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: The goal of this procedure is to grow our “Bacteriophage” (if we have any). We are trying to see if there are bacteriophage present from our soil sample. From Monday’s experiment, we created an agar for the bacteriophage, to test if phage was present.

Procedures and Protocols:

Materials:

• CiDecon
• 70% Ethanol
• Ethanol Burner
• 18 ml LB Broth
• Petri dishes
• Lysate
• PY 2X TA
• Pipettes
• Micropipettor
• 1M Calcium Chloride
• .5ml of Arthrobacter

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.

Procedure:

• Got the enriched solution that was created Friday 8/24 from the tray my TA had.
  • Important side note- my lab partner made my enrichment solution Friday, and she forgot to label the container. She did not label hers as well, but from her notebook, her solution had 13mL of enriched solution. While examining both solutions, we could tell the two solutions were not labeled but had different volumes. She grabbed the one that had the 13mL solution.
• Used a pipette to obtain 1.5mL of my enrichment solution.
• Once the solution was in the pipette, I attached a filter to the bottom of my pipette, and this filter filtered out the “bacteriophage” from the other bacteria that were too big to fit in.
• I set the solution that was just filtered into a small cap, and I sat it down after labeling it “ML Enrichment 8/27”
• Then we got Petri dishes and labeled it with three circles (NC for our Negative Control, E for our test Enrichment, and D for Direct).
• Then we got two orange vial tubes, one for our TA Control, and the other was for our agar solution.
• We then measured out the LB Broth for both the TA Control and our top agar solution. 13.5mL in the top agar and 4.5mL in the TA control.
• Calcium Chloride was then given to us after our math was complete (formula c1v1=c2v2. Calculations listed below). We used 42.75 for the TA control, and 135 for the top agar solution.
• We then put 5mL of TA (top agar) onto our TA control dish from our vial tubes.
  • Note that the solution once poured onto the plate had many bubbles.

TA Control Before 8/27/18

TA Control After 8/29/18 (Note- Showing before and after since the group thought the bubbles would affect the outcome of our TA Control after 48 hours of making the solution)

• We then got the TA (top agar) for our actual plates and added it to our vial tubes.
• We got 1.5mL Arthobacter and mixed it with our solution that added up to 30mL.
• We each poured 10mL onto each of the three plates (one for each lab partner)
• We finished pouring the agar and we added 10 µL of enriched, direct, and phage buffer the appropriate sections.
• Then we put it in the incubator.
• We put in the incubator around 420.
  • We won’t get a full 48 hours but we will be close
    • Side note- the final three procedures were done by my lab partner.

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

C1V1=C2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Results:

We will find out Wednesday to see if our spot test fails. This procedure helped to set the stage for Wednesday 8/29.

Analysis: 

The procedure was well organized, and it went smoothly. My lab partner did mess up with the LB broth/calculations/pipette, but overall we managed to do everything on time. My lab partners had to stay after the lab class in order to finish, and they were able to help me with my results. Next time, I will be sure not to touch the tip of the cap so that the bacteria on my fingertips do not fall into the cap, thus contaminating my experiment.

Future:

If the Spot test fails or succeeds, we would still have to do a plaque assay. The only difference is if there are spots from the spot tests, then you would have to do two plaque assays, one for the direct and enrichment.