Journal of Materials Science: Materials in Medicine

, Volume 22, Issue 9, pp 2045–2051 | Cite as

Quantification of vital adherent Streptococcus sanguinis cells on protein-coated titanium after disinfectant treatment

  • Monika Astasov-Frauenhoffer
  • Olivier Braissant
  • Irmgard Hauser-Gerspach
  • A. U. Daniels
  • Dieter Wirz
  • Roland Weiger
  • Tuomas Waltimo


The quantification of vital adherent bacteria is challenging, especially when efficacy of antimicrobial agents is to be evaluated. In this study three different methods were compared in order to quantify vital adherent Streptococcus sanguinis cells after exposure to disinfectants. An anaerobic flow chamber model accomplished initial adhesion of S. sanguinis on protein-coated titanium. Effects of chlorhexidine, Betadine®, Octenidol®, and ProntOral® were assessed by quantifying vital cells using Live/Dead BacLight, conventional culturing and isothermal microcalorimetry (IMC). Results were analysed by Kruskal–Wallis one-way analysis of variance. Live/dead staining revealed highest vital cell counts (P < 0.05) and demonstrated dose-dependent effect for all disinfectants. Microcalorimetry showed time-delayed heat flow peaks that were proportioned to the remaining number of viable cells. Over 48 h there was no difference in total heat between treated and untreated samples (P > 0.05), indicating equivalent numbers of bacteria were created and disinfectants delayed growth but did not eliminate it. In conclusion, contrary to culturing, live/dead staining enables detection of cells that may be viable but non-cultivable. Microcalorimetry allows unique evaluation of relative disinfectant effects by quantifying differences in time delay of regrowth of remaining vital cells.


