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

The redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) is used in aq uatic sciences as a vital stain for enumeration of respiring bacteria in si tu. Questions concerning its efficacy have been raised. We propose that the abundance of CTC-positive (CTC+) bacteria is a useful parameter in microbi al ecology based on the following information: (1) Taxonomically diverse st rains of aerobic, heterotrophic marine bacteria reduce CTC to its fluoresce nt product. (2) The proportion of CTC+ cells in laboratory cultures and in bacterioplankton assemblages varies in meaningful ways: the proportion of C TC+ cells is greatest for bacteria in log-phase growth, and lowest for bact eria in late stationary phase; particle-associated bacteria in various mari ne environments exhibit a higher percentage of CTC+ cells compared to bacte ria freely suspended in the water column; the proportion of CTC+ cells in a bacterioplankton assemblage can be increased by an order of magnitude or m ore by addition of substrate, in the absence of net change in bacterial num bers. (3) Flow cytometric analysis shows a strong relationship between char acteristics 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 activit y, and that cells which show no apparent reduction of CTC have either low o r no metabolic activity. Some portion of CTC-negative (CTC-) cells may have sufficient RNA content, and/or ability to assimilate labile substrates at dilute concentrations, to be identifiable as 'active' via indices of cell-s pecific rRNA content or of microautoradiography. Quantitative differences i n metabolism between 'highly active' CTC+ cells, and 'less active' CTC- cel ls have yet to be determined.