Strains of A. brasilense used in this study are listed in Table 1. Strains AB103 and BS110 were previously shown to have identical phenotypes including growth, motility, chemotaxis as well as flocculation (Stephens et al., 2006; Bible et al., 2008). Except where noted, all Azospirillum strains were routinely maintained on solid tryptone yeast (TY) medium or on minimal medium for A. brasilense (MMAB; Hauwaerts et al., 2002). Flocculation Selleck ERK inhibitor was performed essentially as described in Sadasivan & Neyra (1985) and modified by Bible et al. (2008). Preliminary experiments identified the following conditions to

allow visualization of bacterial attachment. Azospirillum brasilense strains were cultured in TY medium to logarithmic phase and standardized to an OD600 nm of 1.0 using a phosphate buffer buy LGK-974 (per liter: 1.7 g K2HPO4, 1.36 g KH2PO4, 0.1 mM EDTA). Cells were re-inoculated into Corning 12-well (3.8 cm2) polystyrene containers (Corning, NY Fisher Catalog No. 3512) containing 3 mL liquid TY or MMAB medium, the latter supplemented with combined nitrogen

(NH4Cl or NaNO3, as indicated) when applicable and containing 5 mM fructose and 5 mM sodium malate as carbon sources. Attachment to glass (hydrophilic) or polyvinylchloride (hydrophobic) coverslips (2 × 2 cm; Fisher Scientific, Pittsburgh, PA) was tested by placing surface-sterilized coverslips into the wells of a PVLC 96-wells plates prior to adding cells. Attachment of cells to polyvinylchloride or PVLC (hydrophobic surface)

was equivalent and further experiments were conducted by measuring attachment to the PVLC wells (Corning). Cells were incubated for 1 and 7 days at 28 °C. To stain the biofilms, the culture was removed from the wells and a 0.01% crystal violet solution (w/v) was added and incubated 20 min. Next, the dye was removed and the excess washed by rinsing three times with sterile water. The remaining dye in the wells (representing attached cells as biofilms) was solubilized with 95% ethanol. Attachment was determined by the absorbance at 600 nm of the crystal violet solubilized (Fujishige et al., 2006). Samples were prepared on hydrophobic (polystyrene) and hydrophilic (glass) surfaces with polystyrene chips (2 × 2 cm) and glass coverslips (2.2 × 2.2 cm) as described previously (Edwards et al., 2011). Similar learn more preparations were also used with lentil (LcH; Sigma-Aldrich, St. Louis, MO; specificity for α-mannose and/or α-glucose terminal residues) or wheat germ agglutinin (WGA; Sigma-Aldrich; specificity for N-acetylglucosamine terminal residues) lectins. On cleaned and UV-sterilized surfaces, 200 μL of 100 μg mL−1 LcH or WGA were added and allowed to absorb for 2 h at room temperature. After incubation, the excess lectin was removed and 5 mL of normalized cell suspension was added to the treated surfaces, followed by incubation at 28 °C for 24 h without agitation.