Rationale: Perform a second third round of purification since the first third round failed, since the LB Broth was contaminated. The goal of the purification

Question: Why did the control fail for the last two experiments?

• The last two experiments, the control were both negative.
• One reason why this could have been negative since the same LB broth was used for both experiments. The eight groups in the class labeled there own LB broth so that the groups in the class could see which groups had the contaminated control. This experiment was done on the side, and the class quickly saw which groups had a contaminated LB broth.
• This led to the redo of the third round of purification.

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.

• Picked Plaque from Plaque Assay, which was performed on 9/21/18.
• Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
• Got two more microcentrifuge caps, labeled one cap 10^-1 and the other solution 10^-2.
• Added 90 microliters of PB to both caps.
• Added 10 microliters of the 10^0 solution into the 10^-1 solution.
  • The 10^0 solution has the picked plaque. Transferred this to the 10^0 solution so that this could be added to the other two caps so that three Plaque Assays could be performed.
• Added 10 microliters of the 10^-1 solution to the 10^-2 solution.
• All microcentrifuge caps had plaques, 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 each of the three solutions and one for the control).
    • 20mL LB Booth (x9)
    • 22.5 microliters of Calcium Chloride (x9)
    • 25mL 2X TA (x9)
• Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
• 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:

The experiment performed 9/21/18, the control of the experiment was contaminated. This led to the uncertainty of the plaque assays, whether or not it had plaques or not. Because of the results, the experiment was done again since the LB broth was contaminated from the last experiment. Not everyone’s control was contaminated, but only a few groups.

Next steps/Conclusions:

On Wednesday, determine wheater the plaques are plaques and make a webbed plate. Determine if the plate has a high titer or a low titer simply by doing some calculations. If the results are negative, pick a plaque from the second round of purification, or simply pick new soil.