2 μm filtered distilled water. Also, 125 μL of 5% (w/v) phenol and 625 μL of ∼95% sulfuric acid were added simultaneously to the diluted samples in semi-micro photometer cuvettes (Plastibrand 759015). After 10 min incubation at room temperature and 15 min incubation at 30 °C, absorbance was measured at 490 nm against a glucose standard (0–100 μg mL−1). For bulk DNA measurements, 1 mL
aliquots of the homogenized biofilm-containing liquid cultures were lyzed by three freeze/thaw cycles (5 min at −20 °C and 5 min at 60 °C). DNA contents of the lyzate, and also DNA contents of the EPS extracts, were measured in 200 μL dilution series in black 96-well optical bottom microtiter plates (Nunc 265301). PI was added to a final concentration of 50 μM. Total fluorescence was measured find more on a Beckman–Coulter DTX880 multimode detector using a long-pass emission filter and 535 nm excitation light. Solutions of 0–100 μg mL−1 fish sperm DNA (Sigma 74782) were used as a standard. Nonpurgeable organic carbon contents were determined with a Total Organic Carbon Analyzer (TOC-Vwp; Shimadzu,
Columbia, MD) using a standard protocol. The sensitivity range of the method was 0.5–3500 mg L−1. Dynamic viscosities of cultures and extracts were determined by measuring elution times for draining 50 mL aliquots of the cultures or culture extracts through a glass burette (i.d.=2 mm). Using deionized water as a reference (=1.003 cSt), dynamic viscosities of the suspensions were determined GSK126 concentration with =tC, with t as elution time and C as instrument constant. Exocellular β-glucosidase activity was measured according to Rath & Herndl, 1994. Replicate static cultures were mixed by vortexing, sampled, and supernatants were collected by centrifugation (10 000 g, 1 min) and microfiltration (0.2 μm). 4-Methylumbelliferyl β-d-glucopyranoside
was added (2.5 μM) and fluorescence was read after 30 min (265 nm excitation, 445-nm band pass emission; Cary Eclipse, Varian) against nonspiked control samples. Microfiltered sonication-lyzed P. putida cells were used to check method response. RAS p21 protein activator 1 Live/dead staining was performed as per an established protocol (Nielsen et al., 2009). In brief, replicate static cultures were mixed and two subsamples of 100 μL were removed; each of these was supplemented with 10 μL Sybr Green (Sybr Green I; Molecular Probes, Eugene, OR) from a 100 × stock solution, 10 μL PI (Invitrogen, Carlsbad, CA) from a 0.5 mg mL−1 stock solution, and 10 μL yellow–green fluospheres–carboxylate microspheres (F-8827; Molecular Probes) in a concentration of 2 × 107 mL−1 as internal standards for flow control. The samples were then supplemented with 870 μL MilliQ water containing 5 mM EDTA for outer-membrane permeabilization (Nebe-von-Caron et al., 2000). Samples were then vortexed, incubated in the dark for 15 min, and briefly vortexed again before being analyzed.