TITLE: Phage amplification and concentration
AUTHORS: Adair  Borges, Januka  Athukoralage

[DESCRIPTION]
This protocol details methods to amplify bacteriophages T4 and SPO1, and concentrate using either PEG precipitation or filtration. 

[STEPS]
SECTION: Phage amplification
2. 
Grow a 3 mL overnight culture of phage host at 37 °C with shaking. Escherichia coli strain B (ATCC Strain 11303) should be propagated in Lysogeny Broth (ATCC Medium 1065), and Bacillus subtilis strain 168M (ATCC Strain 27370) is propagated in Enriched Nutrient broth (ATCC Medium 265).


 


 

 

SECTION: Phage amplification
3. Add ~107 PFU of phage T4 (ATCC Strain 11303-b4) to the E. coli subculture, and ~107 PFU of phage SPO1 (ATCC Strain 27370-b1) to the B. subtilis subculture. Return the infected subcultures to the incubator, at 37 °C with shaking. Grow until the culture is cleared (around 5 h). If the culture doesn't completely clear within 5 h, you can let the infection proceed overnight.
SECTION: Phage isolation and concentration
4. Move the phage lysate to a 50 mL conical tube, and add ~1 mL of chloroform. Seal the tube tightly, and shake on a platform shaker for 10–30 min. Transfer to a new conical tube. Then, spin the lysate down in a centrifuge at maximum speed for 30 minutes. 
SECTION: Phage isolation and concentration
5. Carefully pipette off the supernatant from the spun-down culture into a new 50 mL conical tube, avoiding any debris at the chloroform interface. Also avoid the chloroform. Move to a new 50 mL conical tube, and spin again at max speed for another 30 min or until supernatant is clear. Move to a new 50 mL conical tube. 
SECTION: Phage isolation and concentration
6. To concentrate the phage lysate, we have used both PEG precipitation and a filter-concentrator based protocol. The PEG protocol requires an overnight incubation, but the filter-concentrator generally requires more hands-on time. Use whichever phage concentration protocol works best for your circumstances.
SECTION: Phage amplification
1. For each host, prepare a 30 mL subculture at 1:100 dilution (300 µL in 30 mL). Grow for 2 h at 37 °C in a shaking incubator. Subculture media (LB or ENB) should be supplemented with 1 mM CaCl2 and 1 mM MgCl2.
SECTION: Phage isolation and concentration
7. To PEG-precipitate phage, first prepare 5× PEG precipitation solution containing 2.5 M NaCl and 20% w/v PEG8000.
 

SECTION: Phage isolation and concentration
8. Add PEG precipitation solution to the phage lysate to obtain a 1× concentration of 0.5 M NaCl and 4% w/v PEG8000. Mix by inverting the tube several times and refrigerate overnight at 4 °C. 
SECTION: Phage isolation and concentration
7. Before concentrating, we recommend filtering the supernatant with a 0.22-µM Steriflip filter (EMD Millipore SCGP00525). This can help cut down on the carry-over of host nucleic acids into the phage prep. 
SECTION: Phage isolation and concentration
8. To concentrate the phage lysate, use a variation of the "Phage on Tap" method. After equilibrating a 100 kDa MWCO Amicon centrifugal filter unit (EMD Millipore UFC910024) with SM buffer (Bioworld 41920012-1), use the filter unit to concentrate the ~30 mL of phage lysate down to 0.5–3 mL. As the phage is being concentrated, use SM buffer to wash the sides of the filter. Store phage at 4 °C. 
 

SECTION: Phage isolation and concentration
9. After the overnight incubation with PEG precipitation solution at 4 °C, pellet the PEG precipitated phage particles by centrifuging at 19,000 × g for 60 min at 4 °C. A small white pellet should form at the bottom of the 50 mL conical tube.
SECTION: Phage isolation and concentration
10. Remove the supernatant, being careful not to disturb the PEG-precipitated phage pellet.
SECTION: Phage isolation and concentration
11. Resuspend the PEG-precipitated phage pellet in 300 µL SM buffer. Store at 4 °C.