Abstract:

Calculate the titer of the plate to be able to web a plate; count the plaques on the plate, as well as measure the plaques and plate, and then calculate the titer

Procedure:

• Created an aseptic zone to prevent contamination of the experiment from bacteria
• Obtained my previous plaque assay plate (10^0 conducted on 10/1)
• Split the plate into four quadrants to be able to count the plaques
• Counted the plaques in the plate (85 total)
• Brought the plate over to the light microscope to measure the radii of the plaques and plate
  • Measured 10 plaques and then took the average of their diameters; divided the average diameter by two for the average radius (Average radius = .445mm)
  • Measured the diameter of the plate and then divided by two for the radius (Radius of plate = 42.5mm)
• Calculated the titer of the plate by dividing the total plaques by the total lysate (85 plaques/10μL lysate)
  • Multiplied by 1000 to get from μL to mL (Titer = 8.5 X 10^3)
• Calculated the area of the plate ((π[42.6]^2)=5.671X10^3) and area of the plaques ((π[.445]^2)=6.217X10^3)
• Divided plate area by plaque area (5.671 X 10^3/6.217 X 10^3=9.121 X 10^3)
• Divided 9.121 X 10^3 by 8.5 X 10^3 and multiplied by 1000 (μL to mL) and resulted with 1,073 mL to web a plate
• Cut the recipe down since there was not enough 10^0 lysate; added 90μL to 500μL arthrobacter and let sir for 15 minutes
• Obtained a 50mL conical vial and added in 1.9mL LB Broth and 22.5μL CaCl2
• Added 2.5mL 2XTA into the vial and immediate mixed with arthrobacter+lysate and then plated
• Also created a TA control plate (2.5mL 2xTA + 2mL TA + 22.5μL CaCl2)
• Let plates sit for 15 minutes and then incubated

Observations:

The 10^0 with the 85 counted plaques

The 10^-1 with only 3 plaques

The team’s control plate

Calculations for the titer and plate

The 10^0 plate after counting and calculations

Next Steps/Conclusion:

After this titer is created, will be calculating the titer of this titer to see if it is a strong titer. If not, will titer the titer again to create a stronger titer. If the titer is strong enough, will get a webbed plate and will flood the plate