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

  • Ilse Vandecandelaere
  • Heleen Van Acker
  • Tom CoenyeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1333)


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.


Biofilms In vitro model system Microtiter plate Crystal violet staining Resazurin assay Persisters 


  1. 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 95:342–349CrossRefPubMedGoogle Scholar
  2. 2.
    Flemming HC (2002) Biofouling in water systems – cases, causes and countermeasures. Appl Microbiol Biot 59:629–640CrossRefGoogle Scholar
  3. 3.
    Costerton JW (1999) Introduction to biofilm. Int J Antimicrob Ag 11:217–221CrossRefGoogle Scholar
  4. 4.
    Hall-Stoodley L, Stoodley P (2005) Biofilm formation and dispersal and the transmission of human pathogens. Trends Microbiol 13:7–10CrossRefPubMedGoogle Scholar
  5. 5.
    Lewis K (2001) Riddle of biofilm resistance. Antimicrob Agents Chemother 45:999–1007PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Coenye T, Nelis HJ (2010) In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods 83:89–105CrossRefPubMedGoogle Scholar
  7. 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 22:996–1006PubMedCentralPubMedGoogle Scholar
  8. 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 16–23Google Scholar
  9. 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 13–15Google Scholar
  10. 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 1106:157–166CrossRefPubMedGoogle Scholar
  11. 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 67:2531–2537PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Pitts B, Hamilton MA, Zelver N et al (2003) A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods 54:269–276CrossRefPubMedGoogle Scholar
  13. 13.
    Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72:157–165CrossRefPubMedGoogle Scholar
  14. 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 267:5421–5426CrossRefPubMedGoogle Scholar
  15. 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 114:1833–1842CrossRefPubMedGoogle Scholar
  16. 16.
    Vandenbosch D, Braeckmans K, Nelis HJ et al (2010) Fungicidal activity of miconazole against Candida spp. biofilms. J Antimicrob Chemother 65:694–700CrossRefPubMedGoogle Scholar
  17. 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:215–224Google Scholar
  18. 18.
    Ramsugit S, Guma S, Pillay B et al (2013) Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis. Antonie Van Leeuwenhoek 104:725–735CrossRefPubMedGoogle Scholar
  19. 19.
    Dapa T, Unnikrishnan M (2013) Biofilm formation by Clostridium difficile. Gut Microbes 4:397–402PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Vandecandelaere I, Depuydt P, Nelis HJ et al (2014) Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms. Pathog Dis 70:321–331Google Scholar
  21. 21.
    Stepanovic S, Vukovic D, Dakic I et al (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40:175–179CrossRefPubMedGoogle Scholar
  22. 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 60:359–373CrossRefPubMedGoogle Scholar
  23. 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 PubMedGoogle Scholar
  24. 24.
    Sosunov V, Mischenko V, Eruslanov B et al (2007) Antimycobacterial activity of bacteriocins and their complexes with liposomes. J Antimicrob Chemother 59:919–925CrossRefPubMedGoogle Scholar
  25. 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 13:56–62CrossRefPubMedGoogle Scholar
  26. 26.
    Martinez LR, Ibom DC, Casadevall A et al (2008) Characterization of phenotypic switching in Cryptococcus neoformans biofilms. Mycopathologia 166:175–180PubMedCentralCrossRefPubMedGoogle Scholar
  27. 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 160:144–151CrossRefPubMedGoogle Scholar
  28. 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 62:335–340CrossRefPubMedGoogle Scholar
  29. 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 94:192–199CrossRefGoogle Scholar
  30. 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 193:3598–3605PubMedCentralCrossRefPubMedGoogle Scholar
  31. 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 54:1082–1093PubMedCentralCrossRefPubMedGoogle Scholar
  32. 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, e58943PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Keren I, Minami S, Rubin E et al (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2:e00100-00111CrossRefGoogle Scholar
  34. 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 80:245–250PubMedCentralCrossRefPubMedGoogle Scholar
  35. 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 467:1360–1364PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ilse Vandecandelaere
    • 1
  • Heleen Van Acker
    • 1
  • Tom Coenye
    • 1
    Email author
  1. 1.Laboratory of Pharmaceutical MicrobiologyGhent UniversityGhentBelgium

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