Corum:Plaque Counting

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Contents

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

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Digital Signature

  • SC 17:46, 28 June 2012 (EDT):
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