Phosphotungstic acid

With respect to TEM, the samples were prepared by exchanging a high-titer phage lysate in HEPES buffer via Amicon Ultra −0.5 mL Centrifugal Filter Units 100K (Merck, Darmstadt, Germany). The phages then adsorbed on glow discharged formvar-carbon-coated nickel grids (Maxtaform, 200 mesh, Plano, Wetzlar, Germany) for 10 min. Afterwards the samples were stained with a drop of 1% phosphotungstic acid (in aqua dest., adjusted to pH 7.2; Agar Scientific Ltd., Stansted, United Kingdom) and then air-dried before they were examined using a TEM LEO 906 (Carl Zeiss, Oberkochen, Germany), operating at an acceleration voltage of 60 kV. For photography, a wide-angle Dual Speed 2K-CCD-Camera 14 bit (Tröndle, TRS Moorenweis, Germany) and the analysis software IMAGE SP Professional (SISPROG, Tröndle, Moorenweis, Germany) were used. A description of phage morphology, and determination of the size of phage head and tails, were performed based on five selected virions displayed on the electron micrographs (average size ± standard deviation).

Bacteria in their growth medium were allowed to adsorb on formvar-carbon-coated nickel grids (Maxtaform, 200 mesh, Plano, Wetzlar, Germany) for 10 min prior to washing with distilled water. Samples were then stained by placing a drop of 1% phosphotungstic acid (Agar Scientific, Stansted, UK) in distilled water (pH 7.2) onto the grid for a few seconds. The grid edge was carefully laid onto a filter paper to remove the staining solution from the grid. After air drying, samples were examined at the electron microscopy facility of the University Hospital of RWTH Aachen using a Hitachi HT7800 transmission electron microscope operating at an acceleration voltage of 100 kV.

High-titer phage lysate was exchanged in HEPES buffer via Amicon Ultra-0.5 mL Centrifugal Filter Units 100 K (Merck, Darmstadt, Germany). Phages were allowed to adsorb on glow discharged formvar-carbon-coated nickel grids (Maxtaform, 200 mesh, Plano, Wetzlar, Germany) for 10 min. Samples on grids were stained by placing on a drop of 1% phosphotungstic acid (in aqua dest., adjusted to pH 7.2; Agar Scientific Ltd., Stansted, United Kingdom). After air drying, samples were examined using a TEM LEO 906 (Carl Zeiss, Oberkochen, Germany), operating at an acceleration voltage of 60 kV.
Wide-angle Dual Speed 2K-CCD-Camera 14 bit (Tröndle, TRS Moorenweis, Germany) and analysis software IMAGE SP Professional (SISPROG, Tröndle, Moorenweis, Germany) were used to photograph observations.
Description of phage morphology and determination of the size of phage head and tails were performed based on five selected virions displayed on the electron micrographs (average size ± standard deviation is given).