Skip to main content
Hauptrubriken
CME Facharzt-Training Webinare Zeitschriften Bücher e.Medpedia Podcast
Service
Jobbörse Info & Hilfe
Fachgebiete
Anästhesiologie Allgemeinmedizin Arbeitsmedizin Augenheilkunde Chirurgie Dermatologie Gynäkologie und Geburtshilfe HNO Innere Medizin Kardiologie Neurologie Onkologie und Hämatologie Orthopädie und Unfallchirurgie Pädiatrie Pathologie Psychiatrie Radiologie Rechtsmedizin Urologie Zahnmedizin
Themen
Klimawandel und Gesundheit Neues aus dem Markt COVID-19 Praxis und Beruf Seltene Erkrankungen GOÄ & EBM Panorama
Sammlungen
Kasuistiken Algorithmen & Infografiken Blickdiagnosen Arthropedia FlapFinder (für Dermatochirurgen) Videos Kongressberichterstattung Medizin für Apothekerinnen und Apotheker Für Ärztinnen und Ärzte in Weiterbildung Für Medizinstudierende
Erweiterte Suche
Anmelden
nach oben
Clinical Oral Investigations
Erschienen in: Clinical Oral Investigations 3/2013

01.04.2013 | Original Article

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

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

Erschienen in: Clinical Oral Investigations | Ausgabe 3/2013

Einloggen, um Zugang zu erhalten

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.
Literatur
1.
Hannig C, Hannig M, Rehmer O, Braun G, Hellwig E, Al-Ahmad A (2007) Fluorescence microscopic visualization and quantification of initial bacterial colonization on enamel in situ. Arch Oral Biol 52:1048–1056PubMedCrossRef
2.
Hannig C, Hannig M (2009) Enzyme in der Pellikel—eine Synopsis. Dtsch Zahnärztl Z 64:533–547
3.
Lendenmann U, Grogan J, Oppenheim FG (2000) Saliva and dental pellicle—a review. Adv Dent Res 14:22–28PubMedCrossRef
4.
Hannig M, Joiner A (2006) The structure, function and properties of the acquired pellicle. Monogr Oral Sci 19:29–64PubMed
5.
Bowen WH, Koo H (2011) Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 45:69–86PubMedCrossRef
6.
Hannig M, Hannig C (2007) Der initiale orale Biofilm—pathogen oder protektiv? Oralprophylaxe 29:73–82
7.
Hannig M, Hannig C (2007) Does a dental biofilm, free of bacteria, exist in situ? Parodontologie et Implantologie orale 26:187–200
8.
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43:5721–5732PubMedCrossRef
9.
Thurnheer T, Gmur R, Giertsen E, Guggenheim B (2001) Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque. J Microbiol Methods 44:39–47PubMedCrossRef
10.
Al-Ahmad A, Follo M, Selzer AC, Hellwig E, Hannig M, Hannig C (2009) Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization. J Med Microbiol 58:1359–1366PubMedCrossRef
11.
Hannig C, Follo M, Hellwig E, Al-Ahmad A (2010) Visualization of adherent micro-organisms using different techniques. J Med Microbiol 59:1–7PubMedCrossRef
12.
Decker EM (2001) The ability of direct fluorescence-based, two-colour assays to detect different physiological states of oral streptococci. Lett Appl Microbiol 33:188–192PubMedCrossRef
13.
Netuschil L, Weiger R, Preisler R, Brecx M (1995) Plaque bacteria counts and vitality during chlorhexidine, meridol and listerine mouthrinses. Eur J Oral Sci 103:355–361PubMedCrossRef
14.
Netuschil L, Reisch E, Unteregger G, Sculean A, Brecx M (1998) A pilot study of confocal laser scanning microscopy for the assessment of undisturbed dental plaque vitality and topography. Arch Oral Biol 43:277–285PubMedCrossRef
15.
Shapiro HM (2008) Flow cytometry of bacterial membrane potential and permeability. Meth Mol Med 142:175–186CrossRef
16.
Al-Ahmad A, Wiedmann-Al-Ahmad M, Auschill TM, Follo M, Braun G, Hellwig E, Arweiler NB (2008) Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro. Arch Oral Biol 53:765–772PubMedCrossRef
17.
Pan PC, Harper S, Ricci-Nittel D, Lux R, Shi W (2010) In-vitro evidence for efficiency of antimicrobial mouthrinses. J Dent 38:16–20CrossRef
18.
Netuschil L (1983) Vital staining of plaque microorganisms using fluorescein diacetate and ethidium bromide. Dtsch Zahnarztl Z 38:914–917PubMed
19.
