Rationale: Test to see if the bacteriophage is present by performing multiple plaque tests.

Question: Why did 10^0 serial dilution not have any plaque, yet both 10^-1 and 10^-2 have plaques?

• From group four, only Justin had successful plaques in all three serial dilutions. Cooper had only one successful serial dilution, and Michael had two successful serial dilutions.
• Rule out the possibility of contaminations since Group 4 used the same formula for the plating solution.
• Control was contaminated, but all groups had a contaminated solution,
• One reason why plaques are not showing up in the 10^o serial dilution is that the bacteria could be in a lytic cycle, whereas Justin’s bacteria is clearly in a lysogenic phase. The bacteria in both Michael’s and Cooper’s experiment could both be in the lytic cycle, meaning the bacteria are “temperate” and not as expressive as a lysogenic cycle.

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 from the 10^-1, which was performed on 9/17/18.
  • 10^0 did not have any plaque.
  • 10^-1 had three plaques.
  • 10^-2 had one plaque.
    • Side note: Since 10^0 did not have plaque, this experiment performed on 9/19/18 was done to test whether or not phage is present by only performing two 10^0 plaque assays. No serial dilution was done since the purpose of this experiment was to simply see whether or not phage presence from the 10^o.
• Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
• 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 7 plates (two for each of the 10^0 solutions and one for the control).
    • 20mL LB Booth (x7)
    • 22.5 microliters of Calcium Chloride (x7)
    • 25mL 2X TA (x7)
• 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 the control.
  • Added TA and poured that solution onto the last plate.

10^0 Plaque Assays

Control

Observations:

• Group four had successful plaque assays from the experiment performed on 9/17/18.
• Justin had plaques on all three serial dilutions, whereas Cooper and Michael did not. Michael had plaques on his 10^-1 and 10^-2, and Cooper had only one plaque on his 10^-2 dilution.

 10^0 Plaque Assay

10^-1 Plaque Assay

10^-2 Plaque Assay

• control was contaminated.
  • Possible reason: LB broth contamination.

Next Steps/Conclusions:

On Monday, check all two plates to see if any plaques appear. If plaques do appear, perform the experiment again by picking the plaque of one of the plates. If no plaque, simply redo the experiment with the plaques assay that does have plaque (experiment performed on 9/12/18). Overall, the experiment was very easy since the experiment was done on Monday. The hard part was to determine where the plaques were, and whether if or not plaques were actually there, which they were.