MSSBs were formed from stimulated saliva that was collected from donors, pooled and homogenized. Saliva pellicle was formed on hydroxyapatite (HAP) discs held in a custom mount to remain vertical. Saliva was then replaced with saliva/media mixture and incubated statically for 48 hours with media changes twice daily. Biofilms on the HAP discs were treated once for 30 seconds and then immediately neutralized to prevent further bacterial kill and rinsed. Biofilms on discs were stained using a Live/ Dead stain cocktail (Invitrogen). Viability was analyzed using a Leica-SP5 confocal microscope. 3D images were generated and examined using Volocity software to visualize penetration.


Results are reported as Percent Dead.
Results: 3D images qualitatively illustrate the penetration effect of the EO mouthrinse as compared to the CPC and CPC+T containing mouthrinses. The representative images presented demonstrate the ability of the EO‘s to penetrate to the biofilm-HAP interface whereas the cationic antimicrobials (CPC, CPC+T) were less effective in penetration.


These in-vitro results indicate that in the MSSB model, LISTERINE® ZERO™ shows the deepest penetration and greatest biofilm kill via live/dead stain over CPC and CPC+T mouthrinses after one 30 second treatment of a 48 hour old saliva derived biofilm, further supporting the mechanism of EOs to control biofilm growth.

  % Dead
EO 98%
CPC + T 94%
0.05% CPC 60%
0.07% CPC 74%

% Dead = EO> CPC+T>0.07% CPC> 0.05% CPC

Fourre T, Queiroz D, Mordas CJ, Setbiak B. Biofilm Penetration of Anti-Microbial Alcohol-Free Mouthrinses Using Confocal Microscopy. JDR 2013; 92 Spec Iss A Abst #2264.a