Amplification and Recovery of Infectious Bacteriophages

Take a culture of bacteria infected with bacteriophages.

Excise a plaque, a clear zone formed by phage-induced bacterial lysis.

Introduce the plaque into an actively growing bacterial culture and incubate.

Phages deliver their genome, which then replicates and directs the host machinery to synthesize structural proteins.

These then assemble into mature particles and accumulate within the host cells.

Add a DNA-degrading enzyme to remove extracellular bacterial DNA released from lysed cells. Transfer the suspension to a glass bottle. Centrifuge to pellet the bacteria and discard the supernatant.

Introduce a magnesium-rich buffer containing the DNA-degrading enzyme, and resuspend the bacteria.

Transfer the suspension, add chloroform, and agitate to lyse the cells and release the phage particles.

The magnesium stabilizes the phage particles, and the enzyme degrades residual host DNA.

Centrifuge to remove cellular debris, and transfer the supernatant containing infectious phages into a fresh tube. Store at a low temperature for further studies.

Begin this procedure with preparation of phage plaques as described in the text protocol. Use the end of a sterile Pasteur pipette to core and remove a single plaque.

Blow the plaque into the diluted cells. Then incubate at 37 degrees Celsius for two hours.

Next, add DNase before centrifuging the cells at 8,000 times g at four degrees Celsius.

Discard the supernatant, then resuspend the pellet in 10 milliliters of 1X Tris magnesium chloride. Add 500 microliters of chloroform and vortex at high speed to lyse the cells. Clarify by centrifugation at 12,000 times g for 10 minutes at four degrees Celsius.

Then decant the supernatant into a 15 milliliter conical tube and store at four degrees Celsius.