Breeuwer P, Drocourt JL, Bunschoten N, Zwietering MH, Rombouts FM, Abee T (1995) Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product. Appl Environ Microbiol 61:1614–1619PubMed
20.
Guilbault GG, Kramer DN (1965) Fluorometric procedure for measuring the activity of dehydrogenases. Anal Chem 37:1219–1221PubMedCrossRef
21.
Peak E, Chalmers IW, Hoffmann KF (2010) Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability. PLoS Negl Trop Dis 4:e759PubMedCrossRef
22.
Leeder JS, Dosch HM, Harper PA, Lam P, Spielberg SP (1989) Fluorescence-based viability assay for studies of reactive drug intermediates. Anal Biochem 177:364–372PubMedCrossRef
23.
Liminga G, Jonsson B, Nygren P, Larsson R (1999) On the mechanism underlying calcein-induced cytotoxicity. Eur J Pharmacol 383:321–329PubMedCrossRef
24.
Wojcik K, Dobrucki JW (2008) Interaction of a DNA intercalator DRAQ5, and a minor groove binder SYTO17, with chromatin in live cells—influence on chromatin organization and histone–DNA interactions. Cytometry A 73:555–562PubMed
25.
Cassina V, Seruggia D, Beretta GL, Salerno D, Brogioli D, Manzini S, Zunino F, Mantegazza F (2011) Atomic force microscopy study of DNA conformation in the presence of drugs. Eur Biophys J 40:59–68PubMedCrossRef
26.
Monaco RR (2010) Capture of a transition state using molecular dynamics: creation of an intercalation site in dsDNA with ethidium cation. J Nucleic Acids 2010
27.
Jr Olmsted, Kearns DR (1977) Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry 16:3647–3654PubMedCrossRef
28.
Singer VL, Lawlor TE, Yue S (1999) Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test). Mutat Res 439:37–47PubMedCrossRef
29.
Roth BL, Poot M, Yue ST, Millard PJ (1997) Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 63:2421–2431PubMed
30.
Stevenson (1978) A case for bacterial dormancy in aquatic systems. Microb Ecol 4:127–133CrossRef
31.
Kaprelyants AS, Gottschal JC, Kell DB (1993) Dormancy in non-sporulating bacteria. FEMS Microbiol Rev 10:271–285PubMed
32.
Hannig C, Hannig M (2009) The oral cavity—a key system to understand substratum-dependent bioadhesion on solid surfaces in man. Clin Oral Investig 13:123–139PubMedCrossRef
33.
Hannig C, Hoch J, Becker K, Hannig M, Attin T (2005) Lysozyme activity in the initially formed in situ pellicle. Arch Oral Biol 50:821–828PubMedCrossRef
34.
Svensäter G, Bergenholtz G (2005) Biofilms in endodontic infections. Endod Top 9:27–36CrossRef
35.
Oliver JD (1995) The viable but non-culturable state in the human pathogen Vibrio vulnificus. FEMS Microbiol Lett 133:203–208PubMedCrossRef
36.
Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA, Palmer LM (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Nat Biotechnol 3:817–820CrossRef
37.
Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290PubMedCrossRef
38.
Donelli G, Matarrese P, Fiorentini C, Dainelli B, Taraborelli T, Di Campli E, Di Bartolomeo S, Cellini L (1998) The effect of oxygen on the growth and cell morphology of Helicobacter pylori. FEMS Microbiol Lett 168:9–15PubMedCrossRef
39.
Signoretto C, Lleo MM, Canepari P (2002) Modification of the peptidoglycan of Escherichia coli in the viable but nonculturable state. Curr Microbiol 44:125–131PubMedCrossRef
40.
Guggenheim B, Giertsen E, Schüpbach P, Shapiro S (2001) Validation of an in vitro biofilm model of supragingival plaque. J Dent Res 80:363–370PubMedCrossRef
41.
Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A (1999) The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37:1771–1776PubMed
42.
Davey HM, Hexley P (2011) Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide. Environ Microbiol 13:163–171PubMedCrossRef
Metadaten
Titel
Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm
verfasst von
P. N. Tawakoli
A. Al-Ahmad
W. Hoth-Hannig
M. Hannig
C. Hannig
Publikationsdatum
01.04.2013
Verlag
Springer-Verlag
Erschienen in
Clinical Oral Investigations / Ausgabe 3/2013
Print ISSN: 1432-6981
Elektronische ISSN: 1436-3771
DOI
https://doi.org/10.1007/s00784-012-0792-3

