The following protocol is for 12-well plate

• Preparation
  • Plate 12-well plate with cells (85-95% confluent on the day of infection)

• Experiment
  1. Prewarm media, in 37^oC water bath.
  2. Prepare 11 different concentrations of virus by diluting with media.
  3. Carefully remove media from 12-well plate.
  4. Add 250uL of each dilution to the 11 wells. Add only media to the 12th well as a control. Swirl the plate a little to make sure the entire surface of the well is covered evenly with liquid.
  5. Incubate at room temperature for 30 minutes, swirl every ~4mins.
  6. During incubation, prepare agarose.
     • Make 5% low-melt agarose in PBS.
     • Dilute it with warm media to 1%agarose.
     • Add warm MgCl₂ to 40 mM final concentration.
       • (The previous steps should be carried out in the shortest amount of time possible and with warm solutions to prevent agarose from solidifying)
     • Pipette up and down to mix thoroughly.
     • Keep it in 42^oC water bath.
  7. When 30 mins incubation period is over, remove virus.
  8. Slowly add 1mL agarose/media/MgCl₂ solutions to each well to form a uniform layer.
  9. Leave at room temperature for agarose to solidify.
  10. Add 1mL media on top of the agarose to each well.
  11. Keep it in 35^oC incubator.

• Results
  • Observe after 1 to 2 days.
  • Observe plaques (empty spots where cells are detached) by looking at the bottom of the well.
  • Count the no. of plaques at concentrations where individual plaques could be observed.
  • With the concentration known, PFU/mL of the original stock can be calculated.