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Abstract

A method based on fluorescent-antibody labelled bacteria (FALB) was developed to demonstrate the presence of ingested bacteria in the food vacuoles of heterotrophic nanoflagellates. Individual bacterial cells are identified in the food vacuoles by immunofluorescence labelling using strain-specific antibodies after uptake of the live cells. The procedure includes permeabilization of fixed flagellate cells prior to the immunoreaction. The uptake of Pseudomonas fluorescens (strain ON2 and Ag1) and P. putida (strain MM1) by Spumella sp., Bodo saltans and 2 unidentified heterotrophic nanoflagellate species was tested. The ingested bacteria were visualized in the food vacuoles by epifluorescence and laser confocal scanning microscopy. The ability of the polyclonal antibodies to recognize the target bacteria was good and very few cross-reactions with other bacteria or other organic compounds were observed. A linear uptake of live bacteria was recorded during the first 15 min of the feeding period and immunolabelled remains of the bacteria were visible inside and around the food vacuoles after extended feeding periods. Ingestion rates measured by the FALB method were significantly higher than those measured by FLB which confirms conclusions from several recent studies using vial stains to detect grazing on bacteria. It was concluded that the FALB method is useful and suitable to examine feeding of protists on specific bacteria as well as differential uptake of bacteria within a mixture. The FALB method does not involve any manipulations of the bacterial cells before the feeding experiment and thus measures realistic ingestion rates