Trump s fixative solution

L. johnsonii N6.2 cultures were fixed with Trump’s fixative solution (VWR, USA), placed onto 0.2 μm polycarbonate membrane and processed by Pelco BioWave Pro laboratory microwave (Ted Pella, Redding, CA, USA). The samples were washed three times with PBS (pH= 7.2), post-fixed with 1% buffered osmium tetroxide. After the post-fixation step, the samples were washed with distilled water and dehydrated with a graded ethanol series (25%, 50%, 75%, 95%, 100%). The final step was to preserve the surface structures of the samples by drying them in a critical point dryer (Autosamdri-815, Tousimis Research Corp, Rockville MD, USA). The samples were mounted on carbon adhesive tabs on aluminum specimen mounts and Au/Pd sputter coated (DeskV Denton Vacuum, Moorestown, NJ, USA). Digital micrographs were acquired by field-emission scanning electron microscope (SU-5000, Hitachi High Technologies America, Inc. Schaumburg, IL, USA).

L. johnsonii N6.2 was grown in exosome-free MRS for 24 h. Cells were obtained by centrifugation at 7,000 x g for 15 min, at 4°C, washed in exosome-free 0.9% peptone water (w/v) and fixed with 2% glutaraldehyde (v/v) in exosome-free 0.9% peptone water (w/v) at 4°C for transmission electron microscopy (TEM) and Trump’s fixative solution (VWR, USA) for scanning electron microscopy (SEM). All electron microscopy imaging was conducted by the Microscopy core in the University of Florida Interdisciplinary Center for Biotechnology Research.