Header

UZH-Logo

Maintenance Infos

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


Tawakoli, P N; Al-Ahmad, A; Hoth-Hannig, W; Hannig, M; Hannig, C (2013). Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clinical oral investigations, 17(3):841-850.

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.

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.

Statistics

Citations

Dimensions.ai Metrics
66 citations in Web of Science®
68 citations in Scopus®
113 citations in Microsoft Academic
Google Scholar™

Altmetrics

Downloads

547 downloads since deposited on 06 Dec 2012
291 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Center for Dental Medicine > Clinic for Preventive Dentistry, Periodontology and Cariology
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2013
Deposited On:06 Dec 2012 15:20
Last Modified:16 Feb 2018 19:35
Publisher:Springer
Series Name:Clinical Oral Investigations
ISSN:1432-6981
OA Status:Green
Publisher DOI:https://doi.org/10.1007/s00784-012-0792-3
PubMed ID:22821430

Download

Download PDF  'Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm'.
Preview
Content: Accepted Version
Language: English
Filetype: PDF
Size: 1MB
View at publisher