Bordet-Gengou Agar

2. Materials and methods

2.1. Bacterial strains, plasmids and oligonucleotides.

All strains, plasmids and primers used in this work are listed in Table 1.

2.2. Growth conditions

Escherichia coli strains were cultured and/or maintained on Luria-Bertani (LB) broth or agar media. B. bronchiseptica strains were cultured and maintained on Bordet-Gengou agar (BGA, Difco Laboratories, Detroit USA) medium supplemented with 15% v/v defibrinated sheep blood (Instituto Biológico, La Plata, Argentina). For planktonic and biofilm cultures, B. bronchiseptica was grown in Stainer-Scholte (SS) broth. When required, the growth medium was supplemented with the appropriate antibiotic at the following concentrations: streptomycin (Sm), 50 µg/ml; kanamycin (Kan),

A Lab-Tek culture chamber with a 1-mm-thick borosilicate coverglass at the base (NUNC, Thermo Fisher Scientific, Waltham, MA, USA) was used as a system to examine the attachment of B. bronchiseptica RB50 and ΔompQ strains. To visualize bacteria by fluorescence microscopy, cells of both strains were transformed with a medium copy number vector harboring the gene coding for the green fluorescent protein (GFP) under the control of the constitutive tac promoter. For each strain, three chamber wells were inoculated with a 1.5 ml suspension of GFP-expressing bacteria adjusted to an OD650 of 0.05. After incubation for 2 h at 36ºC, bacterial suspension was removed and each chamber well was washed twice with PBS. Bacteria that remained attached to the borosilicate base of the chamber well was visualized at a magnification of 1000x using Leica DMLB fluorescence microscope equipped with a standard Blue/Green/Red filters set (excitation: 400/20, 495/15, 570/50 nm) and with a charge coupled device (CCD) digital camera. At least three independent adhesion experiments were performed for each strain. Quantification of adhered cells was performed by counting attached GFP-expressing bacteria using ImageJ [28] and the ITCN plug-in