November 2

10/29 ~ Attempting to web a plate / plate calculations

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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.


Posted November 2, 2018 by justin_yu1 in category Justin Yu

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