Estimating abundance and single-cell characteristics of respiring bacteria via the redox dye CTC

Abstract

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is used in aquatic sciences as a vital stain for enumeration of respiring bacteria in situ. Questions concerning its efficacy have been raised. We propose that the abundance of CTC-positive (CTC+) bacteria is a useful parameter in microbial ecology based on the following information: (1) Taxonomically diverse strains of aerobic, heterotrophic marine bacteria reduce CTC to its fluorescent product. (2) The proportion of CTC+ cells in laboratory cultures and in bacterioplankton assemblages varies in meaningful ways: the proportion of CTC+ cells is greatest for bacteria in log-phase growth, and lowest for bacteria in late stationary phase; particle-associated bacteria in various marine environments exhibit a higher percentage of CTC+ ceUs compared to bacteria freely suspended in the water column; the proportion of CTC+ cells in a bacterioplankton assemblage can be increased by an order of magnitude or more by addition of substrate, in the absence of net change in bacterial numbers. (3) Flow cytometric analysis shows a strong relationship between characteristics of CTC+ cells (abundance, size, red fluorescence) and rates of leucine incorporation by bacterial assemblages. We suggest that CTC+ cells represent those bacteria characterized by a high level of metabolic activity, and that cells which show no apparent reduction of CTC have either low or no metabolic activity. Some portion of CTC-negative (CTC-) cells may have sufficient RNA content, andlor ability to assimilate labile substrates at dilute concentrations, to be identifiable as 'active' via indices of cell-specific rRNA content or of rnicroautoradiography. Quantitative differences in metabolism between 'highly active' CTC+ cells, and 'less active' CTCcells have yet to be determined.