Rationale: The purpose of this lab is to calculate the amount of lysate needed to create a webbed plate and run a plaque assay.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. The plaques on the 10^-1 dilution plaque assay were counted and 92 were found.
3. The diameter of the plate was measured and found to be 86mm, and the radius was 43mm. The diameter of 10 plaques was found and the average diameter was calculated to be 2.4mm.
4. Next, the titer of the plate was found to be 9.2 x 10^4
5. The area of the plate was calculated to be 1849 pi, and the area of the plaque was found to be 1.44 pi.
6. Next, the area of the plate was divided by the area of a plaque to calculate how many plaques would be needed to web the plate. This calculation determined that 1284.03 plaques would be needed.
7. The amount of lysate needed to web a plate was then calculated by dividing the number of plaques needed by the titer, and changing the units to ul. It was determined that 14.01 ul of lysate would be needed to web the plate.
8. 14.01 ul of the lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
9. Top agar solution was made for three plates. 6 ml of LB broth and 67.5 ul of CaCl2 were added to a tube.
10. 7.5 ml of 2x TA was added to the tube. 4.5 ml of top agar solution was then added to the 0.5 ml of arthrobacter and poured onto a plate labeled LIP 14.01 ul PA 10-31-18. The rest of the top agar was poured onto a plate labeled LIP LJF 10-31-18 Control.
11. The plates were then allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
12. The workstation was cleaned using aseptic technique and materials were properly stored or disposed of.

Observations/Results:

• Observations:
  • Bubbles formed on the plates
• Results:
  • Titer of lysate: 9.2 x 10^4
  • Average area of plaques: 1.44 pi
  • Area of plate:  1849 pi
  • Lysate needed to web a plate: 14.01 ul

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the webbed plate to obtain more lysate.