Rationale: Calculated how much of the titer I needed to web a plate and produce a plaque assay that is webbed.

Procedure:

• Aseptic zone to prevent contamination of plates and bacteria
• Obtained webbed plate from incubation and prepared to count plaques
• Counted plaques present on the plate (231)
• Brought the plate over to the light microscope to measure the diameter(s) the plaques [Avg. Diameter was .8μL]
• Calculated the area of the plate [5.671*10^3] and the area of the plaques [1.12878*10^4]
• Divided plate area by plaque and then divided that number by the titer of the plate to obtain the amount of lysate needed to web a plate [488μL to web]
• Obtained previous lysate from refrigerator and added 488μL to 0.5mL of arthrobacter, let sit for 10 minutes
• Obtained a 50mL conical vial and added in 4mL of LB Broth and 45μL of CaCl2
• Added 5mL of 2XTA to the 50mL conical vial and immediately pipetted 4.5mL into the arthrobacter + lysate vial and plated
• Allowed the plates to sit for 15 minutes and then moved to incubation

Observations:

Previous result of new lysate; counted plaques and calculated webbed plate

A clean control plate!

Calculations for webbing a plate

Next Steps/Conclusion: This plaque assay should provide a webbed plate that can be flooded to obtain another lysate, which will be tested for it’s strength. If the plate is contaminated, will re-run the experiment with the same amount of lysate.