Clostridium difficile strain CD105HE1 ribotype 076 was used to propagate the phage as per the method of Nale et al. [28 (link)] Bacteriophage CDKM9 is a myovirus previously isolated from an environmental water sample as described by Rashid et al. [48 ]
The identity and purity of the phages used for encapsulation was confirmed using transmission electron microscopy (TEM) analysis. 1 ml of phage stock was centrifuged at 10,000 rpm for 1 hour at 4°C and the pellet was washed two times with 0.1 M ammonium acetate (Fisher, UK) and re-suspended in 100 μl of ammonium acetate. 4 μl of sample suspension was placed on glow discharged pioloform/ carbon coated copper grids which were allowed to stand for 5 min for bacteriophage to bind, 1% w/v uranyl acetate was added to stain the sample. 10μl of deionised water was used to rinse the sample and the prepared sample was left to dry for 24 h. The grids were examined using a JEOL 1220 electron microscope with an accelerating voltage of 80 kV. Digital images were captured using SIS Megaview III digital camera with associated analysis software.
The identity and purity of the phages used for encapsulation was confirmed using transmission electron microscopy (TEM) analysis. 1 ml of phage stock was centrifuged at 10,000 rpm for 1 hour at 4°C and the pellet was washed two times with 0.1 M ammonium acetate (Fisher, UK) and re-suspended in 100 μl of ammonium acetate. 4 μl of sample suspension was placed on glow discharged pioloform/ carbon coated copper grids which were allowed to stand for 5 min for bacteriophage to bind, 1% w/v uranyl acetate was added to stain the sample. 10μl of deionised water was used to rinse the sample and the prepared sample was left to dry for 24 h. The grids were examined using a JEOL 1220 electron microscope with an accelerating voltage of 80 kV. Digital images were captured using SIS Megaview III digital camera with associated analysis software.
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