Uronic acid carbazole assay.

Alginate production was measured via carbazole assay (30). Laboratory and clinical isolate strains were grown in PIB from a single colony isolation. The strains were then plated on 150-mm agar plates containing no antibiotic; RFX at concentrations of 10 μM (MIC₅₀), 6 μM (MIC₃₀), 2 μM (MIC₁₀), and 0.2 μM (MIC₁); or TOB at concentrations of 2 μM (MIC₅₀), 1.2 μM (MIC₃₀), 0.4 μM (MIC₁₀), and 0.04 μM (MIC₁) and incubated at 37°C for 48 h. Sterile PBS (30 ml) was added to the plates, and the cells plus their mucoid film were collected into 50-ml conical tubes. A 1-ml sample was taken to measure growth via OD at 600 nm. A 700-μl sample was then added to tubes containing 6 ml of a sulfuric acid-borate solution. A 200-μl aliquot of 0.1% carbazole was then added to the tubes and incubated for 30 m at 55°C. The alginate was then measured via OD at 530 nm and plotted against a standard curve made with d-mannuronic acid (Sigma-Aldrich). The reported values represent an average from three independent alginate collections with standard deviation.