Phage Purification from Borrelia burgdorferi

Take a suspension of Borrelia burgdorferi bacteria infected with a borrelial phage.

Centrifuge the suspension and collect the supernatant containing phage particles and cellular debris.

Add a salt solution and incubate with shaking.

The salt solution enables the separation of residual host-derived macromolecules from the phage particles.

Centrifuge the suspension and collect the supernatant containing the phage particles.

Add polyethylene glycol or PEG, which acts as a molecular crowding agent that facilitates phage particle precipitation.

Incubate on ice and then centrifuge.

Discard the supernatant.

Resuspend the pellet in a buffer, then transfer it to a microcentrifuge tube.

Treat with chloroform to dissolve lipid-based cell debris.

Centrifuge the tube and collect the upper aqueous phase containing the borrelial phages.

Repeat the chloroform addition, mixing, centrifugation, and collection steps to ensure complete removal of contaminants.

Store the isolated phage particles under cold conditions for further use.

Supplement the prepared culture with the appropriate antibiotic concentration described in the manuscript, while incubating the sample at 33 degrees Celsius for 72 to 96 hours. To prepare solutions for PEG precipitation, prepare 500 milliliters of five molar sodium chloride, 500 milliliters of 40% PEG, and 100 milliliters of suspension medium as described in the manuscript. To sterilize, autoclave the solution, cool prior to use, and store at room temperature or four degrees Celsius.

For PEG precipitation of phage from the donor Borrelia burgdorferi clone, after 72 to 96 hours of incubation, centrifuge the samples at 8,000 g for 20 minutes at four degrees Celsius. Decant the supernatant into a clean, 50-milliliter conical tube and dispose of the cell pellet. Add five molar sodium chloride to a final concentration of one molar.

After mixing well, rock gently at room temperature for one hour. Centrifuge the samples at 8,000 g for 10 minutes at four degrees Celsius. After decanting the supernatant into a clean 50-milliliter conical tube as demonstrated previously, add 40% PEG-8000 solution to the supernatant to a final concentration of 10%. Mix well and set on ice for more than one hour, up to overnight.

Centrifuge the samples at 8,000 g for 20 minutes at four degrees Celsius as demonstrated previously. Discard the supernatant and remove as much excess liquid as possible without losing any pellet, which contains the phage particles. Resuspend the pellet in a minimal volume of suspension medium using the suspension medium to wash down the side of the bottle and collect any potential phage particles.

Treat the recovered phage sample with an equal volume of chloroform based on the volume of resuspension. Mix the sample well and centrifuge at 8,000 g for 10 minutes. Then, remove the aqueous layer to a clean tube, avoiding any of the thick interface layers.

After determining the volume recovered following the first chloroform treatment, again treat the sample with an amount of chloroform equal to 10% of that volume. Transfer the aqueous layer to a clean tube while being careful to avoid any of the interface or organic layers. Use the phage immediately or store it at four degrees Celsius.