Determination of Catheter Fibrin Deposition by Scanning Electron Microscopy

The previously described in vitro catheter infection model was used with some minor modifications (Vanassche et al., 2013). Sterile, polyurethane, triple-lumen, central venous catheters were cut into 2-mm fragments. These fragments were placed in a suspension of either wild-type S. aureus Newman or S. aureus Newman vWbp-knockout strains (ΔvWbp) at an OD₆₀₀ of 1.0 and incubated on a shaking platform at 37°C for 30 min. After being rinsed with sterile NaCl (0.9%), the catheters were placed in 1 ml of fresh rabbit plasma spiked with fibrinogen containing heparin with or without ABBA. After 24 h, catheters were rinsed with sterile NaCl (0.9%) and fixed overnight. Following a 2-h post-fixation period in 2% OsO₄, the samples were dehydrated with a series of ethanol concentrations. After overnight immersion in hexamethyldisilazane, the samples were coated with platina and visualized using a JEOL 7401F scanning electron microscope (JEOL Europe, Zaventem, Belgium) at 2.0 kV.