Corum:Plaque Counting

Overview

Phage quantification by E. coli counting plaque forming units (PFU).

Materials

• 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

Procedure

1. Inoculate LB with miniculture.
2. Grow in shaker 37 °C 3-4 hr to OD600 = 1.
3. Liquify soft-agar in microwave. Incubate in 50 °C water bath 10 min.
4. 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
5. Gently swirl the petri dish to homogenize materials.
6. Incubate on bench 10 min.
7. Incubate overnight 37 °C.
8. In the morning, count and average the plaques.
9. Convert to PFU/mL by multiplying the average plaque count by 10 and then by the dilution factor.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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Contact

• Sean P Corum

or instead, discuss this protocol.

Digital Signature

• SC 17:46, 28 June 2012 (EDT):