Em 109t

Cells were harvested with a cell scraper, centrifuged and fixed with 2% glutaraldehyde in PBS for 2 h at 4°C, washed three times with PBS pH 7.2 and subsequently treated with 1% osmium tetroxide (Sigma-Aldrich) for 2 h at 4°C. In the next step, cells were washed again with PBS and sequentially dehydrated in solutions with increasing concentrations of acetone. Finally, samples were included in epoxy resin (Spurr) and ultrathin sections were obtained with an ultramicrotome Ultracut Reichert-Jung E. Sections were contrasted with uranyl acetate/acetone for 3 min, washed with distilled water and colored with lead citrate for 2 min before observation with the Zeiss EM 109T equipped with a Gatan ES1000W digital camera.

The morphology of S. vacuolatus was evaluated in vivo from cultures grown in BBM liquid medium that contained 0, 4, 6 or 8 mg L -1 of glyphosate. Lugol staining was applied to highlight the cell starch granules. The observation and photomicrography were performed with a Leica DM 500 light microscope equipped with a Leica ICC50 digital camera.
The ultrastructure from control and treated cells was analyzed by transmission electron microscopy. The ultrathin sections were observed and photographed with a Zeiss EM 109 T electron microscope equipped with a Gatan ES100W digital camera at the Instituto de Biología Celular y Neurociencias (LANAIS -MIE), Facultad de Medicina, Universidad de Buenos Aires.
The number of nuclei and the cell wall thickness of at least 40 randomly chosen control and trated (8 mg L -1 ) cells were measured in the ultrathin sections photographed. The percentage of cells with more than one nucleus and the average cell wall thickness were calculated.
Additional details on procedure for ultrathin section preparation are presented in Supplementary material.