The biovolume of the cells was calculated using the following formula for a prolate spheroid: V = (π/4)W2(L – W/3) where W = cell width and L = cell
length. A conversion factor of 0.35 pgC μm−3 ( Bjørnsen 1986) was used to calculate the carbon biomass from the biovolume selleck compound of the cells. To obtain total bacterial cell counts (TCC), fixed samples (1% formaldehyde) were incubated for 30 min with 5 mM (final conc.) EDTA (for dissolving aggregates), stained for 15 min with SYBR Green (1x, Sigma Aldrich, USA) and analysed with a BDbiosciences FACS Calibur flow cytometer. The flow cytometer was equipped with an argon ion laser (15 mW) and the 488 nm emission line was used as the light source. Right-angle light scatter (SSC) was selleck chemicals detected with a 488/10 nm band-pass filter and fluorescence (FL1) with a 530/30 nm band-pass filter. The system threshold was set to FL1 and SSC. The sample flow was calibrated by weighing three sets of water samples before and after each set of samples. The salinities (densities) of the samples were included in the calculations. 10 ml water samples were fixed with filtered formaldehyde (final conc. 1%), filtered on polycarbonate white filters (Osmonics INC., Poretics, 0.2 μm pore size, diameter 47 mm), rinsed with 100 ml sterile
distilled water, dried and stored at –20°C. CARD FISH hybridisation was performed according to the protocol of Pernthaler et al. (2004). Oligonucleotide probes with horse-radish peroxidase were used to specifically stain bacterial populations ( Table S1, see page 853). CARD-FISH preparations were evaluated on an epifluorescence microscope from Zeiss Axiophot.
The photomicrographs were taken using an Axio Vision Camera (Carl Zeiss, Jena, Germany), and the bacteria were counted manually by ImageJ ( Collins 2007). Oxalosuccinic acid At least 1000 DAPI-stained cells per sample were counted. The nonEUB counts were non- or individual (1–2) cells per filter and therefore neglected. Relative numbers were based on DAPI counts. In the case of Bacteria, Alphaproteobacteria, Betaproteobacteria and Actinobacteria, the mean percentage of hybridised cells were calculated from two filters. The bacterial biomass production was determined by the 3H-leucine uptake method (Kirchman et al. 1985), using a mixture of radioactive leucine (8.3 nmol l−1, specific activity 60 Ci mmol−1) and non-radioactive leucine (100 nM) (Hoppe et al. 1998). Triplicates and a negative control (fixed with 1% formaldehyde, final concentration) were incubated at the in situ temperature for one hour. The incubation was stopped by adding sterile filtered formaldehyde (final conc. 1%). The protein production (BPP) was calculated based on the equation of Simon & Azam (1989), assuming an intracellular leucine isotope dilution of two. The cell-specific exponential growth u was calculated with the equation u = ln((BBM + BPP)/BBM). The doubling time (DT) was calculated with the equation DT = ln(2)/u ( Crump et al. 2004).