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Phage quantification by E. coli counting plaque forming units (PFU).
- 50 μL overnight E. coli miniculture (Host B for T7 phage (Carolina 124300), Host C for ΦX174 (Carolina 12440))
- 5 ml warmed LB broth
- Sterilized soft agar (5 g LB broth powder + 1.1 g Bacto-agar in 200 mL water)
- 3 warmed LB plate
- Diluted phage sample
- Inoculate LB with miniculture.
- Grow in shaker 37 °C 3-4 hr to OD600 = 1.
- Liquify soft-agar in microwave. Incubate in 50 °C water bath 10 min.
- Add the following in order to the LB plates in parallel triplicate:
- 100 μL diluted phage sample
- 200 μL OD600 = 1 cell culture
- 3 mL soft agar
- Gently swirl the petri dish to homogenize materials.
- Incubate on bench 10 min.
- Incubate overnight 37 °C.
- In the morning, count and average the plaques.
- Convert to PFU/mL by multiplying the average plaque count by 10 and then by the dilution factor.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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- SC 17:46, 28 June 2012 (EDT):