Merlin scanning electron microscope

Bacteria were grown at 37°C with a constant agitation at 200 rpm overnight. 1 ml of cell suspension was placed into a sterile 1.5 ml tube and centrifuged for 15 min at 4,000 rpm at 15°C. Pelleted cells were washed in PBS twice. After centrifugation, collected cells were resuspended in 1% glutaraldehyde/PBS, pH 7.4, and incubated overnight at 22°C. Then cells were rinsed three times with PBS and dehydrated in ethanol at the concentrations of 30%, 40%, 50%, 70%, 80%, and 90% (for 15 min each). Next, cells were suspended in 96% ethanol and centrifuged for 20 min at 5,000 rpm. The procedure was repeated three times. The resulting cell suspension was spread over the glass coverslip surface and dried. After the drying, glass coverslips were coated with gold palladium (Quorum Q150T ES vacuum coater, UK) and imaged using MERLIN scanning electron microscope (Carl Zeiss, Germany) in a high vacuum mode at 15 kV. Scanning electron micrographs of planktonic cells were taken at 10,000x magnification.

Fibrin clots, formed by adding CaCl₂ (24 mM final concentration) without thrombin to platelet-free plasma, were allowed to form for 2 h at room temperature in a humid chamber. The clots were washed in 0.05 M sodium cacodylate buffer (pH 7.4), then fixed in 2% glutaraldehyde in the same buffer, dehydrated in ethanol, dried with HMDS, and sputter-coated with gold/palladium. The samples were imaged with a Merlin scanning electron microscope (Carl Zeiss, Oberkochen, Germany). Fiber thickness was measured using ImageJ (remeasurement of the original data sets) at randomly selected areas for 100 fibers per clot.