Ems 550 automated sputter coater

Overnight cultures of ΔSSA_0351 and S. sanguinis SK36 were diluted 1 : 100 in BHI and grown to late log phase. Bacterial samples were deposited onto a 0.1 µm disposable Millipore filter to remove medium. Samples were fixed using 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min, followed by 1 % osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4). The samples embedded in the filters were then dehydrated in ethanol followed by PBS and allowed to air dry. The filters were sectioned and mounted onto stubs and coated with gold for 3 min (EMS– 550 Automated Sputter Coater, Electron Microscopy Sciences, Hatfield, PA, USA). Micrographs were taken at 10 000 and 20 000× total magnification using a Zeiss EVO 50 XVP scanning electron microscope (Carl Zeiss, Peabody, MA, USA).

Following overnight incubation, 0.5 ml of each culture was deposited onto a 0.1 µm disposable Millipore filter to remove BHI broth. The samples were fixed using 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer, followed by 1 % osmium tetroxide in 0.1 M sodium cacodylate buffer. Samples embedded in the filters were then dehydrated in ethanol and hexamethyldisilazane (HMDS) and finally allowed to air-dry. The filters were sectioned and mounted. Prior to visualization samples were coated with gold for 3 min (EMS-550 Automated Sputter Coater; Electron Microscopy Sciences). Micrographs were taken at ×20 000, ×30 000 and ×40 000 total magnification using a ZEISS EVO50 XVP scanning electron microscope (Carl Zeiss).

Untreated or treated P. gingivalis cells were deposited onto a 0.1 μm disposable Millipore filter to remove medium, and samples were fixed using 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min, followed by 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4). Samples embedded in the filters were then dehydrated in ethanol followed by hexamethyldisilazane (HMDS) and allowed to air-dry. The filters were sectioned and mounted onto stubs and coated with gold for three minutes (EMS– 550 Automated Sputter Coater, Electron Microscopy Sciences, Hatfield, PA). Micrographs were taken at 30,000× total magnification using a Zeiss EVO 50 XVP scanning electron microscope (Carl Zeiss, Peabody, MA).