Enriching and isolating phages in liquid culture

Set up cultures of your target host strains. We use this protocol for cheese bacterial isolates, which grow best in LB at 25 °C with shaking at 200 rpm, and reach high density after two days.

After cells have grown to high density, prepare your infection media. We used LB supplemented with 1 mM MgSO₄ and 1 mM CaCl₂ (final concentrations). Add your phage extracts to this media, either individually for many parallel enrichments, or pooled together.

Aliquot your infection media into 3 mL glass culture tubes, or deep-well 96-well plates. We generally use 0.25–1 mL of media for these enrichments.

Subculture your hosts into the infection media at a 1:100 dilution. For instance, you would add 10 µL of host culture to 1 mL of infection media.

Incubate your enrichments at 25 °C with shaking at 200 rpm overnight.

Harvest infected subcultures: Move infected cultures into 1.5 mL tube + 50 µL CHCl₃. Spin down for 5 min at max speed, then transfer the supernatant to fresh 1.5 mL tube + 50 µL CHCl₃.

Use uninfected parent cultures to pour phage plates using the double agar overlay method. Add 200 µL of culture to 3 mL of molten (but not overly hot) top agar. Pour top agar onto a room-temperature LB plate, swirling to evenly distribute the agar across the plate before it cools.

Once the plate is cooled, spot the phage-enriched supernatant onto the phage plate. If you only have one enrichment per host, you can use a larger volume (like 10 µL) in one large drop that you can gently swirl with the pipette tip to distribute. If you have many enrichments per host, you should pipette smaller, 2–3 µL spots onto the host in a grid pattern.

Incubate plates at room temperature. Plaques will show up in 24–72 hours.

Pick plaques using a pipette tip, and transfer into 300 µL of SM buffer with 50 µL of CHCL₃.

Purify these phages by diluting the picked plaque down to a concentrate that will give single plaques, then infecting 200 µL of host culture with 10 µL of diluted phage, and finally plating out using the double agar overlay method. Repeat 2×.