Rationale:

Due to the limited amount of agar plates, a plaque assay for the enriched will be conducted rather than both enriched and direct.

Scientific Question:

Does the presence of Arthrobacterphage appear more dominant in one oak tree species than the others? If so, in this species is there a correlation between the presence of Arthrobacterphage and the presence of oak wilt fungus growth?

Procedure:

• To prevent contamination, wipe the table with CiDecon and ethanol and also set up an aseptic zone.
• Remove the spot test plate from the incubator to check for plaques.

Plaque

• Use a pipette to add the enriched lysate to 0.5 mL of Arthrobacterphage, label it Culture 1, and allow it to sit for 15 minutes.
• Add 8mL of LB Broth to a 50 mL conical vial.
• Add 90 uL of CaCl2 to the 50 mL conical vial using 10uL – 100 uL pipette.
• When the 15 minutes for “Culture 1” is almost over, add 10 mL 2X Top Agar to the solution of LB Broth and CaCl2 in the 50 mL vial.
• Shake the vial several times to get an even mixture of the solution.
• Using a pipette, add 4.5 mL of the top agar into a new vial.
• 1 mL of the top agar was added to the top agar control plate (shared by four tables.)
• Pour “Culture 1” onto the agar plate and label it “PA 1 (enriched).”

Plaque Assay 1

Results and Analysis:

• The materials need to create the top agar was multiplied by four.
• It was a little difficult to get an exact amount of LB Broth and Top Agar due to the difficulty of seeing the marks.

Conclusion and Future Plans:

• Due to the presence of plaque in my spot test which indicated the presence of phage, the plaque assay provided a way to further prove that there are phages within soil sample 1.
• In the future (9/5/18), I will complete the plaque assay for the direct isolation lysate if there are enough agar plates.