TITLE: Enriching and isolating phages on agar plates
AUTHORS: Adair Borges

[DESCRIPTION]
This protocol explains how we isolate phage from microbial communities on solid agar plates. For a protocol on liquid enrichment and isolation, check out our companion protocol.

[STEPS]
SECTION: Solid-plate phage isolation
1. 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.
SECTION: Solid-plate phage isolation
2. Prepare your phage extracts to infect your host strains. There are a couple different ways to go about setting up your experiment: 

- If you have one extract per strain that you want to test, we recommend using between 10–100 µL of phage and pre-infecting the bacterial culture before plating it to spread plaques across the full plate. (See pre-infection step-case below)

- If you have many extracts per strain and you do not care about which plaques come from which source, you can pool your phage extracts and use 10–100 µL of pooled phage per host. You can then use this pooled extract to pre-infect the bacterial culture before plating it to spread plaques across the full plate. (See pre-infection step-case below)

- If you have many extracts per strain and you want to be able to track which plaques come from which source, we recommend preparing a 96-well plate with each concentrated extract in one well, and using 3 µL of phage per host and spotting in a grid pattern across the plate. (See spotting step-case below)
SECTION: Solid-plate phage isolation
3. Add 10–100 µL of phage (pooled or individual extracts) to 200 µL of host cells. 
SECTION: Solid-plate phage isolation
4. Add the infected sample 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. 

 

SECTION: Solid-plate phage isolation
5. Incubate plates at room temperature. Plaques will show up in 24–72 hours. 
SECTION: Solid-plate phage isolation
6. Pick plaques using a pipette tip, and transfer into 300 µL of SM buffer with 50 µL of CHCl3. 
SECTION: Solid-plate phage isolation
7. Purify these phages by diluting the picked plaque down to a concentration 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×.
SECTION: Solid-plate phage isolation
3. Use uninfected host cultures to pour phage plates, using the double agar overlay method. Add 200 µL of culture to 3 mLs 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.

 

SECTION: Solid-plate phage isolation
4. Once the plate has cooled, spot 2–3 µL of phage concentrate onto the phage plate. If you have many samples, use a multichannel pipette to make regular 2–3 µL spots onto the host in a grid pattern.
SECTION: Solid-plate phage isolation
5. Incubate plates at room temperature. Plaques will show up in 24–72 hours. 
SECTION: Solid-plate phage isolation
6. Pick plaques using a pipette tip, and transfer into 300 µL of SM buffer with 50 µL of CHCl3. 
SECTION: Solid-plate phage isolation
7. Purify these phages by diluting the picked plaque down to a concentration 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×.