Leo 912 omega tem

Virus particle suspensions were prepared according to the ammonium acetate method as described by Ackermann (2009 ). Three microliters taken from the 140 ml concentrate were pipetted onto carbon coated 200 mesh copper grids and stained with 2% aqueous uranyl acetate for 30 s. The samples were viewed using a LEO 912 Omega TEM (Zeiss, Oberkochen, Germany) at 120 kV. Images were collected using a ProScan CCD camera.

Prior to extraction of phage DNA, all samples were preliminary screened for the presence of phage particles by fluorescent microscopy. 10 µl aliquots of supernatant were transferred to sterile 1.5 ml Eppendorf tubes and 0.3 µl of undiluted SYBR GOLD was added and incubated at 80 °C for 10 min. 5 µl of the total volume was placed on a glass slide and examined under 1000x magnification using a Zeiss Axioplan II fluorescence microscope fitted with filter set 25. To further visualize phage particles, 1 ml of the initial enrichment culture was centrifuged at 5000 RPM for 10 min to pellet bacteria. The supernatant was centrifuged at 25000 × g for 2 hours in a fixed angle rotor to pellet phage particles. The pellet was re-suspended in 50 µl 100 mM ammonium acetate (pH 7.2) and left to re-suspend with shaking at room temperature for 16 hours^{41 (link)}. The wash was repeated a second time with fresh buffer. The final pellet was re-suspended in 5 µl 100 mM ammonium acetate and visualized by TEM. Three microliters of each sample were pipetted onto carbon-coated 200-mesh copper grids and stained with 2% aqueous uranyl acetate. The samples were viewed using a LEO 912 Omega TEM at 120 kV (Zeiss, Oberkochen, Germany) housed at the University of Cape Town Physics Department. Images were collected using a ProScan CCD camera.

The mean size of the ternary complex, polydispersity, zeta potential, and stability, were measured by dynamic light scattering (DLS), using a DelsaNano C particle analyzer (Beckman Coulter). The complexes were diluted 1:3 with distilled DNAse-RNAse free water for measurement. The stability of the ternary complexes was determined by size measurement at the time-point of 30 minutes, 1 hour, 2 hours and 4 hours. Morphology and size of the complexes were further characterized by transmission electron microscopy (TEM) (Zeiss LEO 912 Omega TEM) at an accelerating voltage of 120 kV. The prepared samples was settled on a CF-400-Cu square mesh copper grid (Electron Miscroscopy Sciences, USA) and stained with 2% uranil acetate solution. ImageJ software was used to create a statistic of the size of the complexes.