Chlorhexidine Microcalorimetry Vital Cell Betadine Vitality Percentage 
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  1. 1.
    Auschill TM, Hein N, Hellwig E, Follo M, Sculean A, Arweiler NB. Effect of two antimicrobial agents on early in situ biofilm formation. J Clin Periodontol. 2005;32:147–52.CrossRefGoogle Scholar
  2. 2.
    Cousido MC, Tomás Carmona I, García-Caballero L, Limeres J, Alvarez M, Diz P. In vivo substantivity of 0.12% and 0.2% chlorhexidine mouthrinses on salivary bacteria. Clin Oral Investig. 2010;14:397–402.CrossRefGoogle Scholar
  3. 3.
    Decker EM, Maier G, Axmann D, Brecx M, von Ohle C. Effect of xylitol/chlorhexidine versus xylitol or chlorhexidine as single rinses on initial biofilm formation of cariogenic streptococci. Quintessence Int. 2008;39:17–22.Google Scholar
  4. 4.
    Hope CK, Wilson M. Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob Agents Chemother. 2004;48:1461–8.CrossRefGoogle Scholar
  5. 5.
    McBain AJ, Bartolo RG, Catrenich CE, Charbonneau D, Ledder RG, Gilbert P. Effects of a chlorhexidine gluconate-containing mouthwash on the vitality and antimicrobial susceptibility of in vitro oral bacterial ecosystems. Appl Environ Microbiol. 2003;69:4770–6.CrossRefGoogle Scholar
  6. 6.
    Pan PC, Harper S, Ricci-Nittel D, Lux R, Shi W. In vitro evidence for efficacy of antimicrobial mouthrinses. J Dent. 2010;38(Suppl 1):S16–20.CrossRefGoogle Scholar
  7. 7.
    Rohrer N, Widmer AF, Waltimo T, Kulik EM, Weiger R, Filipuzzi-Jenny E, et al. Antimicrobial efficacy of 3 oral antiseptics containing octenidine, polyhexamethylene biguanide, or Citroxx: can chlorhexidine be replaced? Infect Control Hosp Epidemiol. 2010;31:733–9.CrossRefGoogle Scholar
  8. 8.
    Tandjung L, Waltimo T, Hauser I, Heide P, Decker EM, Weiger R. Octenidine in root canal and dentine disinfection ex vivo. Int Endod J. 2007;40:845–51.CrossRefGoogle Scholar
  9. 9.
    Weiger R, de Lucena H, Decker E, Löst C. Vitality status of microorganisms in infected human root dentine. Int Endod J. 2002;35:116–71.CrossRefGoogle Scholar
  10. 10.
    Decker E-M. The ability of direct fluorescence-based, two-colour assays to detect different physiological states of oral streptococci. Lett Appl Microbiol. 2001;33:188–92.CrossRefGoogle Scholar
  11. 11.
    Bandara HM, Yau JY, Watt RM, Jin LJ, Samaranayake LP. Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation. J Med Microbiol. 2009;58:1623–31.CrossRefGoogle Scholar
  12. 12.
    Kim J, Pitts B, Stewart PS, Camper A, Yoon J. Comparison of the antimicrobial effects of chlorine, silver ion, and tobramycin on biofilm. Antimicrob Agents Chemother. 2008;52:1446–53.CrossRefGoogle Scholar
  13. 13.
    Shen Y, Stojicic S, Haapasalo M. Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture. J Endod. 2010;36:1820–3.CrossRefGoogle Scholar
  14. 14.
    Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwell RR. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb Ecol. 1982;8:313–23.CrossRefGoogle Scholar
  15. 15.
    Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev. 2010;34:415–25.Google Scholar
  16. 16.
    Higuera-Guisset J, Rodrıguez-Viejo J, Chacon M, Munoz FJ, Vigues N, Mas J. Calorimetry of microbial growth using a thermopile based microreactor. Thermochim Acta. 2005;427:187–91.CrossRefGoogle Scholar
  17. 17.
    Wadsö I. Isothermal microcalorimetry in applied biology. Thermochim Acta. 2002;394:305–11.CrossRefGoogle Scholar
  18. 18.
    Braissant O, Wirz D, Goepfert B, Daniels AU. Use of isothermal microcalorimetry to monitor microbial activities. FEMS Microbiol Lett. 2010;303:1–8.CrossRefGoogle Scholar
  19. 19.
    Braissant O, Wirz D, Goepfert B, Daniels AU. Biomedical use of isothermal microcalorimeters. Sensors. 2010;10:9369–83.CrossRefGoogle Scholar
  20. 20.
    Bäckmann P, Wadsö I. Cell growth experiments using a microcalorimetric vessel equipped with oxygen and pH electrodes. J Biochem Biophys Methods. 1991;23:283–93.CrossRefGoogle Scholar
  21. 21.
    Bunker JC, James AM. Microcalorimetric studies on the effects of media and environmental conditions on the growth of bacteria. Microbios. 1986;47:177–88.Google Scholar
  22. 22.
    Ruming Z, Yi L, Zhixiong X, Ping S, Songsheng Q. A microcalorimetric method for studying the biological effects of La3+ on Escherichia coli. J Biochem Biophys Methods. 2000;46:1–9.CrossRefGoogle Scholar
  23. 23.
    Sand W, von Rège H. Evaluation and quantification of bacterial attachment, microbial activity, and biocide efficacy by microcalorimetry. Methods Enzymol. 1999;310:361–74.CrossRefGoogle Scholar
  24. 24.
    Postollec F, Norde W, van der Mei HC, Busscher HJ. Enthalpy of interaction between coaggregating and non-coaggregating oral bacterial pairs—a microcalorimetric study. J Microbiol Methods. 2003;55:241–7.CrossRefGoogle Scholar
  25. 25.
    Boling EA, Blanchard GC, Russell WJ. Bacterial identification by microcalorimetry. Nature. 1973;241:472–3.CrossRefGoogle Scholar
  26. 26.
    Lopez D, Vinas M, Loren JG, Bermudez J. Analysis of microcalorimetric curves for bacterial identification. Can J Microbiol. 1987;33:6–11.CrossRefGoogle Scholar
  27. 27.
    Monk P, Wadsö I. The use of microcalorimetry for bacterial classification. J Appl Bacteriol. 1975;38:71–4.Google Scholar
  28. 28.
    Vine GJ, Bishop AH. The analysis of microorganisms by microcalorimetry in the pharmaceutical industry. Curr Pharm Biotechnol. 2005;6:223–38.CrossRefGoogle Scholar
  29. 29.
    Hauser-Gerspach I, de Freitas PS, Dan Daniels AU, Meyer J. Adhesion of Streptococcus sanguinis to glass surfaces measured by isothermal microcalorimetry (IMC). J Biomed Mater Res. 2008;85:42–9.CrossRefGoogle Scholar
  30. 30.
    Gompertz B. On the nature of the function expressive of the law of human mortality, and on a new mode of determining the value of life contingencies. Philos Trans R Soc London. 1825;115:513–85.CrossRefGoogle Scholar
  31. 31.
    Zwietering MH, Jongenburger I, Rombouts FM, van’t Riet K. Modeling of the bacterial growth curve. Appl Environ Microbiol. 1990;56:1875–81.Google Scholar
  32. 32.
    Cho SB, Nakanishi K, Soga N, Ohtsuki C, Nakamura T, Kitsugi T, et al. Defence of apatite formation on silica gel on its structure: Effect of heat treatment. J Am Ceram Soc. 1995;78:1769–74.CrossRefGoogle Scholar
  33. 33.
    Hauser-Gerspach I, Kulik EM, Weiger R, Decker EM, von Ohle C, Meyer J. Adhesion of Streptococcus sanguinis to dental implant and restorative materials in vitro. Dental Mater J. 2007;26:361–6.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Monika Astasov-Frauenhoffer
    • 1
  • Olivier Braissant
    • 2
  • Irmgard Hauser-Gerspach
    • 1
  • A. U. Daniels
    • 2
  • Dieter Wirz
    • 2
  • Roland Weiger
    • 3
  • Tuomas Waltimo
    • 1
  1. 1.Institute of Preventive Dentistry and Oral Microbiology, School of Dental MedicineUniversity of BaselBaselSwitzerland
  2. 2.Laboratory of Biomechanics and Biocalorimetry, c/o Biozentrum/PharmazentrumUniversity of BaselBaselSwitzerland
  3. 3.Clinic for Periodontology, Endodontology and Cariology, University of BaselBaselSwitzerland

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