Rationale:

Calculate the titer of the most recent plaque assay, as well as calculate how much lysate is needed to web a plate. The end goal is to create a high titer

Procedure:

• Created an aseptic zone to prevent contamination from bacteria
• Obtained my 10/8 plaque assay and control plate
• Drew quadrants onto the plate to count the amount of plaques (570 plaques total)
• Brought the plate over to the light microscope to measure the average radii of the plaques and radius of the plate
  • Measured 10 plaques of various sizes, and then took the average of their diameters; divided the average diameter by two to get the average radii of the plaques (Avg. radii = 0.5675 mm)
  • Measured the diameter of the plate and divided by two to get the radius (Radius of plate = 42.5 mm)
• Calculated the titer of the plate by dividing number of plaques by the total lysate (570/10μL)
  • Then multiplied that number of 1000 to convert from μL to mL (Titer = 5.7 X 10^4)
• Calculated the area of the plate ((π[42.6]^2)=5.671X10^3) and area of the plaques ((π[.5675]^2)=3.2205 X 10^-1
• Divided the plate area by plaque area (5.671 X 10^3 / 1.709 X 10^4=1.7609 X 10^4
• Divided 1.7609 X 10^4 by 5.7 X 10^4 and multiplied by 1000 (converting from μL to mL) and resulted with 308 mL to web a plate
• Consulted with Lathan, who advised to flood the current plate
• Obtained a 50mL conical vial of PB, and pipetted 6 mL PB into the plaque assay plate
• Parafilmed the plate and then left to refrigerate

Observations:

The positive 10/8 control plate

The 10/8 plaque assay w/ numerous plaques

The drawn quadrant on the plate for counting the plaques (Total 570)

Parafilmed plate after 6mL PB was added in

Calculations to find the titer and how much lysate to web the plate

Conclusion/ Next Steps:

Will be going to open lab on 10/12 to check on the flooded plate, and will be running the PB through a 22μL filter to isolate the bacteriophage. Then will be performing another plaque assay to confirm a high titer