Rationale: Pick Plaque from the plaque assay and to perform serial dilutions. Performed several plaque assays to isolate phage, and made a sample control to go along with our experiment.

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 9/12/18.
• 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.
  • This formula was used to make our solution for 10 plates (three for each of the three solutions and one for the control).
    • 20mL LB Booth (x10)
    • 22.5 microliters of Calcium Chloride (x10)
    • 25mL 2X TA (x10)
• 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:

• Group 4 had plaques on all plaque assays, but only Justin had a plaque from his spot tests.
• On the plate of the plaque assay, it was very hard to identify a solid plaque but contained multiple little spots that could well be possible plaques.

 Plaque Assay with Plaque

• Spot Tests had zero plaque but had air bubbles. Justin had spots form group four.

Additional Questions:

Question 1. Group four all had plaques from their plaque assays, but only Justin had a plaque from his spot test.  This could have resulted from the soil since all three group members got soil from three different trees. Justin’s soil could have different phage from his group members, resulting in Justin’s better-defined plaques. The soil samples from group 4 at least have some similarities, given everyone in the group had a successful plaque assay.

Question 2. 

Lathan needs 4.01 microliters to web his plate (work is shown above).

Next Steps/Conclusions:

On Wednesday, check all three plates to see if any plaques appear. If plaques do appear, perform the experiment again by picking the plaque of all three plates. If no plaque, simply redo the experiment with the plaques assay that does have plaque. Overall, the experiment was very easy since Lathan’s lecture video basically covered everything that was needed to be known for this week lab. The hard part was to determine where the plaques were, and whether if or not plaques were actually there, which they were.