AQUATIC MICROBIAL ECOLOGY Aquat Microb Ecol Vol. 44: 85–96, 2006 Published August 16 INTRODUCTION Studies in which phagotrophy is addressed are still seriously hampered by tedious and often time-consum- ing methods (Riemann et al. 1995). Over the last decades, many methodologies have been developed to detect phagotrophy in heterotrophic and mixotrophic phytoplankton species. Visually detected and manu- ally counted tracer-based techniques are the most common approaches for estimating phagotrophy in algae (Havskum & Riemann 1996, Granéli & Carlsson 1998, Legrand et al. 1998, 2001, Pålsson 2001). Surro- gate prey, including dye and starch particles, fluores- cent microspheres (Smalley et al. 1999), fluorescently labeled bacteria and algae have been used to investi- gate feeding behaviors and to measure ingestion rates of planktonic protists (Li et al. 1996). Although the use of fluorescently labeled prey has resulted in an increased understanding of phagotro- phy, advances in this field have been slow (Granéli & Carlsson 1998). Usually a large number of cells must be counted manually with the help of a microscope in order to achieve statistically significant ingestion rates, a monotonous and lengthy procedure. In addition, epi- fluorescence microscopy has a low resolution with regard to detection of ingested fluorescent prey, espe- © Inter-Research 2006 · www.int-res.com *Email: wanderson.carvalho@hik.se Acidotropic probes and flow cytometry: a powerful combination for detecting phagotrophy in mixotrophic and heterotrophic protists Wanderson F. Carvalho*, Edna Granéli Marine Science Department, University of Kalmar, 391 82 Kalmar, Sweden ABSTRACT: Studies with phagotrophic organisms are hampered by a series of methodological con- straints. To overcome problems related to the detection and enumeration of mixotrophic and hetero- trophic cells containing food vacuoles, we combined flow cytometry and an acidotropic blue probe as an alternative method. Flow cytometry allows the analysis of thousands of cells per minute with high sensitivity to the autofluorescence of different groups of cells and to probe fluorescence. The method was first tested in a grazing experiment where the heterotrophic dinoflagellate Oxyrrhis marina fed on Rhodomonas salina. The maximum ingestion rate of O. marina was 1.7 prey ind. –1 h –1 , and the fre- quency of cells with R. salina in the food vacuoles increased from 0 to 2.4 ± 0.5 × 10 3 cells ml –1 within 6 h. The blue probe stained 100% of O. marina cells that had R. salina in the food vacuoles. The acidotropic blue probe was also effective in staining food vacuoles in the mixotrophic dinoflagellate Dinophysis norvegica. We observed that 75% of the D. norvegica population in the aphotic zone pos- sessed food vacuoles. Overall, in cells without food vacuoles, blue fluorescence was as low as in cells that were kept probe free. Blue fluorescence in O. marina cells with food vacuoles was 6-fold higher than in those without food vacuoles (20 ± 4 and 3 ± 0 relative blue fluorescence cell –1 , respectively), while in D. norvegica cells were 4.5-fold brighter than the ones without food vacuoles (291 ± 155 and 64 ± 23 relative blue fluorescence cell –1 , respectively). The use of acidotropic probes can prevent fix- ation artifacts such as regurgitation of food vacuoles and changes in the cellular characteristics. The combination of flow cytometry and an acidotropic probe proved to be an efficient tool in detecting phagotrophy in mixotrophic and heterotrophic marine phytoplankton species. KEY WORDS: Mixotrophy · Phagotrophy · Acidotropic probes · Flow cytometry Resale or republication not permitted without written consent of the publisher