Folia Microbiologica

, Volume 60, Issue 4, pp 335–342 | Cite as

A flow cytometric approach to quantify biofilms

  • Monique Kerstens
  • Gaëlle Boulet
  • Marian Van kerckhoven
  • Sofie Clais
  • Ellen Lanckacker
  • Peter Delputte
  • Louis Maes
  • Paul Cos
Article
First Online:
Received:
Accepted:

Abstract

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.

Keywords

Crystal Violet Staining Flow Cytometric Assay Microtiter Plate Assay Thiazole Orange Viable Plate Counting 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

A

Absorbance

C. albicans

Candida albicans

CFU/mL

Colony forming units per milliliter

CV

Crystal violet

E. coli

Escherichia coli

FCM

Flow cytometry

PBS

Phosphate buffered saline

TP3

TO-PRO®-3 iodide

VBNC

Viable but non-culturable cell

VPC

Viable plate count

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Notes

Acknowledgments

This work was supported by the Research Foundation—Flanders (FWO; grant 24761), the University of Antwerp (BOF/GOA grant 25624), and the agency for Innovation by Science and Technology (IWT-SBO grant 120005). Monique Kerstens is a Ph.D. fellow of the FWO—Flanders.

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015

Authors and Affiliations

  • Monique Kerstens
    • 1
  • Gaëlle Boulet
    • 1
  • Marian Van kerckhoven
    • 1
  • Sofie Clais
    • 1
  • Ellen Lanckacker
    • 1
  • Peter Delputte
    • 1
  • Louis Maes
    • 1
  • Paul Cos
    • 1
  1. 1.Laboratory of Microbiology, Parasitology and Hygiene (LMPH), S7, Faculty of Pharmaceutical, Biomedical and Veterinary SciencesUniversity of AntwerpWilrijkBelgium

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