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 and cellular debris.

Filter-sterilize the resulting supernatant using a 0.22-micron nylon filter and collect the filtrate to a new 50-milliliter tube. Add 0.1 volume of chloroform to the filtered T4 phage lysate to kill the remaining bacteria and prevent bacterial growth. Vortex briefly and incubate the lysate at room temperature for 10 minutes.

Centrifuge the lysate at 4,000 g for 5 minutes to separate the chloroform from the lysate. Using a serological pipette, carefully transfer the top lysate layer into a new 50-milliliter tube. Add 13 milliliters of phage lysate to the upper reservoir of 100 kilodalton centrifugal filter device.

Concentrate the lysate at 4,000 g for 5 minutes or until most of the lysate has passed through the filter. Using a P200 or P1000 pipette, gently pipette the remaining 2 milliliters of lysate up and down within the upper reservoir to unclog the filter membrane. Discard the filtrate from the lower reservoir into a waste container.

Repeat concentration and retain approximately 2 milliliters of concentrated phage in the upper reservoir. After the last spin, gently pipette the remaining lysate up and down in the upper reservoir. To wash the phage lysate, add 12 milliliters of saline magnesium or SM buffer to the upper reservoir and centrifuge at 4,000 g for 10 minutes.

After the second wash, resuspend the remaining 2 milliliters of lysate in the SM buffer to a final volume of 10 milliliters. Transfer the lysate to a 50-milliliter tube and store it at 4 degrees Celsius.