4.3. Determination of Minimum Inhibitory Concentration (MIC)

For antimicrobial activity screening, the minimum inhibitory concentration (MIC) of the seven oils was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institutes [74]. Five percent solutions were initially prepared in 5% (v/v) of dimethylsulfoxide (DMSO) (Sigma Aldrich, San Luis, MO, USA). Subsequently, two-fold serial dilutons of the oil-containing solutions were made in culture broths (BHI, TS and SD), in order to produce ten oil concentrations, ranging from 2.5 to 0.0048% (v/v). One hundred microliters of each concentration were placed into 96-well plates, in duplicate, (TPP, Trasadingen, Switzerland). Then, 10 μL of microorganism suspension (10⁷ CFU/mL for bacteria; 10⁵ CFU/mL for yeast) were added to each serial dilution and incubated at 37 °C for 24 h. Positive control and negative controls, respectively, were obtained by adding, or not adding the standardized microbial inoculum to culture medium without oil supplement. An additional control with two-fold serial dilutions of DMSO were prepared in order to verify the effect of the diluent on inhibiting the microorganism growth.

Microbial growth was evaluated by turbidity or absence of turbidity the culture medium. The MIC was determined as being the lowest concentration of the oil or DMSO to result in no microbial growth (Table 6).

Minimum Inhibitory Concentration (MIC) of oils against S. aureus, S. mutans and C. albicans.

To confirm growth inhibition, 20 μL of the suspension from each well were dropped onto a BHI, TS and SD agar surface. Microbial inhibition was categorized as strong (MIC < 0.5 mg/mL), moderate (0.5 ≤ MIC ≤ 1.5 mg/mL), and weak (MIC > 1.5 mg/mL) as previously reported [75].

The C. officinalis, E. citriodora, R. communis, M. alternifólia and P. strobus oils promoted strong growth inhibition of S. aureus and S. mutans. The E. citriodora and M. alternifólia essential oils promoted strong growth inhibition of C. albicans. Since these oils presented relevant antimicrobial activity, they were selected for the formulation of experimental dentifrices.