Colorimetric microdilution assay: Validation of a standard method for determination of MIC, IC50%, and IC90% of antimicrobial compounds

The emergence of multiresistant bacteria directly impacts on the search for new compounds with antimicrobial activity, and it is important the improvement of new techniques are able to determine the minimum inhibitory concentration (MIC) of antimicrobial compounds. The microdilution technique is widely used for saving culture media, reagents and compounds to be tested. However, the literature does not describe a colorimetric method capable of correlating absorbance with concentration of viable microorganisms (CFU-1). Therefore, the novelty of this work was the standardization and validation of a colorimetric and quantitative method capable of determining the MIC of several compounds with antimicrobial activity and the conversion of absorbance values to CFU mL(-1). The conditions carried out for the method were: the use of 0.125% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC) solution added after 22 h of incubation at 35 degrees C, followed by 2 more hours of incubation and subsequent reading in a spectrophotometer. The tested microorganisms were: Staphylococcus aureus (ATCC 6538), Escherichia soli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Candida albicans (ATCC 10231). The method was validated and showed linearity (R-2 > 0.95), precision (RSD < 26%), accuracy (75% to 122%) and robustness (p > 0.05). The validated parameters ensured the harmonization of methodology to determine not only MIC as well as inhibitory concentrations of 50% (IC50%) and 90% (IC90%) of the antimicrobial compounds.