Rationale: Now that we have collected soil metadata I will run a spot test to see if there is arthrobacter phage present in the soil taken from tree B. This way we can start putting together data comparing the presence of arthrobacter in trees that have been sprayed with pesticides versus those which have not.

Procedure:

1. Spun down tube of enriched sample at 3000g for 5 minutes
2. Took a plate and found that it was contaminated so I grabbed another plate. I

   labeled one side enriched and the other side PB control.

3. Used a syringe and a 22μL filter to filter out about 3mL of my enriched

   lysate.

4. I then made my top agar by mixing 2.0mL of LB broth, 22.5μL of CaCl2, 0.5mL of arthrobacter, and 2.5mL of 2x TA in that order.
5. I pipetted up and down for 30 seconds before pouring into my plate. I swirled around until the top agar covered the whole plate and let sit for 10 minutes.
6. I then pipetted 10μL of my enriched lysate onto the point labeled for enriched lysate and 10μL of phage buffer on the control side. I then let it sit for 15 minutes.
7. I placed the plate without inverting it in the incubator to develop over the weekend.

Observations:

Plaque assay turned out negative.

Next Steps:
Next class I will conduct a plaque assay and using the results of these two assays I will compare against the other tree’s sample from Cameron park and the tree samples from the Baylor campus. My table can start compiling a spreadsheet of our results, so we can start analyzing whether or not the presence of pesticides is affecting the presence of arthrobacter phage.