5.3. Resazurin Microplate Assay

The Resazurin Microplate Assay (REMA) was performed to determine the minimum inhibitory concentration (MIC) values of essential oils and meropenem of bacterial strains studied via broth microdilution as detailed in CLSI M07-A8. However, Tween 80, at final concentration of 10%, was incorporated into the MHB in order to enhance the solubility of cinnamon bark essential oil (CBO) whereas resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide), at a final concentration of 0.02% was used to improve the visualization. Two-fold dilutions were performed in each test well to yield final well volumes composed of 50 μL of test compound, 40 μL of bacterial suspension at approximately 1 × 10⁵ cfu/mL and 10 μL of resazurin at a final concentration of 0.02%. E. coli ATCC 25922 was used for antibiotic (meropenem) positive control in this assay apart from negative and growth controls. All assays were performed in triplicates and incubated at 37 °C with shaking at 200 rpm for 20 h. The MICs of essential oils and meropenem were determined qualitatively and quantitatively via the color change in resazurin and relative fluorescent unit.