Purification of Bacteriophages Using a Sucrose Density Gradient

Begin with a suspension containing bacteriophages and host bacterial debris.

To purify the bacteriophages, sequentially add high-density and low-density sucrose solutions into an ultracentrifuge tube.

Using a gradient-forming instrument, mix the sucrose solutions by tilt-tube rotation to create a continuous density gradient.

Remove a small volume of sucrose gradient from the top, then carefully add the bacteriophage-debris mixture without disturbing the gradient.

Ultracentrifuge to facilitate differential migration of bacteriophages and bacterial debris based on their densities.

The bacteriophages localize as a thick, milky band halfway down the gradient.

Using a blunt cannula-assembled syringe, aspirate the phage band and transfer it to an ultracentrifuge tube.

Add buffer and mix gently.

Ultracentrifuge again to pellet the bacteriophages. Discard the supernatant.

Add buffer and incubate under cold conditions to allow resuspension of bacteriophages.

The purified bacteriophages are ready for further studies.

Prepare four 5 to 45 % sucrose gradients by pipetting 2.5 milliliters of 45 % sucrose into four ultracentrifuge tubes. Top the layer with approximately 2.6 milliliters of 5 % sucrose, stop