A colorimetric assay using tetrazolium salts with an electron mediator to evaluate yeast growth in opaque dispersed systems

Abstract

Background: traditional microbiological enumeration techniques such as colony count are time consuming. Visual evaluation of turbidity growth lacks objectivity and precision. On the other hand, the accuracy of the spectrophotometric readings can be limited by media opacity and/or additives. In this context, colorimetric methods based on tetrazolium salts are an alternative to traditional methods due to their ability to generate endpoints based on color change. The objective of this study was to develop a colorimetric assay based on two different tetrazolium salts and an electron mediator in order to replace the conventional method of viable plate count or absorbance for microbial growth detection in opaque dispersed systems. Methods: a colorimetric method to evaluate yeast growth in broth and opaque dispersed systems using two tetrazolium salts, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) and an electron mediator, 2-methoxy-1,4-Naphthoquinone (MNQ) was developed. In addition, the minimum inhibitory concentrations against Zygosaccharomyces bailii for natamycin and lemongrass essential oil were determined by the proposed method and compared with those obtained using the conventional broth microdilution and plate count methods.Results: showed that the mixture of INT/MNQ, was more effective than TTC/MNQ. In opaque structure systems, the later showed to be more sensitive than absorbance However, in broth it was as sensitivity as absorbance to detect yeast growth. Besides, the INT/MNQ reagent functioned adequately to determine the minimum inhibitory concentrations of antimicrobials in opaque structured systems. Conclusions: These results demonstrated that the proposed method functioned adequately to determine the minimum inhibitory concentrations of antimicrobials in opaque structured systems showing an excellent agreement with the plate count method and the proposed technique allowed the evaluation of yeast growth with lower population levels than the ones required through absorbance readings. Moreover, this colorimetric method is easy to perform and less time consuming.