Background

The TTC salt is a water-soluble and colorless compound that can be reduced to formazan, a highly colored compound. The irreversible formation of formazan can be quantified using a spectrophotometer. Owing to its property and its low reduction potential, this tetrazolium salt is widely used in both eukaryotes and prokaryotes as an indicator of cell redox activity, viability, drug susceptibility and substrate utilization assays (Byth et al., 2001; Hayashi et al., 2003; Raut et al., 2008; Lin et al., 2008). The net positive charge on tetrazolium salts facilitates cellular uptake due to the membrane potential, allowing their intracellular reduction (Berridge et al., 2005). In prokaryotes, the main studies of TTC reduction have concerned the Gram-negative respiring bacterium Escherichia coli, while only a few studies have been reported for members of the Rhizobiacea family. In this protocol, one of the best genetically characterized members of this family, the S. meliloti 1021 rhizobium strain, was used. The respiratory activity, expression of cytochrome terminal oxidases, of this strain was analysed using TTC as an indicator of cell redox potential.

To enable the development of a measurable color intensity and, at the same time, to avoid any possible inhibition of bacterial growth, the bacteria were incubated in the presence of TTC for an appropriate period of time compared to those described by other authors (Tengerdy et al., 1967; Byth et al., 2001; Tachon et al., 2009).