Rationale: We are spot testing in order to see if we found a bacteriophage in our soil sample B from tree A that can infect arthrobacter.

Procedure:

1. I started out by cleaning off the table with CiDecon and then letting 70% ethanol dry onit. Once complete I lit a burner to create an aseptic zone.
2. I then filled a syringe with 2mL of my enriched lysate from the previous lab whileholding both the syringe and my tube within the aseptic zone.
3. I used a 22μm filter to filter out 1.5mL of lysate into a small tubule.
4. In order to start making top agar I first placed 18mL of LB broth in a 50mL tube. Thenpipetted out 4.5mL into a different 50mL tube so that I can make some TA to use as a

   control.

5. Added 135μL of CaCl2 to the tube with 13.5mL of LB broth and 42.75μL to the tubewith 4.5mL of LB Broth.
6. Added 1.5mL of arthrobacter to the non-control tube then added 15mL of 2X Top Agarto the tube and pipetted up and down. Afterwards I pipetted 10mL into my plate.
7. Poured 5mL of 2X Top Agar into the control tube and shook then poured into plate.Note: I wasn’t supposed to shake the tube, but it shouldn’t affect the control much other

   than leaving bubbles in the plate.

8. After waiting 10 minutes for top agar to dry I pipetted 10μL of my enriched lysate ontothe plate on the designated enriched zone which I marked with sharpie on then back. Note: The drop I pipetted onto the plate rolled from the spot I dropped it down to the middle.
9. Then I pipetted 10μL of my direct solution from the previous lab, which was stored in the fridge, onto the marked area.
10. Lastly, I pipetted 10μL of phage buffer onto the negative control marked area.
11. Let sit for 12 minutes, then stored in the incubator for 48 hours.

Observations:
I found a small plaque in the center where my enriched lysate had rolled down to. Everyone in my group had found a plaque and we all took our sample from the same place. 9 of the 24 students found a plaque.

My Plate:

Control Plate:

Interpretations and Next Steps:
This could possibly mean that my sample may have contained a bacteriophage able to infect arthrobacter. Since our tree seemed to be a sick red oak and only 9 of the 24 students got a plaque and all three members of my group, who all took a sample from the same tree, got a plaque there may be a correlation between the presence of a bacteriophage and trees getting sick. Further tests would need to be done to see if I truly did find a bacteriophage. Also, I would need to test more trees to see if the correlation is truly a correlation and not a fluke of some sort.