Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO(®)-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO(®)-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.

Laboratory of Microbiology Parasitology and Hygiene S7 Faculty of Pharmaceutical Biomedical and Veterinary Sciences University of Antwerp Universiteitsplein 1 2610 Wilrijk Belgium

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Antimicrob Agents Chemother. 2010 Sep;54(9):3746-55 PubMed

Clin Med Res. 2005 Aug;3(3):176-85 PubMed

Curr Pharm Des. 2010;16(20):2279-95 PubMed

J Med Microbiol. 2011 Dec;60(Pt 12):1717-24 PubMed

Lett Appl Microbiol. 2014 May;58(5):447-53 PubMed

Water Res. 2010 Aug;44(15):4325-39 PubMed

J Appl Microbiol. 2010 Jul;109(1):91-8 PubMed

Appl Environ Microbiol. 2007 Nov;73(21):7023-8 PubMed

J Small Anim Pract. 2014 Mar;55(3):153-9 PubMed

J Funct Biomater. 2012 Jun 01;3(2):418-31 PubMed

J Microbiol Methods. 2008 Feb;72 (2):157-65 PubMed

J Microbiol Methods. 2007 Feb;68(2):421-3 PubMed

Int J Antimicrob Agents. 2009 Jun;33(6):525-31 PubMed

Appl Microbiol Biotechnol. 2002 Sep;59(6):629-40 PubMed

J Med Microbiol. 2010 Jan;59(Pt 1):1-7 PubMed

Clin Microbiol Rev. 2002 Apr;15(2):155-66 PubMed

BMC Microbiol. 2013 Dec 04;13:278 PubMed

BMC Ecol. 2006 Jan 16;6:1 PubMed

Methods Mol Biol. 2012;835:121-32 PubMed

Antimicrob Agents Chemother. 2006 Nov;50(11):3839-46 PubMed

J Microbiol Methods. 2010 Nov;83(2):89-105 PubMed

Clin Microbiol Infect. 2013 Feb;19(2):107-12 PubMed

FEMS Microbiol Ecol. 2009 Jan;67(1):6-20 PubMed

Can J Microbiol. 2011 Oct;57(10):850-6 PubMed

J Microbiol Methods. 2000 Sep;42(1):97-114 PubMed

J Antimicrob Chemother. 2009 Jan;63(1):129-35 PubMed

J Appl Microbiol. 2004;96(5):1067-73 PubMed

FEMS Immunol Med Microbiol. 2011 Mar;61(2):133-40 PubMed

Trends Microbiol. 2005 Jan;13(1):34-40 PubMed

J Microbiol Methods. 2014 Mar;98:31-4 PubMed

Lett Appl Microbiol. 2008 Feb;46(2):249-54 PubMed

Appl Environ Microbiol. 1999 Aug;65(8):3502-11 PubMed

Indian J Microbiol. 2013 Sep;53(3):269-75 PubMed

J Periodontal Res. 2013 Aug;48(4):517-26 PubMed

J Microbiol Methods. 2013 Feb 15;92(2):189-91 PubMed

PLoS One. 2011 Mar 29;6(3):e14786 PubMed

J Biosci Bioeng. 2006 Jan;101(1):1-8 PubMed

J Dent. 2013 Jun;41(6):521-7 PubMed

FEMS Microbiol Rev. 2010 Jul;34(4):554-87 PubMed

J Antimicrob Chemother. 2003 Nov;52(5):782-9 PubMed

Appl Environ Microbiol. 2012 Aug;78(15):5138-42 PubMed