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

Abstract

Fluorescently labeled tracers (FLT) are often used to estimate the loss rates of picoplank- ton to grazers. These tracers are commonly enumerated by epifluorescence microscopy, although flow cytometry is a viable alternative in the detection of FITC (fluorescein+isothiocyanate)- or DTAF (5-((4,6-dichlorotriazin-2-yl)amino)-fluorescein)-stained bacteria1 tracers. However, the bacterivory measured with FLT has hardly been applied to routine monitoring of oceanic waters, partly because of the time-consuming preparation of the tracers and other problems associated with the long-term incu- bations needed to generate detectable rates of tracer change. In addition, these long-term incubations make samples especially sensitive to the unwanted addition of nutrients carried over with the tracers. Here we present some experiments designed to ease the estimation of grazing rates on bacteria with this technique. Two bacteria1 strains and 2 fluorescent dyes were tested: Escherichia coli minicells (0.065 pm3) and Pseudomonas diminuta (0.064 pm3), 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 asso- ciated with phosphorus enrichment of the sample that at times can occur in oligotrophic water samples. The FITC- or DTAF-stained tracers can be observed with either epifluorescence microscopy or flow cytometry. However, FITC- or DTAF-stained P. diminuta were more easily resolved with the flow cytometer than stained minicells. Flow cytometric detection of P. diminuta tracers, prepared in bicar- bonate-buffer and stained with FITC, is a fast protocol for the estimation of the grazing loss rates of bacteria in oceanic environments.