In situ neutralisation of the antibacterial effect of 0.2% Chlorhexidine on salivary microbiota: Quantification of substantivity

Arch Oral Biol. 2015 Aug;60(8):1109-16. doi: 10.1016/j.archoralbio.2015.04.002. Epub 2015 Apr 24.


Objective: To quantify the substantivity of a single 0.2% Chlorhexidine mouthwash in saliva after its neutralisation with tooth-brushing and 1% acetic acid, in order to identify the effect of Chlorhexidine substantivity in regard to the re-growing period of the salivary bacteria.

Methods: Unstimulated saliva samples were collected from a group of 15 healthy individuals at baseline (BS), and then 30s and 1, 3, 5 and 7h after the following protocols were performed: a single sterile water mouthwash (M-WATER) (negative control), a single 0.2% Chlorhexidine mouthwash (M-0.2% CHX) (positive control) and a single 0.2% Chlorhexidine mouthwash followed by a complete and detailed tooth-brushing, and a single 1% acetic acid mouthwash (M-0.2% CHX+NP). The samples were analysed using an epifluorescence microscope in combination with LIVE/DEAD(®) BacLight™ fluorescence solution.

Results: After the M-0.2% CHX treatment, the bacterial vitality was significantly lower than BS until 7h (87.6 ± 6.5% vs. 73.6 ± 8.8%; p<0.001). However, after M-0.2% CHX+NP, the bacterial vitality remained significantly lower until 3h with regard to BS (81.4 ± 3.8% vs. 68.1 ± 10.6%; p=0.001), increasing at 5 and 7h (no differences from BS).

Conclusion: The immediate antibacterial effect of a single 0.2% Chlorhexidine mouthwash is so potent that the bacterial population needs more than 3h to return to baseline bacterial vitality levels. The substantivity of a 0.2% Chlorhexidine mouthwash is a property that significantly increases its antibacterial activity from the first hour and contributes to extend the duration of its effect by at least double.

Keywords: Acetic acid; Chlorhexidine; Epifluorescence; Mouthwash; Neutralisation; Substantivity.

Publication types

  • Randomized Controlled Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Anti-Infective Agents, Local / pharmacology*
  • Chlorhexidine / pharmacology*
  • Colony Count, Microbial
  • Cross-Over Studies
  • Double-Blind Method
  • Female
  • Healthy Volunteers
  • Humans
  • Male
  • Microbiota / drug effects*
  • Microscopy, Fluorescence
  • Middle Aged
  • Mouthwashes / pharmacology*
  • Saliva / microbiology*
  • Toothbrushing


  • Anti-Infective Agents, Local
  • Mouthwashes
  • Chlorhexidine