SummaryWe present a new procedure for effectively detaching particle-associated bacteria by 10% (v/v) methanol and sonication which is particularly suitable for samples with a high particle load and sediments. We also optimized the sample preparation by applying the highly dsDNA-specific fluorescent stain SybrGreen I together with an optically brilliant mounting medium (polyvinylalcohol 4-88, 'moviol') in one step. The new protocol allows a much faster, easy and less toxic handling of samples as compared to other methods. Cells are stained directly on a black Nuclepore filter and show an intensive fluorescence signal with low background. The detachment procedure was optimized with respect to the temperature of the 10% methanol solution (35 ∞ ∞ ∞ ∞ C), ultrasonication and centrifugation. The application of the new method in comparison with detachment procedures with pyrophosphate and Tween-80 with various types of marine samples including sediments always yielded higher numbers and/or higher fractions of particle-associated cells. Staining and mounting the samples with the moviol-SybrGreen I solution allowed an accurate and highly reproduceable enumeration of bacteria also in samples with high concentrations of SPM. Fixation of bacteria by glutardialdehyde resulted in a brighter fluorescence as compared to fixation by formalin. Because of the high specificity to dsDNA and bright fluorescence of SybrGreen I, the fast and easy handling and the possibility to store stained samples for at least several months at ----20 ∞ ∞ ∞ ∞ C without any loss in fluorescence intensity, the newly developed method is also an attractive alternative to DAPI staining of aquatic bacteria.

An indoor mesocosm used to determine the effect of warming on microbial communities. Inset: Recycling of organic matter inside the mesocosm is mediated by the interaction of phytoplankton and bacteria.

A microcosm experiment was conducted to investigate the interactive effects of rising sea-surface temperature and altered nutrient stoichiometry on the biogeochemical cycling of organic matter in a pelagic algal-bacterial assemblage. Natural seawater, containing a mixed bacterial community, was inoculated with an axenic culture of the bloom-forming diatom species Skeletonema costatum. A factorial combination of three temperatures, simulating weak to strong warming as projected for the end of the 21st century, and either nitrogen (N)-replete or -deficient growth conditions were applied. Depending on the type of nutrient limitation, the mixed algal-bacterial communities displayed pronounced differences in the accumulation and microbial utilization of organic matter in response to warming. Under N-deficient conditions, the build-up of organic matter occurred, irrespective of temperature, dominantly in the particulate pool, and only small amounts of dissolved material accumulated. The subsequent bacterial consumption of organic matter was low, as indicated by measurements of bacterial secondary production and extracellular enzyme activities, and remained also largely unaffected by an increase in temperature from 4uC up to 12uC. Contrastingly, warming resulted in a distinct temperature-dependent increase in the accumulation of dissolved organic carbon compounds under N-replete growth conditions. Moreover, rising temperature notably stimulated the bacterial activity, indicating an enhanced flow of organic matter through the microbial loop. These findings suggest that there will be strong shifts in the biogeochemical cycling of organic matter in the upper ocean in response to increased temperature and nutrient loading that will affect pelagic foodweb structures and the biological sequestration of organic matter.