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A new fluorescent dye, cyanoditolyl tetrazolium chloride (CTC), is suggested for use in grazing studies to prepare fluorescently labeled bacteria (FLB). As food tracers, CTC-stained bacteria appeared to have advantages over FLBs prepared in accordance with previously published protocols. The CTC is a vital dye, non-toxic to the examined bacteria; in our study, the CTC-stained bacteria grew at the same rate as non-stained bacteria. This is an advantage over the commonly used DTAF-employing staining protocol, which results in heat-killed or immobilized prey particles. The CTC-stained bacteria were not toxic to the studied bacterial grazers; a test ciliate grew on FLBs at the same rate as with non-stained prey. The CTC-stained bacteria fluoresced bright red as opposed to the green fluorescence produced by other protocols. This made it easier to distinguish and count the consumed tracers inside protozoan and, potentially, metazoan grazers, both of which appeared green upon glutaraldehyde fixation. CTC-stained bacteria were compared to other food tracers (FLBs stained with fluorescent dyes according to previously published protocols as well as fluorescent latex microspheres) in grazing experiments. The former were consumed by a test bacterivorous ciliate at rates 2.5to 4-fold higher than the latter. Two factors may be responsible for these rates. First, CTC-stained bacteria might better mimic the extant prey. Second, ingested CTC-stained bacteria might be easier to count due to the color of their fluorescence, contrasting with the glutaraldehyde-induced fluorescence of the grazers.

Methodology of in situ grazing experiments:evaluation of a new vital dye for preparation of fluorescently labeled bacteria