Purification of Bacteriophages from Bacterial Lysate

Samenvatting

Source: Pett, N., et al. T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo. J. Vis. Exp. (2024)

This video demonstrates the purification of bacteriophages from bacterial cell lysates using sequential centrifugation, membrane filtration, chloroform extraction, centrifugal filtration, and buffer exchange to obtain a purified phage preparation.

Protocol

1. Preparation of experimental phage lysates
   1. Culture E. coli in 5 mL of LB medium in sterile polystyrene or glass culture tubes (with caps). Incubate tubes overnight at 37 °C with shaking at 200 rpm, until the stationary phase is reached.
   2. Subculture the overnight E. coli culture 1:50 in 100 mL LB medium in a glass conical flask. Incubate at 37 °C with shaking at 200 rpm until the bacteria have reached early to mid-exponential phase (approx. 1.5 h).
      NOTE: An initial growth curve can be performed to determine the optical density at 600 nm (OD₆₀₀) range at which the bacterial culture is in exponential phase. Culturing in a glass conical tube is suggested as recent evidence has found that phages are able to adhere to plastics such as polypropylene.
   3. Add 100 µL of the high titer T4 phage stock into the E. coli subculture and incubate at 37 °C with shaking for 3 h, or until the new lysate is no longer cloudy. Collect the T4 phage lysate into 50 mL conical tubes and either proceed directly to clean-up steps or, if required, store at 4 °C until the next day.
      NOTE: A larger phage burst size (indicating a higher average number of virions released per replication cycle) necessitates a longer incubation period for the bacterial subculture. This provides increased bacterial density prior to spiking with phage stocks.
   4. Centrifuge lysates at 4000 x g for 20 min at room temperature to pellet any remaining bacteria and cellular debris. Filter-sterilize the resulting supernatant using a 0.22 µm nylon filter and transfer the filtrate to new 50 mL conical centrifuge tubes.
   5. Add 0.1 volumes of chloroform to each volume of filtered T4 phage lysate to kill any remaining bacteria and prevent bacterial growth. Vortex briefly to mix and incubate at room temperature for 10 min.
      NOTE: Lipid enveloped phages are sensitive to chloroform, which may reduce phage titer. Skip this step if required.
      CAUTION: Chloroform is a toxic organic solvent that is dangerous when inhaled, ingested, or absorbed through the skin. Use a fume hood and appropriate personal protective equipment (PPE) when working with chloroform. Use glass instead of polystyrene serological pipettes when working with chloroform, as it is not compatible with most plastics. Chloroform can be placed in polypropylene conical centrifuge tubes, but should not be stored long-term in plastic tubes.
   6. Centrifuge the lysate at 4000 x g for 5 min at room temperature to separate the chloroform from the lysate. Use a serological pipette to carefully transfer the top lysate layer into a new 50 mL conical tube without disturbing the underlying chloroform layer. Discard the chloroform waste into the appropriate hazardous liquid waste container. Store lysates at 4 °C until concentration and buffer exchange the following day.
   7. Concentrate phage lysates in a 100 kDa centrifugal filter device by adding 13 mL of phage lysate to the upper reservoir of the device and centrifuging at 4000 x g for 5 min, or until most of the lysate has passed through the filter into the lower reservoir.
      NOTE: Aim to have approximately 2 mL of lysate at the end of the spin time. As the phage concentration increases in the filter with the addition of lysate, spin times will likely increase. Centrifugal filter devices have a physical dead stop to prevent them from spinning dry. Do not let the filter membrane dry out if continued use is intended (i.e., by removing all liquid from the upper reservoir). Spin times may vary based on phage type and titer.
   8. Using a P200 or P1000 pipette, gently pipette the remaining ~2 mL of lysate up and down within the upper reservoir to unclog the filter membrane after each spin. Discard the filtrate from the lower reservoir into a waste container, leaving the concentrated phage in the upper reservoir.
   9. Repeat steps 1.7-1.8 until the entire volume of phage lysate has been passed through the filter device, with ~2 mL of concentrated phage retained in the upper reservoir after each spin.
   10. Unclog the filter membrane after the last spin by pipetting the remaining lysate (~2 mL) in the upper reservoir up and down. Wash the phage (buffer exchange) by adding 12 mL of Saline magnesium (SM) buffer to the upper reservoir and centrifuge at 4000 x g for 10 min, or until most of the buffer has passed through the filter.
   11. Discard the filtrate and repeat the wash step (step 1.10). Resuspend the remaining 2 mL of lysate in the SM buffer to a final volume of 10 mL (or less), for long-term storage. Transfer the lysate to a 50 mL conical centrifuge tube and store at 4 °C.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
50 ml PES Steriflip Sterile Disposable Vacuum Filter Units	Millipore Sigma	SCGP00525
Amicon® 100kDa Ultra-15 centrifugal filter device, Ultracel-100	Millipore Sigma	UFC910008
BD Microtainer® Tubes, SST	BD Medical	365967
Chloroform (Ethanol as Preservative/Certified ACS)	Fisher	C298-500
MaxQ 6000 Incubated Shaker	Thermo Scientific	8354-30-0009
Microbiology Media: LB Broth (Powder) - Lennox	Fisher BioReagents	BP1427-500
Microcentrifuge Tubes with Locking Snap Cap, 2ml	Fisher	14-666-315
Phage stocks	Carolina Biological Supply	n/a