Research Question:

Does the presence of Arthrobacteriophage appear more dominant in one oak tree species compared to another? If so, in this species, is there a correlation between the presence of Arthrobacteriophage and the presence of Oak Wilt Fungus growth?

Objective:

From our Spot Test Monday 8/27/18, we were all hoping to find a plaque, meaning our Bacteriophage were present from our soil collection. Our group was the only full group (1/8)that possibly had plaque present. So the purpose of the Plaque Assay is to solidify our results form our spot test. It will confirm our results from our spot testing.

Materials:

• Lysate
• PY Broth
• PY 2X TA
• 1M Calcium Chloride
• .5mL of Arthobacter host
• Pipettes
• Agar Plate

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.

We then started our Plaque Assay

• We got our Spot Test Dishes from the incubator. We then checked to see if any plaque had formed. Dr. Adair and one of the TA’s said that our Spot Test could potentially have Bacteriophage.
• Instead of doing two Plaque Assays (since we did have plaque growth form our Spot Test, we were supposed to perform two plaque assays), we did one Plaque Assay since we did not have petri dishes for the entire class. We created a Plaque Assay from our Enriched Lysate, to test if our Spot Test was accurate.
  • Formula “recipe” (figure 1).
    • .5mL Arthobacter + 10 microliter Lysate
    • 2.0 LB Broth
    • 2.5mL 2X TA
    • 22.5 microliter of Calcium Chloride
      • side note this formula x4 since we have three partners plus our group control plate, which the control was split four ways due to the fact we did not have enough petri dishes for the entire class.
• We then got 0.5mL Arthobacter from the hood, and we then added 10 microliters of our Lysate that was stored in a cap, that was in the refrigerator from the Spot Test. We then let this Solution set for about 12 minutes +/- one minute.
• We then started to make our agar solution for all of the plates, including the TA control.
• Added 8mL LB Broth into our 50mL vial.
• Added 10 microliters of Top Agar.
• Added 4.5mL of this solution we made in our 50mL vial onto our individual dishes.
  • Side note: each transferred the solution using different pipettes, but this was done outside of the aseptic zone. Result being possible contaminations.
• We then let the solution sit for about 10 minutes.
• We then put our dishes in the incubator, upside down.
• We cleaned our lab space, and one of the TA’s took our Spot Tests to let them grow.
  • Side note- Friday during open lab, we will know if both the spot test grew more plaque, and to see if our Plaque Assay results help support our spot test results.

Plaque Assay Steps (figure. 1)

Spot Test Results

TA Control for Four groups

Results:

We had a small plaque appear from the Spot Test, and hopefully the same from the Plaque assay that was done in the lab Wednesday 8/29/18. Results will be known Friday 8/31/18.

Analysis:

This procedure was not hard at all after knowing what to expect from having done the Spot Test a few days before. It was a joy to see the small plaque and to know that group 6 all had successful results. The only thing that was hard for us was to ensure everything thing was done in an aseptic zone. This lab was better, but it could have been better regarding the usage of the pipettes in an aseptic zone.

Future:

We will use the data from the Plaque Assay to determine whether or not we actually have Phage present or not. If not, then we would just repeat the Plaque Assay, and see if our plaque from the Spot Test had grown or not. If not, then ultimately we would have to recollect soil and do this whole procedure again.