Orius sc200d 1

Transmission electron microscopy (TEM) was performed using 400-mesh formvar and carbon copper grids (ProSciTech, Townsville, Australia). Bacteriophage stocks (> 10⁷ PFU/ mL) were allowed to adsorb to the grid for one min before excess solution was removed using filter paper. The grids were then negatively stained three times with 2% (w/v) uranyl acetate for 20 s. Excess stain was removed by filter paper and grids were air-dried for 20 min before examination under a JEOL JEM-2100 transmission electron microscope. This was operated at an accelerating voltage of 200 kV and high-resolution digital images were recorded on a Gatan Orius SC200D 1 wide angle camera with Gatan Microscopy Suite and Digital Micrograph (Version 2.32.888.0) imaging software. Virions were measured using ImageJ software [42 (link)] (Version 1.8.0_112).

Bacteriophage particles were visualized by Transmission Electron Microscopy using a JEOL JEM-2100 transmission electron microscope (TEM) at 200 kV. Bacteriophage lysate was adsorbed onto 400-mesh formvar and carbon coated copper grids (ProSciTech, Australia) for 1 min. Grids were rinsed with milli-Q water and adsorbed phage particles were negatively stained twice using 2% (W/V) uranyl acetate (Sigma-Aldrich^®, Australia) for 20 s. Excess stain was removed using filter paper and grids air dried for 30 min. Images were captured on a Gatan Orius SC200D 1 wide-angle camera using the Gatan Microscopy Suite and Digital Micrograph Imaging software (version 2.3.2.888.0). The images obtained were further analyzed using ImageJ (version 1.8.0_112).

Tissue sample collected from pox lesions were suspended in 1:10 in phosphate buffered saline (PBS) and chopped with a stainless-steel scalpel, followed by grinding with sterile round glass body homogeniser. Suspensions were clarified by centrifugation at 14,000 g for 5 min, followed by filtration of the supernatant through a 0.45 μm filter. The filtrate was then adsorbed onto a 400-mesh copper EM grid coated with a thin film of carbon for 5 minutes. Excess solution was removed using 3MM filter paper (Whatman) and the grid rinsed briefly with distilled water before negative staining with three 10 sec applications of 2% [w/v] uranyl acetate (Electron Microscopy Sciences, PA, USA) followed by removal of excess stain on filter paper after each application. Grids were air-dried for 20 minutes before examination under a JEOL JEM-2100 transmission electron microscope (TEM) operated at an accelerating voltage of 200 kV. High resolution digital images were recorded on a Gatan Orius SC200D 1 wide angle camera with Gatan Microscopy Suite and Digital Micrograph (Version 2.32.888.0) imaging software. Viruses were measured using ImageJ software.