Wheat germ agglutinin wga oregon green 488

To determine the composition of the biofilm matrix, multi-species biofilms were prepared as described above and stained with FilmTracer FM 1-43 (Invitrogen, Eugene, OR), FilmTracer LIVE⁄DEAD Biofilm Viability Kit (Invitrogen), Wheat Germ Agglutinin (WGA-Oregon Green 488, Molecular Probes; which binds N-acetyl-D-glucosamine [PGA] and N-acetylneuraminic acid residues), FilmTracerTM SYPRO® Ruby biofilm matrix stain (Molecular Probes; binds proteins) or BOBO^TM-3 iodide (Molecular Probes; label extracellular DNA or eDNA) as prescribed by the manufacturer. After 30 min incubation at room temperature, the fluorescent marker solution was removed, and the biofilms were washed with water. The wells were then filled with 100 μL of water or PBS for WGA-stained biofilms. The stained biofilms were visualized by CLSM (FV1000 IX81; Olympus, Markham, ON, Canada) and images were acquired using Fluoview software (Olympus).

Biofilms were prepared under static conditions as described above and were stained with FilmTracer™ FM®1-43 Green biofilm cell stain (Molecular Probes, Eugene, OR, USA) according to manufacturer’s instructions. To determine the composition of the biofilm matrix, biofilms were stained with Wheat Germ Agglutinin (WGA-Oregon Green 488, Molecular Probes; binds to N-acetyl-D-glucosamine and N-acetylneuraminic acid residues), FilmTracer™ SYPRO® Ruby biofilm matrix stain (Molecular Probes; labels most classes of proteins) or BOBO™-3 iodide (Molecular Probes; stains extracellular DNA) according to manufacturer’s instructions. After a 30 min incubation at room temperature, the fluorescent marker solution was removed, biofilms were washed with water and the wells were then filled with 100 μL of water or PBS for WGA-stained biofilms. Stained biofilms were visualized by CLSM (Olympus FV1000 IX81, Markham, ON, Canada).

In order to study the morphology of mono and two-species biofilms, E. coli biofilms with or without A. pleuropneumoniae 719 were prepared as described above and stained with FilmTracer FM 1-43 (Invitrogen, Eugene, OR), Wheat Germ Agglutinin (WGA-Oregon Green 488, Molecular Probes), Film Tracer TM SYPRO® Ruby biofilm matrix stain (Molecular Probes), or BOBOTM-3 iodide (Molecular Probes) according to manufacturer's instructions (fluorescent markers stain bacterial membranes, N-acetyl-Dglucosamine [PGA] and N-acetylneuraminic acid residues, proteins and extracellular DNA or eDNA, respectively). After 30 min of incubation at room temperature, the fluorescent marker solution was removed, and the biofilms were washed with water. After that, the biofilms were observed by confocal laser scanning microscopy (CLSM; LMS 700 ZEISS; Carl Ziess Microscopy, Jena, Germany) and images were acquired using Zen Black 2012 (black edition) software (ZEISS).