An improved resazurin MIC assay to determine the MIC of actives Cetylpyridinium Chloride, Triclosan, Grape Seed Oil, Tea Tree Oil, and La’Act O used in oral care formulations

Abstract

Antimicrobial agents such as Cetylpyridinium Chloride (CPC), Triclosan, and natural extracts are widely investigated for oral health applications. However, accurate determination of minimum inhibitory concentrations (MICs) for water-insoluble compounds remains challenging due to solubility and inoculum-related issues. An improved resazurin MIC assay was developed using 0.15% (w/v) agar as a stabilizer to ensure consistent contact between test materials and microorganisms. CPC, Triclosan, Grape seed extract, Tea Tree oil, and La’Act O were tested against representative oral microorganisms. CPC and Triclosan demonstrated broad-spectrum activity with MIC values ?0.005% against all tested microorganisms. Grape seed extract inhibited Escherichia coli, Actinomyces viscosus, Streptococcus salivarius, and Streptococcus sanguinis at 10%, but showed no effect against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, and Streptococcus mutans. Tea Tree oil inhibited growth at 0.31-0.63%, depending on the organism. La’Act O was effective at 0.08-0.16% against most Gram-positive bacteria but showed no inhibition of E. coli. This improved assay enables accurate MIC determination for water-insoluble antimicrobials. CPC and Triclosan exhibited the highest potency, while Tea Tree oil and La’Act O showed moderate, selective activity. Grape seed extract demonstrated limited antimicrobial potential. The method provides a reliable platform for evaluating insoluble compounds in oral care research.