Background: Regular phytoplankton monitoring in Dutch coastal waters is performed as an indicator of the ecological state of these waters. The monitoring program is focused on temporal and spatial changes of species composition and abundance. Flow cytometry has been introduced to provide additional information, to improve ecosystem understanding, and to increase the efficiency of analysis and reportage. Methods: Phytoplankton community abundance and composition were routinely determined by flow cytometry and microscopy at six locations in the North Sea over three annual cycles between 2000 and 2003. Supplementary measurements were also made for fluorescence (chlorophyll-a and other pigments) and, in combination with flow cytometric and microscopic data, were used to determine phytoplankton abundance and composition as a function of their size distribution. Real-time imaging of species was also used to identify species on the basis of their flow cytometric optical characteristics. Results: Flow cytometric analysis took 15 min on average. Analysis including data processing, and Web site reportage took less than 1 h. Phytoplankton concentrations (cells/ml), biomass (fluorescence/ml), and concentration of phycoerythrin-or phycocyanin-containing cells (cells/

SUMMARY: The developments in the combination of flow cytometry and immunology as a tool to identify, count and examine marine phytoplankton cells are reviewed. The concepts of immunology and flow cytometry are described. A distinction is made between quantitative and qualitative immunofluorescence. Quantitative immunofluorescence, the identification and enumeration of phytoplankton cells, is the research area that has advanced rapidly in the past decade, and is reviewed extensively. Key steps of quantitative immunofluorescence, fixation and immunolabel intensity, are discussed in more detail. Qualitative immunofluorescence is a new, hardly explored but highly interesting development in which qualitative -physiological-variables related to e.g. nutrient limitation or primary production are measured in individual cells instead of phytoplankton populations as a whole. Several combinations of immunological probes, both for species identification and for physiological measurements, are proposed. A special case of qualitative immunofluorescence is the measurement of phytoplankton toxins in single cells from natural populations. It is anticipated that the future use of semiconductor nanocrystals or "quantum dots" as fluorophores will greatly enhance signal detection in flow cytometry, and hence in both quantitative and qualitative immunofluorescence applications.