Assessing metabolic activity of yeast biofilm-forming cells on nanofibrous materials using MTT assay

Nanofibrous materials (NMs), widely used in medical and food industry applications, are highly susceptible to colonisation by microorganisms, including yeasts. Although yeasts can form dense biofilms, methods for studying their metabolic activity remain limited. This study is the first to apply the MTT assay, a standardised method for assessing cell metabolic activity, to evaluate the metabolic activity of yeast biofilm-forming cells on electrospun NMs. First, the biofilm formation of Candida albicans and Saccharomyces cerevisiae on NMs electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Then, key parameters of the MTT assay were systematically evaluated: (i) the addition of glucose to the MTT solution, (ii) the presence of menadione in the MTT solution, and (iii) the incubation time with the MTT solution. The addition of glucose was not proven necessary; however, in some cases, it may help distinguish the number of metabolically active cells. Based on this study, we recommend incubation with an MTT solution containing menadione for 2 h. To verify the protocol, colony-forming unit (CFU) enumeration was employed as a reference method. As differences between the results of these two methods were observed, the MTT assay should be complemented by other standardised methods. Nevertheless, the refined protocol offers a solid basis for investigating interactions between NMs and yeasts.