Purpose: To devise a method for artificial biofilm formation using Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Streptococcus gordonii, as well as a method for evaluating the effects of various ingredients on the artificial biofilm. Methods: An artificial biofilm was developed using P. gingivalis, T. forsythia, T. denticola, and S. gordonii, which was then observed using scanning electron microscopy and evaluated by microflora analysis. The artificial biofilm was exposed to chlorhexidine gluconate and stained with a fluorescent dye. Then, the fluorescent-stained biofilm was observed using a confocal laser microscope and measured using a fluorescent microplate reader. Results: The microflora analysis confirmed that the culture medium developed was capable of culturing four different bacterial species at the same time. The distribution of dead bacteria differed according to the difference in the concentration of exposed chlorhexidine gluconate. Moreover, the rate of attachment of viable cells decreased in a concentration-dependent manner. Many bacteria were detached from the biofilm in the group exposed to 0.09% chlorhexidine gluconate. Exposure to chlorhexidine gluconate induced a concentration-dependent decrease in living microorganisms and an increase in dead microorganisms in the biofilm. Clinical significance: The results of this study revealed that S. gordonii, P. gingivalis, T. forsythia, and T. denticola could be used to develop artificial biofilms. The effects of chlorhexidine gluconate on the biofilm showed that evaluating the change in the artificial biofilm caused by the component effect in the experiments was possible via exposure to chlorhexidine gluconate. This method can efficiently evaluate the component effect and has a high potential for use as an indicator. This study demonstrated that this simulation could help develop preventive measures.
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