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Comparative Study
. 2013 Apr;17(3):841-50.
doi: 10.1007/s00784-012-0792-3. Epub 2012 Jul 21.

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

P N Tawakoli  1 , A Al-Ahmad, W Hoth-Hannig, M Hannig, C Hannig
Affiliations
Free article
Comparative Study

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

P N Tawakoli et al. Clin Oral Investig. 2013 Apr.
Free article

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 × 10(4) 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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