Estimation of bacterioplankton activity in Tasmanian coastal waters and between Tasmania and Antarctica using stains

Abstract

Various stains have recently become available that detect bacteria with compromised cell membranes and high esterase or respiratory activity. However, the validity of these stains for determining concentrations of live, dead and active marine bacteria is the subject of debate. We used BacLight™ (Molecular Probes) to stain bacteria with intact membranes green, due to the fluorescence of SYTO®9, while 'leaky' bacteria that were permeable to propidium iodide (PI) stained red. Bacteria with high metabolic activity were stained using 6-carboxy fluorescein diacetate (6CFDA) or 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Total bacteria concentration was determined using 4′, 6 diamidino-2-phenylindole (DAPI) and BacLight™ (SYTO®9 + PI-stained bacteria). Comparison of stains in Tasmanian coastal waters showed that total concentrations of bacteria obtained using DAPI were not significantly different from those using BacLight™ at the concentrations used in our field study. Lower concentrations of BacLight™ reduced the concentration of PI-stained bacteria but not that of those stained by SYTO®9. Concentrations of metabolically active bacteria obtained using CTC and 6CFDA were equivalent to those of viable, culturable bacteria obtained using most probable number. Bacterioplankton activity in the Southern Ocean between Tasmania and East Antarctica during austral winter 1999 and summer 2001 was investigated using these stains. SYTO®9-stained cells commonly comprised <30% of total bacteria, while the remainder stained with PI. CTC- or 6CFDA-stained bacteria comprised around half of the SYTO®9-stained bacterial concentration. Significant positive correlation was observed between concentrations of total bacteria, seawater temperature (mainly due to the correlation between temperature and concentrations of PI-stained bacteria) and in situ chlorophyll fluorescence (mainly due to the correlation between in situ chlorophyll fluorescence and concentrations of highly active bacteria). Thus, bacterioplankton in the Southern Ocean are not a homogeneous community of active cells. As bacterial metabolism determines the extent to which bacteria are involved in respiration, remineralisation and the microbial loop, our findings are of major significance for understanding pelagic carbon flow and nutrient cycling in the Southern Ocean.