Viability of bacteria from different aquatic habitats. II. Cellular fluorescent markers for membrane integrity and metabolic activity

Abstract

We applied different types of fluorescent markers to natural bacterioplankton from different aquatic systems to investigate microscopically the percentage of viable bacteria. To characterise viable bacteria, cell-specific respiration was measured by cyanoditolyltetrazolium chloride (CTC) reduction. Membrane integrity was investigated with 3 'dead cell' stains (SYTOX ® Green, propidium iodide and ethidium homodimer-2). Cellular enzyme activity was detected by artificial substrate analogs with a high cell retention (CellTracker™ Green CMFDA for cellular esterase and 7-amino-4-chloromethylcoumarin L-leucine amide, hydrochloride (CMAC-Leu) for cellular peptidase). The percentage of impermeable, i.e. morphologically intact, cells accounted for 22 to 81% of the total cell number at all locations. Al- though up to 48% of all bacteria were respiring, they averaged between 10 and 14% in freshwater, es- tuarine waters and in the Baltic Sea. The portion of esterase-positive cells correlated significantly with the concentrations of dissolved (DOC) and particulate organic carbon (POC) as well as with chlorophyll a (chl a) content. Cellular esterase was shown by this labelling technique in only 9% of freshwater, 12% of estuarine and 5% of Baltic Sea bacteria, . The percentages of bacteria with cellular peptidase were even lower with 6, 5 and 3%, respectively. The different amounts of intact and respiring bacteria as well as those with cellular hydrolytic enzyme activities require not only correct operational definitions of active and viable bacteria, but also the appropriate choice of fluorescent markers regarding the goals of investigation. Fluorescent labels for cellular hydrolytic enzymes will also provide a new tool to localise active cells in aggregates or on sediment particles, where, besides the respiration of organic carbon, hydrolysis of organic substances is an important conversion process.