Cytometric evidence reconciling the toxicity and usefulness of CTC as a marker of bacterial activity

To understand the opposing views on the utility of the CTC method, we examined bacterial abundance over incubations with the fluorogenic tetrazolium dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and inspected the flow cytometric signatures of bacteria dually labeled with a DNA stain (Syto13) and CTC. Incubation of a marine plankton sample with CTC produced positive cells up to a stable value, reached in < 1 h, while the red fluorescence of the granules increased for at least an extra hour. Incubation also produced a decrease in cell abundance of 22% after 30 min in the presence of 5 mM CTC. The decrease was a function of CTC concentration and incubation time, and particularly affected bacteria with high nucleic acid content. Flow cytometric inspection of a double-stained (CTC and Syto13) sample showed that after 15 min of incubation, particles appeared having both red (CTC+) and green (DNA+) staining. Afterwards, other particles appeared that maintained the same light scatter properties and the red fluorescence, but that lost all green fluorescence. While the number of particles with double staining stabilized after about 1 h, particles with red but without DNA staining increased for at least 100 min. Simultaneously, the classic determination of CTC+ cells (observing only the red signal of the particles) increased as reported elsewhere. We interpreted these patterns as evidence of CTF (the formazan derivative of CTC) particles growing in the bacterial cells until they are so large that they break up the cells, after which they remain present as CTF granules with no associated cellular material. Microscopic or flow cytometric enumeration of red particles might still be a good indication of the percentage of bacterial cells having taken up and reduced the activity probe, but flow cytometric cell sorting of red particles based only on scatter and red fluorescence signals will select CTF particles without associated cellular material. Our results help reconcile the ecologically sound results and the CTC toxicity evidence currently reported in the literature, and lead to a warning against interpretations of cell sorting of CTC+ particles for phylogenetic or activity studies based only on red or orange fluorescence.