Lab 10 Procedure

Calculate the Plaque Forming Units (PFU) of the Mutant Phage

• Working in a group of 2, obtain your mutant phage plates from the last lab.
• Take a picture of the plates for your lab report.
• Determine which dilution of phages produces 30-300 plaques on the agar plate.
• Count (or estimate if counting is not possible) the number of plaques on the agar plate and record the number in your lab notebook.

• Calculate the plaque forming unit in 1 mL of the original sample using the formula below:
  • Plaque counts X plate dilution X 10*
  • *10 is needed because 0.1 mL of phage was used for plating. Since the calculation is PFU/ mL, we need to multiply the value by 10
  • For example, if the plaque number is 50 on a plate with dilution 10^-4, then the PFU/ mL is 50 X 10⁴  X 10 = 5,000,000 or 5 X 10⁶ PFU/ mL.
• Record the result in PFU/ mL in your lab notebook and on the side of the microfuge tubes containing the original mutant phage.
• This value tells you the concentration of phage particles (in PFU/mL) in the original sample.
• 1 PFU is generally considered as 1 phage particle.

Phage elution

• Insert a 1 mL pipette tip into center of a discrete plaque and remove a plug of soft agar.
• Place the plug in a microfuge tube containing 0.6 mL of sterile saline.
• Use a pipette tip to break up agar.
• Repeat with 1 more plaque.
• Let the two tubes sit at room temperature for 15 minutes to allow phage to diffuse out of the agar.
• Label 2 columns with the mutation name.
• Load the content of the phage/agar/saline solution to the columns.
• Spin down the column for 1 minute at 14,000g.
• Remove the supernatant (that contains phages) to a new, well-labelled tube.
• Store the phages in your freezer boxes as backups.