Flow Cytometric Analysis of 5-Cyano-2,3-Ditolyl Tetrazolium Chloride Activity of Marine Bacterioplankton in Dilution Cultures

Abstract

The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We 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 flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total 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 maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation 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 actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.

Near‐zero growth kinetics of Pseudomonas putida deduced from proteomic analysis

“…S6), (ii) transmission electron microscopy of the cellular cross-sections revealing fine internal structure ( Fig. 2), (iii) flow cytometry with 5-cyano-2,3ditolyltetrazolium chloride (CTC) (Figs 3 and S11), selectively staining the active respiring cells (Rodriguez et al, 1992;Sieracki et al, 1999;Bartosch et al, 2003;Creach et al, 2003) and (iv) optical microscopy giving overview of size and shape variation ( Fig. S10).…”

“…A flow cytometry analysis was done with SYTO 9 (5 μM) and CTC (1.8 mM), as described (Rodriguez et al, 1992;Sieracki et al, 1999;Creach et al, 2003). The only modification was that we used lower CTC concentration to avoid possible toxicity and longer incubation times (up to 8 h) to increase the CTC signal from starving cells.…”

Current and future applications of flow cytometry in aquatic microbiology

“…The £uorogenic tetrazolium salts such as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) have been increasingly used during recent years as a measure of cell-speci¢c metabolic activity in bacteria with and without £ow cytometry [68,101^104] since it acts as an electron acceptor in the electron transport system of bacterial cells. As such, it is taken to be a direct measure of abundance of those cells that are actively engaged in respiration [104]. The CTC assay remains controversial since the interpretation of results is often di¤cult.…”

“…Therefore, there is clear evidence that the CTC assay cannot be considered a viability assay. Furthermore, respiring cell counts are also often lower than those obtained by other methods used for activity assessment such as microautoradiography or the detection of esterase activity and membrane potential [104,107]. Discrepancies are due to the fact that each assay addresses di¡erent cellular functions and has speci¢c limitations.…”

Quantifying the metabolic activities of human-associated microbial communities across multiple ecological scales

“…Active microbial cells can also be quantified with the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride CTC (Rodriguez et al, 1992;Sherr et al, 1999;Sieracki et al, 1999). This salt competes with oxygen as an electron donor and is reduced by the active electron transport system of respiring cells to an insoluble red compound, formazan (Kaprelyants & Kell, 1993;Smith & McFeters, 1997).…”