Assessing tetrazolium and ATP assays for rapid in situ viability quantification of bacterial cells entrapped in hydrogel beads

Abstract

Entrapped bacteria are used in several applications including food and beverage production, antibiotic production, and wastewater treatment. To date in order to determine the viability of entrapped bacteria, they have to be de-entrapped from the matrix first. However, the cell de-entrapment procedures such as matrix dissolution by acid or heating at high temperatures, may affect the viability of the cells. In this study, the uses of two quantitative approaches for in situ viability estimation of calcium alginate and phosphorylated poly(vinyl) alcohol (PPVA) entrapped Escherichia coli were investigated. Bioreducible tetrazolium salt (XTT) and adenosine triphosphate (ATP) based assays were used to determine microbial viability without the dissolution of the matrices (spherical beads). The data from both assays showed linearity and strong correlations between the viability signals and number of beads in which each bead contained a similar number of live cells. An application of XTT assay on the PPVA entrapped bacterial beads was an exception to these results. Effects of the acid and heat dissolution de-entrapment procedures on cell viability were also evaluated by using both assays and a traditional plate count method. The heating process showed the greatest reduction in bacterial viability when compared to the other de-entrapment procedures. The ATP assay is a more sensitive and less time consuming approach for viability estimation when compared to the XTT assay and traditional plate count method. Both XTT and ATP assays have potential for use in quantifying the viability of entrapped bacteria.