Rationale: Perform three plaque assays to pass the third round of purification. The entire lab had to redo their experiments since everyone had a negative control.

Procedure:

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
3. We then got an ethanol burner, and our aseptic zone was created.

• Used the 10^0 lysate that was made on Monday 9/24/18 since this passed the second round of purification.
• Added the 10^0 lysate to the 90 microliter PB into the 10^-1 solution, and pipetted/mixed well through the microcentrifuge. Labeled this solution as the 10^-1 solution on the microcentrifuge.
• Got one more microcentrifuge caps, labeled one cap 10^-2.
• Added 90 microliters of PB to the 10^-2 solution.
• Added 10 microliters of the 10^-1 solution to the 10^-2 solution.
• All microcentrifuge caps had the lysate solution, then added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2).
• Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
  • The formula below was used to make the solution for 9 plates (three for Michael and Justin, two for Cooper, and one for the control).
    • 2.1mL LB Booth (x9)
    • 22.5 microliters of Calcium Chloride (x9)
    • 2.5mL 2X TA (x9)
    • 400 microliters of Arthro
• Added the 2XTA last to the 5omL vials, shook the vial, and quickly pipetted the solution onto each vial containing the Arthro/lysate solutions.
• Sat each plate for about 15mins to the solution solidify.
• The remaining solution that was left in the 50mL vial was used for our control.
  • Added TA and poured that solution onto the last plate.
    • Side note: the control solidified <15 minutes.

Observations:

All groups had negative controls, which cause every group to redo their experiments from Monday. The experiment was not hard, but the hardest part was probably ensuring everything was done safely with no contaminations.

Group #4 Control 10^0

 10^-1

Conclusions/Next Steps:

Determine if the plaques on the plates are plaques (if plaques are present). Determine if the experiment was a high titter/low titter. If low, do calculations to determine how much lysate will need to completely web the plate. Next step is to make a webbed plate.