Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures

The respiratory activity of marine bacteria is an important indication of t he ecological functioning of these organisms in marine ecosystems. The redo x dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellul arly in respiring cells to an insoluble, fluorescent precipitate. This prod uct is detectable and quantifiable by flow cytometry in individual cells. W e describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by how cytometry than by vi sual epifluorescence microscopy. Samples can be stored refrigerated or froz en in liquid nitrogen for at least 4 weeks without a significant loss of to tal cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the max imum number within 2 to 10 min. The proportion of active cells is correlate d with the growth rate, while the amount of fluorescence per cell varies in versely with the growth rate. The CTC reduction kinetics in assemblages bub bled with nitrogen and in assemblages bubbled with air to vary the oxygen a vailability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more C TC-positive cells, and the amount of fluorescence per cell,vas greater. Act ivity may have been reduced by bubble-induced turbulence. Addition of an ar tificial reducing agent, sodium dithionite, after CTC incubation and fixati on resulted in a greater number of positive cells but did not "activate" a majority of the cells, This indicated that some of the negative cells actua lly transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to st udy single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.