Acidotropic probes and flow cytometry:: a powerful combination for detecting phagotrophy in mixotrophic and heterotrophic protists

Studies with phagotrophic organisms are hampered by a series of methodological constraints. To overcome problems related to the detection and enumeration of mixotrophic and heterotrophic 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 frequency of cells with R. salina in the food vacuoles increased from 0 to 2.4 +/- 0.5 x 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 possessed 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 fixation 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.