms formed in 96-well microtiter plates. J Microbiol Methods 95:342–349

    Article  CAS  PubMed  Google Scholar 

  2. Flemming HC (2002) Biofouling in water systems – cases, causes and countermeasures. Appl Microbiol Biot 59:629–640

    Article  CAS  Google Scholar 

  3. Costerton JW (1999) Introduction to biofilm. Int J Antimicrob Ag 11:217–221

    Article  CAS  Google Scholar 

  4. Hall-Stoodley L, Stoodley P (2005) Biofilm formation and dispersal and the transmission of human pathogens. Trends Microbiol 13:7–10

    Article  CAS  PubMed  Google Scholar 

  5. Lewis K (2001) Riddle of biofilm resistance. Antimicrob Agents Chemother 45:999–1007

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Coenye T, Nelis HJ (2010) In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods 83:89–105

    Article  CAS  PubMed  Google Scholar 

  7. Christensen GD, Simpson WA, Younger JJ et al (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006

    PubMed Central  CAS  PubMed  Google Scholar 

  8. Heersink J (2003) Basic biofilm analytical methods. In: Hamilton M, Heersink J, Buckingham-Meyer J, Goeres D (eds) The biofilm laboratory: step-by-step protocols for experimental design, analysis, and data interpretation. Cytergy Publishing, Bozeman, pp 16–23

    Google Scholar 

  9. Heersink J, Goeres D (2003) Reactor design considerations. In: Hamilton M, Heersink J, Buckingham-Meyer J, Goeres D (eds) The biofilm laboratory: step-by-step protocols for experimental design, analysis, and data interpretation. Cytergy Publishing, Bozeman, pp 13–15

    Google Scholar 

  10. Waters EM, McCarthy H, Hogan S et al (2014) Rapid quantitative and qualitative analysis of biofilm production by Staphylococcus epidermidis under static growth conditions. Methods Mol Biol 1106:157–166

    Article  PubMed  Google Scholar 

  11. Gomez-Suarez C, Busscher HJ, van der Mei HC (2001) Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces. Appl Environ Microbiol 67:2531–2537

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Pitts B, Hamilton MA, Zelver N et al (2003) A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods 54:269–276

    Article  CAS  PubMed  Google Scholar 

  13. Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72:157–165

    Article  CAS  PubMed  Google Scholar 

  14. O’Brien J, Wilson I, Orton T et al (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426

    Article  PubMed  Google Scholar 

  15. Brackman G, De Meyer L, Nelis HJ et al (2013) Biofilm inhibitory and eradicating activity of wound care products against Staphylococcus aureus and Staphylococcus epidermidis biofilms in an in vitro chronic wound model. J Appl Microbiol 114:1833–1842

    Article  CAS  PubMed  Google Scholar 

  16. Vandenbosch D, Braeckmans K, Nelis HJ et al (2010) Fungicidal activity of miconazole against Candida spp. biofilms. J Antimicrob Chemother 65:694–700

    Article  CAS  PubMed  Google Scholar 

  17. Braem A, Van Mellaert L, Mattheys T et al (2013) Staphylococcal biofilm growth on smooth and porous titanium coatings for biomedical applications. J Biomed Mater Res A 102A:215–224

    Google Scholar 

  18. Ramsugit S, Guma S, Pillay B et al (2013) Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis. Antonie Van Leeuwenhoek 104:725–735

    Article  PubMed  Google Scholar 

  19. Dapa T, Unnikrishnan M (2013) Biofilm formation by Clostridium difficile. Gut Microbes 4:397–402

    Article  PubMed Central  PubMed  Google Scholar 

  20. Vandecandelaere I, Depuydt P, Nelis HJ et al (2014) Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms. Pathog Dis 70:321–331

