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Comparison of Different live/dead Stainings for Detection and Quantification of Adherent Microorganisms in the Initial Oral Biofilm

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

Clin Oral Investig. 2013 Apr;17(3):841-50.

PMID: 22821430

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP148504341	Calcein-AM		148504-34-1	Price

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