Weitere Artikel der Ausgabe 3/2013

Clinical Oral Investigations 3/2013 Zur Ausgabe

Original Article

The randomized shortened dental arch study: tooth loss over five years

Original Article

The arrangement of the interproximal interfaces in the human permanent dentition

Original Article

Postoperative tooth sensitivity with a new self-adhesive resin cement—a randomized clinical trial

Original Article

Four-year clinical evaluation of a self-adhesive luting agent for ceramic inlays

Original Article

Comparison of biogenerically reconstructed and waxed-up complete occlusal surfaces with respect to the original tooth morphology

Original Article

Relationship between oral health, diabetes management and sleep apnea

Neu im Fachgebiet Zahnmedizin

Parodontalbehandlung verbessert Prognose bei Katheterablation

19.04.2024 Vorhofflimmern Nachrichten

Werden Personen mit Vorhofflimmern in der Blanking-Periode nach einer Katheterablation gegen eine bestehende Parodontitis behandelt, verbessert dies die Erfolgsaussichten. Dafür sprechen die Resultate einer prospektiven Untersuchung.

Invasive Zahnbehandlung: Wann eine Antibiotikaprophylaxe vor infektiöser Endokarditis schützt

11.04.2024 Endokarditis Nachrichten

Bei welchen Personen eine Antibiotikaprophylaxe zur Prävention einer infektiösen Endokarditis nach invasiven zahnärztlichen Eingriffen sinnvoll ist, wird diskutiert. Neue Daten stehen im Einklang mit den europäischen Leitlinienempfehlungen.

Zell-Organisatoren unter Druck: Mechanismen des embryonalen Zahnwachstums aufgedeckt

08.04.2024 Zahnmedizin Nachrichten

Der Aufbau von Geweben und Organen während der Embryonalentwicklung wird von den Zellen bemerkenswert choreografiert. Für diesen Prozess braucht es spezielle sogenannte „Organisatoren“. In einer aktuellen Veröffentlichung im Fachjournal Nature Cell Biology berichten Forschende durch welchen Vorgang diese Organisatoren im Gewebe entstehen und wie sie dann die Bildung von Zähnen orchestrieren.

Die Oralprophylaxe & Kinderzahnheilkunde umbenannt

11.03.2024 Kinderzahnmedizin Nachrichten

Infolge der Umbenennung der Deutschen Gesellschaft für Kinderzahnheilkunde in Deutsche Gesellschaft für Kinderzahnmedizin (DGKiZ) wird deren Mitgliederzeitschrift Oralprophylaxe & Kinderzahnheilkunde in Oralprophylaxe & Kinderzahnmedizin umbenannt. Aus diesem Grunde trägt die erste Ausgabe in 2024 erstmalig den neuen Titel.

Newsletter

Bestellen Sie unseren kostenlosen Newsletter Update Zahnmedizin und bleiben Sie gut informiert – ganz bequem per eMail.

Newsletter bestellen
Bildnachweise