Basic Research| Volume 37, ISSUE 1, P31-35, January 2011

Resazurin Metabolism Assay for Root Canal Disinfectant Evaluation on Dual-species Biofilms

Published:November 15, 2010DOI:



      Endodontic infections are caused by polymicrobial biofilms. Therefore, novel root canal disinfectants should be evaluated not only on single-species biofilms but also on dual- or mixed-species biofilms. A simple, high-throughput assay is urgently needed for this. In this study, the application of the resazurin metabolism assay was investigated for the evaluation of a root canal disinfectant on dual-species biofilms.


      Enterococcus faecalis with or without Streptococcus mutans in biofilms were formed in an active attachment biofilm model for 24 hours. Subsequently, the biofilms were treated with various concentrations of NaOCl for 1 minute. After resazurin metabolism by both organisms was confirmed, treatment efficacies using 0.0016% resazurin were evaluated.


      During NaOCl treatments, resazurin metabolism displays a clear dose response, not only in single-species E. faecalis (or S. mutans) biofilms but also in dual-species biofilms. Notably, the assay revealed that the resistance of dual-species biofilms to NaOCl was 30-fold higher than in single-species E. faecalis biofilms. Viability counts on a selected NaOCl treatment (0.004%) confirmed this result and showed the increased resistance of E. faecalis in dual-species biofilms.


      Clearly, the high-throughput and low cost resazurin metabolism assay has a great potential for testing novel root canal antimicrobial agents in mixed-species biofilms.

      Key Words

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Endodontics
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Donlan R.M.
        • Costerton J.W.
        Biofilms: survival mechanisms of clinically relevant microorganisms.
        Clin Microbiol Rev. 2002; 15: 167-193
        • Costerton J.W.
        • Lewandowski Z.
        • Caldwell D.E.
        • et al.
        Microbial biofilms.
        Annu Rev Microbiol. 1995; 49: 711-745
        • Hall-Stoodley L.
        • Costerton J.W.
        • Stoodley P.
        Bacterial biofilms: from the natural environment to infectious diseases.
        Nat Rev Microbiol. 2004; 2: 95-108
        • Svensäter G.
        • Bergenholtz G.
        Biofilms in endodontic infections.
        Endod Top. 2004; 9: 27-36
        • Peeters E.
        • Nelis H.J.
        • Coenye T.
        Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.
        J Microbiol Methods. 2008; 72: 157-165
        • Peeters E.
        • Nelis H.J.
        • Coenye T.
        Evaluation of the efficacy of disinfection procedures against Burkholderia cenocepacia biofilms.
        J Hosp Infect. 2008; 70: 361-368
        • Sreenivasan P.K.
        • Tambs G.
        • Gittins E.
        • et al.
        A rapid procedure to ascertain the antimicrobial efficacy of oral care formulations.
        Oral Microbiol Immunol. 2003; 18: 371-378
        • O’Brien J.
        • Wilson I.
        • Orton T.
        • et al.
        Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.
        Eur J Biochem. 2000; 267: 5421-5426
        • Sarker S.D.
        • Nahar L.
        • Kumarasamy Y.
        Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals.
        Methods. 2007; 42: 321-324
        • Deng D.M.
        • ten Cate J.M.
        • Crielaard W.
        The adaptive response of Streptococcus mutans towards oral care products: involvement of the ClpP serine protease.
        Eur J Oral Sci. 2007; 115: 363-370
        • Shiloh M.U.
        • Ruan J.
        • Nathan C.
        Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.
        Infect Immun. 1997; 65: 3193-3198
        • Mariscal A.
        • Lopez-Gigosos R.M.
        • Carnero-Varo M.
        • et al.
        Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm.
        Appl Microbiol Biotechnol. 2009; 82: 773-783
        • Chavez de Paz L.E.
        • Bergenholtz G.
        • Dahlen G.
        • et al.
        Response to alkaline stress by root canal bacteria in biofilms.
        Int Endod J. 2007; 40: 344-355
        • Chai W.L.
        • Hamimah H.
        • Cheng S.C.
        • et al.
        Susceptibility of Enterococcus faecalis biofilm to antibiotics and calcium hydroxide.
        J Oral Sci. 2007; 49: 161-166
        • Giardino L.
        • Ambu E.
        • Savoldi E.
        • et al.
        Comparative evaluation of antimicrobial efficacy of sodium hypochlorite, MTAD, and Tetraclean against Enterococcus faecalis biofilm.
        J Endod. 2007; 33: 852-855
        • Chavez de Paz L.E.
        Redefining the persistent infection in root canals: possible role of biofilm communities.
        J Endod. 2007; 33: 652-662
        • Deng D.M.
        • Hoogenkamp M.A.
        • Exterkate R.A.
        • et al.
        Influence of Streptococcus mutans on Enterococcus faecalis biofilm formation.
        J Endod. 2009; 35: 1249-1252
        • Ozok A.R.
        • Wu M.K.
        • Luppens S.B.
        • et al.
        Comparison of growth and susceptibility to sodium hypochlorite of mono- and dual-species biofilms of Fusobacterium nucleatum and Peptostreptococcus (micromonas) micros.
        J Endod. 2007; 33: 819-822
        • Rocas I.N.
        • Hulsmann M.
        • Siqueira Jr., J.F.
        Microorganisms in root canal-treated teeth from a German population.
        J Endod. 2008; 34: 926-931
        • Munson M.A.
        • Pitt-Ford T.
        • Chong B.
        • et al.
        Molecular and cultural analysis of the microflora associated with endodontic infections.
        J Dent Res. 2002; 81: 761-766
        • da Silva L.A.
        • Nelson-Filho P.
        • Faria G.
        • et al.
        Bacterial profile in primary teeth with necrotic pulp and periapical lesions.
        Braz Dent J. 2006; 17: 144-148
        • Duggan J.M.
        • Sedgley C.M.
        Biofilm formation of oral and endodontic Enterococcus faecalis.
        J Endod. 2007; 33: 815-818
        • Ceri H.
        • Olson M.E.
        • Stremick C.
        • et al.
        The Calgary Biofilm Device: New technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.
        J Clin Microbiol. 1999; 37: 1771-1776
        • Hoogenkamp M.A.
        • Crielaard W.
        • ten Cate J.M.
        • et al.
        Antimicrobial activity of vanadium chloroperoxidase on planktonic Streptococcus mutans cells and Streptococcus mutans biofilms.
        Caries Res. 2009; 43: 334-338
        • Vogel A.I.
        A Text-book of Quantitative Inorganic Analysis, Including Elementary Instrumental Analysis.
        3rd ed. Longmans, London1962
        • Kara D.
        • Luppens S.B.
        • Cate J.M.
        Differences between single- and dual-species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine.
        Eur J Oral Sci. 2006; 114: 58-63
        • Gabrielson J.
        • Hart M.
        • Jarelov A.
        • et al.
        Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates.
        J Microbiol Methods. 2002; 50: 63-73
        • Ito A.
        • Taniuchi A.
        • May T.
        • et al.
        Increased antibiotic resistance of Escherichia coli in mature biofilms.
        Appl Environ Microbiol. 2009; 75: 4093-4100
        • Davies D.
        Understanding biofilm resistance to antibacterial agents.
        Nat Rev Drug Discov. 2003; 2: 114-122