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PIXE spectra from these areas were fitted using a full non-linear deconvolution procedure (Ryan et?al. 1990a,b). The required information on thickness and matrix composition of each root cross-section was obtained from analysis of the corresponding BS spectra using a RUMP simulation package (Doolittle 1986) with non-Rutherford cross-sections for C, O, N. The reported errors were extracted from the error matrix generated in the fit (Ryan et?al. 1990a), whereas the minimum detection limits were calculated using the Currie formula (Currie 1968). Small pieces of find more fresh roots from yeast-inoculated and control plants were fixed under vacuum in 3% (vol/vol) glutaraldehyde in sodium cacodylate buffer (pH 7.2) at room temperature for 24?h. The samples were then rinsed in 0.05 m sodium cacodylate buffer, post-fixed in 2% (vol/vol) osmium tetraoxide at 4?��C for 24?h and dehydrated in a graded ethanol series. The specimens were subsequently infiltrated in increasing concentrations of Spurr's resin (Spurr 1969), and embedded at 60?��C for 48?h. For light microscopy, semithin transverse sections (0.5�C2??m in thickness) were cut using glass knives Selleck PF-06463922 attached to a Reichert OMU3 ultramicrotome (C Reichert, Vienna, Austria). Sections were heat-fixed onto glass slides, stained with Azur II and toluidine blue and examined with a Nikon Eclipse light microscope (Nikon, Tokyo, Japan) to detect endophytic C. laurentii CAB 578 cells. For TEM, ultrathin sections (100�C150?nm in thickness) were mounted onto copper grids, stained with 2% (vol/vol) uranyl acetate followed by lead citrate (Reynolds 1963) and examined with a Jeol 1200 transmission electron microscope (Tokyo, Japan) operating at 80?kV. For the detection of Casparian bands, free-hand cross-sections of fresh roots from both yeast-inoculated and control plants were stained according to the method of Brundrett, Enstone & Peterson (1988). The epifluorescence facility of the Nikon Eclipse 50i light microscope, equipped with a UV filter assembly (excitation wavelength, 365?nm; beam splitter, 395?nm; barrier filter, 420?nm), was used to locate apoplastic barriers in root tissues of both treatments. To determine the significance of concentration differences between the yeast-inoculated and control treatment FMO5 means, a Student's t-test (Schefler 1979), independent by variables was used (STATISTICA, version 7.1; Statsoft Inc., Tulsa, OK, USA [1]). Only significant differences (P?