    Google Scholar 

  21. Stepanovic S, Vukovic D, Dakic I et al (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40:175–179

    Article  CAS  PubMed  Google Scholar 

  22. Herczegh A, Gyurkovics M, Agababyan H et al (2013) Comparing the efficacy of hyper-pure chlorine-dioxide with other oral antiseptics on oral pathogen microorganisms and biofilm in vitro. Acta Microbiol Immunol Hung 60:359–373

    Article  CAS  PubMed  Google Scholar 

  23. Delattin N, De Brucker K, Vandamme K et al (2013) Repurposing as a means to increase the activity of amphotericin B and caspofungin against Candida albicans biofilms. J Antimicrob Chemother. doi:10.1093/jac/dkt1449

    PubMed  Google Scholar 

  24. Sosunov V, Mischenko V, Eruslanov B et al (2007) Antimycobacterial activity of bacteriocins and their complexes with liposomes. J Antimicrob Chemother 59:919–925

    Article  CAS  PubMed  Google Scholar 

  25. Messiaen AS, Nelis H, Coenye T (2013) Investigating the role of matrix components in protection of Burkholderia cepacia complex biofilms against tobramycin. J Cyst Fibros 13:56–62

    Article  PubMed  Google Scholar 

  26. Martinez LR, Ibom DC, Casadevall A et al (2008) Characterization of phenotypic switching in Cryptococcus neoformans biofilms. Mycopathologia 166:175–180

    Article  PubMed Central  PubMed  Google Scholar 

  27. Brackman G, Hillaert U, Van Calenbergh S et al (2009) Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia. Res Microbiol 160:144–151

    Article  CAS  PubMed  Google Scholar 

  28. Ahiwale S, Tamboli N, Thorat K et al (2011) In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage. Curr Microbiol 62:335–340

    Article  CAS  PubMed  Google Scholar 

  29. Moreira JM, Gomes LC, Araujo JDP et al (2013) The effect of glucose concentration and shaking conditions on Escherichia coli biofilm formation in microtiter plates. Chem Eng Sci 94:192–199

    Article  CAS  Google Scholar 

  30. Luidalepp H, Joers A, Kaldalu N et al (2011) Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. J Bacteriol 193:3598–3605

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Fung DK, Chan EW, Chin ML et al (2010) Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development. Antimicrob Agents Chemother 54:1082–1093

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Van Acker H, Sass A, Bazzini S et al (2013) Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species. PLoS One 8, e58943

    Article  PubMed Central  PubMed  Google Scholar 

  33. Keren I, Minami S, Rubin E et al (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2:e00100-00111

    Article  Google Scholar 

  34. Bjerkan G, Witso E, Bergh K (2009) Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthop 80:245–250

    Article  PubMed Central  PubMed  Google Scholar 

  35. Kobayashi H, Oethinger M, Tuohy MJ et al (2009) Improved detection of biofilm-formative bacteria by vortexing and sonication: a pilot study. Clin Orthop Relat Res 467:1360–1364

    Article  PubMed Central  PubMed  Google Scholar 

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Author notes

    Authors and Affiliations

    1. Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, Ghent, 9000, Belgium

      Ilse Vandecandelaere, Heleen Van Acker & Tom Coenye

    Authors
    1. Ilse Vandecandelaere
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    2. Heleen Van Acker
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    3. Tom Coenye
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    Corresponding author

    Correspondence to Tom Coenye .

    Editor information

    Editors and Affiliations

    1. Department of Microbial and Molecular Systems, KU Leuven - University of Leuven, Heverlee, Belgium

      Jan Michiels

    2. Department of Microbial and Molecular Systems, KU Leuven - University of Leuven, Heverlee, Belgium

      Maarten Fauvart

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    Vandecandelaere, I., Van Acker, H., Coenye, T. (2016). A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification. In: Michiels, J., Fauvart, M. (eds) Bacterial Persistence. Methods in Molecular Biology, vol 1333. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2854-5_5

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    • DOI: https://doi.org/10.1007/978-1-4939-2854-5_5

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-2853-8

    • Online ISBN: 978-1-4939-2854-5

    • eBook Packages: Springer Protocols

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