Establishing microdilution for determination of minimum inhibitory concentrations of essential oils in routine diagnostics

Introduction Essential oils (EO) are volatile multicomponent plant products with antimicrobial activity which can be applied topically against bacterial and yeast infections in animals. The antimicrobial activity of EO can be tested in vitro with agar disc diffusion (aromatogram) by determination of the size of inhibition zones, which amongst other aspects depends on diffusion of the various EO constituents in the agar.

Objective Aim of this study was to establish a routine suited half-automated microdilution of EO for the in vitro determination of their minimal inhibitory concentration (MIC) using a pipetting robot.

Methods For microdilution of lipophilic EO in hydrophilic bouillon five emulsifiers (Tween^®20, Tween^®80, dimethylsulfoxide, ethanol, agar) were tested for their ability to maintain a homogeneous suspension overnight and thus ensure direct contact between pathogen and EO. The efficacy of 12 EO against Escherichia coli was analyzed. A program for a pipetting robot (BioTek Precision XS) for generation of a serial dilution of EO in bouillon and following pathogen inoculation in 96-well plate format was developed. A dilution series pipetted by hand was set as the gold standard. After incubation of plates for 20 hours at 37°C the MIC of EO was determined visually with color indicator resazurin. During establishment several parameters, e. g. mixing cycles or pipetting speed, had to be adjusted until the program could be evaluated as successful. Different concentrations and incubation times of resazurin were tested.

Results Tween^®20 proved to be best suited and was chosen as emulsifier for essential oil-microdilution. For the final pipetting-program a low aspiration and high dispensing rate as well as ten resuspension steps per well were defined. Color indicator resazurin gave best results added after overnight-incubation of plates (20 µl per well, 0.01%). A clearly visible color change was detected after an additional 2 hour incubation at room temperature.

Conclusion Within this study, the half-automated microdilution was successfully established as a new method for the in vitro determination of MIC of EO in routine diagnostics.

Publication History

Article published online:
27 April 2020