Purification of Bacteriophages from Bacterial Lysate

Begin with a bacterial lysate containing bacteriophages, host bacterial debris, and residual unlysed bacteria.

Centrifuge to pellet residual bacteria and larger debris.

Collect the supernatant and filter it through a membrane to remove remaining larger contaminants.

Transfer the filtrate to a fresh tube and add chloroform.

Vortex to mix and incubate. Chloroform kills residual bacteria and prevents bacterial growth.

Centrifuge to separate the chloroform from the aqueous layer containing bacteriophages.

Transfer the aqueous layer into a centrifugal filter device and centrifuge.

The filter allows liquid and smaller contaminants to flow out, while retaining bacteriophages within the device.

Continue centrifugation until the liquid level reduces, concentrating the bacteriophages in a smaller volume.

Discard the filtrate. Then, pipette repeatedly to resuspend the concentrated bacteriophages.

Add buffer and centrifuge again to remove excess buffer.

Add fresh buffer and collect the purified bacteriophages in a fresh tube for further studies.

Once the T4 phage lysate is clear, collect them in a 50-milliliter conical tube. Centrifuge the lysate at 4,000 g for 20 minutes to pellet any remaining bacteria