Direct estimate of active bacteria: CTC use and limitations

Abstract

During the last 10 years, the dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used to determine the in situ number of "active" bacteria in different ecosystems. A part of this success is due to a simple protocol, which does not require sophisticated equipment. However, it has not been established whether the method determines viable cells, e.g. those capable of growth and cell division, as opposed to cells that are active in the sense of having some detectable metabolic activity. In this study, the number of CTC-positive cells through the growth stages of Escherichia coli was estimated and compared to counts of the total number of bacteria, the culturability (CFU counts) and respiratory activity (CO(2) evolution). There was a good correlation between the number of CTC-positive cells and the CFU count, regardless of the growth phase. However, CTC could still be reduced by a large part of the population during the first hours of stationary phase even if the bacteria were no longer releasing CO(2). Thus, the reduction of CTC is a good estimator for cell viability, rather than cell activity. Additionally, a review of the literature showed that there is presently no standardized protocol for using CTC, which makes difficult at present the comparison of active bacterial numbers in different samples from different sites.