Fisherbrand syringe filter

Minimal inhibitory concentrations (MICs) were determined using a modified conventional macrobroth two-fold dilution bioassay [11 (link), 20 (link)] with MHB as diluent. MHB stock solutions of novobiocin sodium (Sigma-Aldrich Co., St Louis, MO), rifamycin SV (Sigma-Aldrich Co.), and polymyxin B sulfate (Sigma-Aldrich Co.) were prepared to desired concentrations, filter sterilized (0.22 μm Fisherbrand Syringe Filter; Thermo Fisher Scientific Inc., Pittsburgh, PA), and stored at 4°C until needed. A stock solution (1,280 μg/mL) of triclosan was prepared in 95% ethanol and diluted to a concentration of 128 μg/mL in sterile MHB in a manner such that the final ethanol concentration never exceeded 0.4%. Minimal bactericidal concentrations (MBCs) were determined by inoculating MHA plates with 100 μl from concentrations greater than those contained in the MIC endpoint cultures and incubating for 24 h at 37°C before visually assessing colonial growth.

The obtained HBBT, HMW, and LMW fractions were subjected to HPLC to identify the differences in their phenolic profile. All tested samples were concentrated by rotary evaporation (Fisher Scientific; Hanover Park, IL, USA) to complete dryness and were re-dissolved in 1 mL of acetone. To 800 μL of each sample we added 400 μL of distilled water and the resulting solutions were filtered using a 0.45 μm Fisherbrand^® syringe filter (Thermo Fisher Sci, Pittsburg, PA, USA). An injection volume of 20 μL of each sample was injected and analyzed using a reverse phase C-18 column (Agilent ZORBAX Extend C-18 column, 250 × 4.6 mm id.d, 5 μm particle size). The mobile phase consisted of solvent A (4% phosphoric acid) and solvent B (acetonitrile). Gradient elution was used under linear gradient conditions starting with 95% A and decreasing linearly to 65% A over a 70 minute time period at a flow rate of 0.75 mL/min. Phenolic profiles were observed at 254 nm and 280 nm using a UV-VIS detector.