Microwave-Assisted SEM Preparation

Sorted trichomonads or cecum from mono-colonized mice were prepared for scanning electron microscopy (SEM) via a Biowave microwave-assisted (Ted Pella, Inc.) protocol as described in (Jackson et al. 2018 (link)). Briefly, samples were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, 0.05% alcian blue in 0.1 M Sorenson’s phosphate buffer (Karnovsky’s Fixative, Electron Microscopy Sciences) and stored at 4 °C until further processed. Sorted cells were adhered to poly-L-lysine coated silicon wafer chips for 30 minutes. Both sorted cells and cecum pieces were post-fixed in 0.5% OsO₄, 0.8% K₄Fe(CN)₆ in 0.1 M sodium cacodylate buffer for 1 hour. The samples were rinsed in dH₂0, stained with 1% aqueous tannic acid for 1 hour, rinsed with dH₂0, and finally fixed with a second round of 0.5% OsO₄, 0.8% K₄Fe(CN)₆ in 0.1 M sodium cacodylate buffer for 1 hour to enhance conductivity under the scanning electron beam. Samples were rinsed with dH₂0, then dehydrated using a graduated ethanol series into 100% ethanol, critical point dried using a Bal-Tec CPD 030 (Leica Microsystems, Buffalo Grove, IL), placed on a stub using carbon sticky tape, and sputter coated with 10Å of iridium (EMS300TD, Electron Microscopy Sciences). Samples were placed in a Hitachi SU8000 scanning electron microscope operating at 5.0 kV, 10 μA, and a working distance of ~8 mm using a secondary electron detector.