MEASUREMENT OF BACTERIVORY BY HETEROTROPHIC NANOFLAGELLATES USING IMMUNOFLUORESCENCE LABELING OF INGESTED CELLS

A method based on fluorescent-antibody labelled bacteria (FALB) was de veloped to demonstrate the presence of ingested bacteria in the food v acuoles of heterotrophic nanoflagellates. Individual bacterial cells a re identified in the food vacuoles by immunofluorescence labelling usi ng strain-specific antibodies after uptake of the live cells. The proc edure 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 u nidentified heterotrophic nanoflagellate species was tested. The inges ted bacteria were visualized in the food vacuoles by epifluorescence a nd laser confocal scanning microscopy. The ability of the polyclonal a ntibodies to recognize the target bacteria was good and very few cross -reactions with other bacteria or other organic compounds were observe d. A linear uptake of live bacteria was recorded during the first 15 m in of the feeding period and immunolabelled remains of the bacteria we re visible inside and around the food vacuoles after extended feeding periods. Ingestion rates measured by the FALB method were significantl y higher than those measured by FLB which confirms conclusions from se veral recent studies using vial stains to detect grazing on bacteria. It was concluded that the FALB method is useful and suitable to examin e feeding of protists on specific bacteria as well as differential upt ake of bacteria within a mixture. The FALB method does not involve any manipulations of the bacterial cells before the feeding experiment an d thus measures realistic ingestion rates.