Clinical Oral Investigations

, Volume 18, Issue 2, pp 625–634 | Cite as

Penetration kinetics of four mouthrinses into Streptococcus mutans biofilms analyzed by direct time-lapse visualization

  • Rika Wakamatsu
  • Shoji TakenakaEmail author
  • Tatsuya Ohsumi
  • Yutaka Terao
  • Hayato Ohshima
  • Takashi Okiji
Original Article



The aim of this study was to determine whether different antiseptic mouthrinses show different penetration kinetics into Streptococcus mutans biofilms.

Materials and methods

The biofilms, grown on glass-based dishes, were exposed to one of four mouthrinses containing chlorhexidine digluconate, essential oil, cetylpyridinium chloride, or isopropylmethylphenol. Then, penetration velocities were determined by monitoring fluorescence loss of calcein AM-stained biofilms with time-lapse confocal laser scanning microscopy. Bactericidal activity was assessed with fluorescent bacterial viable cell (Live/Dead) staining and viable cell counts. Bacterial detachment after the mouthrinse exposure was determined by measuring fluorescence reduction of SYTO9-stained biofilms.


The essential oil-containing mouthrinse showed significantly faster penetration velocity than the other mouthrinses (ANCOVA and Bonferroni test, p < 0.05). However, even 5 min of exposure left the biofilm structure almost intact. After 30 s (consumer rinsing time) of exposure, the essential oil-containing mouthrinse showed the highest log reduction of viable cells (2.7 log CFU) measured by Live/Dead staining, and the mean reduction of total viable cells was 1.41 log CFU measured by viable cell count.


The essential oil-containing mouthrinse showed the best penetration. Within 30 s of exposure, however, no mouthrinses injured all the microorganisms and all mouthrinses left the biofilm structure nearly intact.

Clinical relevance

The mouthrinses tested showed different levels of biofilm penetration. The essential oil rinse was superior to other rinses by all three of the in vitro measurements performed.


Biofilm Time-lapse observation Penetration kinetics Mouthrinse 



This investigation was supported in part by a Grant-in-Aid for Scientific Research (C) (no. 23592795) from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Young Scientists (B) (no. 22791830) from the JSPS, and the JSPS Institutional Program for Young Researcher Overseas Visits.

Conflict of interest

The authors declare that they have no conflicts of interest.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Rika Wakamatsu
    • 1
  • Shoji Takenaka
    • 1
    Email author
  • Tatsuya Ohsumi
    • 1
  • Yutaka Terao
    • 2
  • Hayato Ohshima
    • 3
  • Takashi Okiji
    • 1
  1. 1.Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health ScienceNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  2. 2.Division of Microbiology and Infectious Diseases, Department of Oral Health ScienceNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  3. 3.Division of Anatomy and Cell Biology of the Hard Tissue Department of Tissue Regeneration and ReconstructionNiigata University Graduate School of Medical and Dental SciencesNiigataJapan

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