Measuring the grazing losses of picoplankton: methodological improvements in the use of fluorescently labeled tracers combined with flow cytometry

Fluorescently labeled tracers (FLT) are often used to estimate the loss rat es of picoplankton to grazers. These tracers are commonly enumerated by epi fluorescence microscopy, although flow cytometry is a viable alternative in the detection of FITC (fluorescein-5-isothiocyanate)- or DTAF (5-([4,6-dic hlorotriazin-2-yl)amino]-fluorescein)-stained bacterial tracers. However, t he bacterivory measured with FLT has hardly been applied to routine monitor ing of oceanic waters, partly because of the time-consuming preparation of the tracers and other problems associated with the long-term incubations ne eded to generate detectable rates of tracer change. In addition, these long -term incubations make samples especially sensitive to the unwanted additio n of nutrients carried over with the tracers. Here we present some experime nts designed to ease the estimation of grazing rates on bacteria with this technique. Two bacterial strains and 2 fluorescent dyes were tested: Escher ichia coli minicells (0.065 mu m(3)) and Pseudomonas diminuta (0.064 mu m(3 )), stained with DTAF or with FITC. In addition, instead of the common use of pyrophosphate buffer during the staining protocol, the use of carbonate- bicarbonate buffer and cells scraped directly from solid media is suggested to avoid the problems associated with phosphorus enrichment of the sample that at times can occur in oligotrophic water samples. The FITC- or DTAF-st ained tracers can be observed with either epifluorescence microscopy or flo w cytometry. However, FITC- or DTAF-stained P, diminuta were more easily re solved with the flow cytometer than stained minicells. Flow cytometric dete ction of P. diminuta tracers, prepared in bicarbonate-buffer and stained wi th FITC, is a fast protocol for the estimation of the grazing loss rates of bacteria in oceanic environments.