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Clinical Oral Investigations

Published in:

01-04-2013 | Original Article

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

Authors: P. N. Tawakoli, A. Al-Ahmad, W. Hoth-Hannig, M. Hannig, C. Hannig

Published in: Clinical Oral Investigations | Issue 3/2013

Abstract

Objectives

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.

Results

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.

Conclusion

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.

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Title: Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm
Authors: P. N. Tawakoli
A. Al-Ahmad
W. Hoth-Hannig
M. Hannig
C. Hannig
Publication date: 01-04-2013
Publisher: Springer-Verlag
Published in: Clinical Oral Investigations / Issue 3/2013
Print ISSN: 1432-6981
Electronic ISSN: 1436-3771
DOI: https://doi.org/10.1007/s00784-012-0792-3

At a glance: The STEP trials

Springer Medicine

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

Abstract

Objectives

Materials and methods

Results

Conclusion

Clinical relevance

At a glance: The STEP trials

Springer Medicine

Abstract

Objectives

Materials and methods

Results

Conclusion

Clinical relevance

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