Clinical Oral Investigations

, Volume 17, Issue 3, pp 841–850

Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm

  • P. N. Tawakoli
  • A. Al-Ahmad
  • W. Hoth-Hannig
  • M. Hannig
  • C. Hannig
Original Article

DOI: 10.1007/s00784-012-0792-3

Cite this article as:
Tawakoli, P.N., Al-Ahmad, A., Hoth-Hannig, W. et al. Clin Oral Invest (2013) 17: 841. doi:10.1007/s00784-012-0792-3



The aim of the present study was to investigate different fluorescence-based, two-color viability assays for visualization and quantification of initial bacterial adherence and to establish reliable alternatives to the ethidium bromide staining procedure.

Materials and methods

Bacterial colonization was attained in situ on bovine enamel slabs (n = 6 subjects). Five different live/dead assays were investigated (fluorescein diacetate (FDA)/propidium iodide (PI), Syto 9/PI (BacLight®), FDA/Sytox red, Calcein acetoxymethyl (AM)/Sytox red, and carboxyfluorescein diacetate (CFDA)/Sytox red). After 120 min of oral exposure, analysis was performed with an epifluorescence microscope. Validation was carried out, using the colony-forming units for quantification and the transmission electron microscopy for visualization after staining.


The average number of bacteria amounted to 2.9 ± 0.8 × 104 cm−2. Quantification with Syto 9/PI and Calcein AM/Sytox red yielded an almost equal distribution of cells (Syto 9/PI 45 % viable, 55 % avital; Calcein AM/Sytox red 52 % viable, 48 % avital). The live/dead ratio of CFDA/Sytox red and FDA/Sytox red was 3:2. An aberrant dispersal was recorded with FDA/PI (viable 34 %, avital 66 %). The TEM analysis indicated that all staining procedures affect the structural integrity of the bacterial cells considerably.


The following live/dead assays are reliable techniques for differentiation of viable and avital adherent bacteria: BacLight, FDA/Sytox red, Calcein AM/Sytox red, and CFDA/Sytox red. These fluorescence-based techniques are applicable alternatives to toxic and instable conventional assays, such as the staining procedure based on ethidium bromide.

Clinical relevance

Differentiation of viable and avital adherent bacteria offers the possibility for reliable evaluation of different mouth rinses, oral medication, and disinfections.


Live/dead staining Viability Adherent bacteria Initial biofilm CFU TEM 

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • P. N. Tawakoli
    • 1
  • A. Al-Ahmad
    • 2
  • W. Hoth-Hannig
    • 3
  • M. Hannig
    • 3
  • C. Hannig
    • 4
  1. 1.Clinic of Preventive Dentistry, Periodontology and Cariology, Center for Dental MedicineUniversity of ZurichZurichSwitzerland
  2. 2.Department of Operative Dentistry and PeriodontologyAlbert Ludwigs UniversityFreiburgGermany
  3. 3.Clinic of Operative Dentistry, Periodontology and Preventive DentistrySaarland UniversityHomburg/SaarGermany
  4. 4.Clinic of Conservative Dentistry, Faculty of Medicine Carl Gustav CarusTechnical University of DresdenDresdenGermany

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