The instrument has a 100-µm multi-purpose large scanner and was operated in contact

mode with speeds ranging from 0.5 to 1.0 Hz and 512 pixels per line scan. A Veeco MLCT-E cantilever with a resonant frequency ranging from 26 to 50 kHz and a nominal spring constant of 0.5 N m−1 NVP-BGJ398 molecular weight was used for imaging. Scans were acquired with sizes ranging from 10 to 75 µm for all samples. Sterile 55-mm glass bottom petri dishes (MatTek Corp., Columbia, MD) were prepared with lectin prior to inoculation. LcH and WGA lectins, diluted to a final concentration of 100 µg mL−1 in PBS, were added to the glass bottom dishes and incubated for 2 h at room temperature. Next, the liquid was removed and 3 mL of overnight cell cultures in TY, diluted to OD600 nm of 1.0 (approximately Selleckchem 3-deazaneplanocin A 106 CFU mL−1) were immediately placed on the wet glass surface of the petri dish. Dishes were incubated statically at 28 °C for 24 h. SYTO 9 dye (1 µL) (Molecular Probes, Invitrogen Inc., Eugene, OR) was then added for 15 min in the dark to fluorescently label

the cells. Images were acquired with laser intensity and gain held constant using a Leica TCS SP2 scanning confocal microscope equipped with a Leica HCX PL APO 63×/1.40–0.60 oil objective lens and Leica LCS software (version 1537, Leica Microsystems Inc., Buffalo Grove, IL). The number of attached cells was assessed using the imagej software to convert the images to a binary format. The pixel area corresponding to the fluorescent cells was identified Exoribonuclease and calculated as a percentage of the total image area (http://rsb.info.nih.gov/ij). Wheat seeds (Triticum aestivum cv. Jagger) were surface-sterilized and allowed to germinate as described (Greer-Phillips et al., 2004). For the wheat root attachment assay, A. brasilense strains were cultured in TY liquid overnight (28 °C, 200 rpm) and the cultures were normalized to an OD600 nm of 1.0 using sterile phosphate buffer. A volume of 200 μL of each strain prepared as described above was inoculated, in triplicate, into glass tubes containing 9.8 mL sterile phosphate buffer and 0.5 g of sterile roots isolated from 1-week-old

plantlets and allowed to incubate for 2 h with shaking. The excised roots were then collected and washed three times with 5 mL of buffer with gentle shaking. Root material was then homogenized in 5 mL of fresh buffer and aliquots of the homogenized slurry were serially diluted and inoculated in triplicate on MMAB plates to determine colony forming units. The fraction of root-attached cells was expressed as percent of total cells inoculated. Wheat colonization assays were performed as described previously (Greer-Phillips et al., 2004) with cultures inoculated at comparable levels (107 cells mL−1) into 15 mL molten semi-soft (0.4% agar) Fahraeus medium (Zamudio & Bastarrachea, 1994) modified with traces of sodium molybdate.