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Use of green fluorescent protein for online, single cell detection of bacteria introduced into activated sludge microcosms Leo Eberl, Aldo Ammendola, Otto Geisenberger, Renate Schulze, Michael Givskov, Claus Sternberg, S Ren Molin and K.H. Schleifer Abstract A derivative of broad host-range plasmid RP4 was tagged with the green fluorescent protein (GFP) and this construct was used to mark various Gram-negative bacteria. Video recording of combined phase contrast and epifluorescence microscopy was employed to track the fate of individual marked cells after introduction into a sludge microcosm. Whether the introduced bacteria were laboratory strains or activated sludge isolates. they were found to be rapidly eliminated from the microcosms as a result of intense predation by protozoa. Furthermore, a method was developed that allows detection of GFP-tagged bacteria in fixed samples that are simultaneously used for in situ hybridisation with a eukaryote-specific rRNA-targeted oligonucleotide probe for visualisation of protozoa. Keywords green fluorescent protein, in situ hybridisation, protozoan grazing, activated sludge

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