Aqueous uranyl acetate

The virion morphology characteristics were visualized by transmission electron microscopy (TEM) using the negative staining technique. A volume of 15 µL of phage solution was dropped onto parafilm prior transferal onto a Ni-mesh grid (G2430N; Plano GmbH, Wetzlar, Germany) that has previously been carbon-coated and glow discharged (Leica MED 020, Leica Microsystems, Wetzlar, Germany). The grid is then let to adsorb for 10–15 min at room temperature and then washed three times with Aquadest and treated with 1% aqueous uranyl acetate (SERVA Electrophoresis GmbH, Heidelberg, Germany) for 20 s for negative staining. Afterwards, excess staining was removed with filter paper. Grids were air dried and then imaged by TEM using a Zeiss EM 906 microscope (Carl Zeiss Microscopy Deutschland, Oberkochen, Germany) at a voltage of 80 kV. The image processing program ImageJ [27 ] was used for phage size measurements.

For the negative staining, carbon-coated mesh grids were hydrophilized with Alcian Blue (Sigma-Aldrich, St. Louis) solution (1% in 1% acetic acid in water) for 10 min, followed by washing steps on drops of distilled H₂O. The grids were placed on 15 μL of particle solution for 10 min, followed by removing the solution by a filter paper, washing steps on drops of distilled H₂O and finally placed on a drop of freshly prepared solution of 1% aqueous uranyl acetate (Serva, Heidelberg, Germany) for 20 s. Finally, the drop on the grid was sucked off with a filter paper and the grid dried in a grid box. The images were acquired on a Zeiss Leo 906 electron microscope (Carl Zeiss, Oberkochen, Germany) at 80 kV acceleration voltage equipped with a slow scan 2K CCD camera (TRS, Moorenweis, Germany).