Rationale: Today we are washing the soil samples we collected so we may test the lysate produced in future labs for the presence of bacteriophages.

Before starting this procedure, we retrieved a soil sample from a tree in our assigned quadrant. The tree was located at about 31.544310, -97.119052 latitude and longitude. The tree had a circumference at 137cm above ground of 50.7cm, a canopy diameter of 386 cm, and a height of 500 cm. The tree had a few damaged and browning leaves on it, and was missing leaves on large chunks of branches.

This is where I collected my sample from which was located right next to a thick root right at the base of the trunk:

Procedure:

1. Wiped down the table with CiDecon, then poured 70% ethanol on table and let dry. Lit a small burner to create an aseptic zone to minimize contamination.
2. Sample started at about 12mL, filled tube with LB broth until 35mL within the aseptic zone.
3. Shook tube for 14 minutes to mix. Note: I dropped the tube on the floor while shaking.
4. Spun at 3000g for 5 minutes. Noticed that after spinning my solution was still very

   opaque, almost milky.

5. Filtered using a 22μm filter and a vacuum until I filled the new tube to 5mL. Took a

   really long time because the extra stuff in my supernatant was clogging up the filter.

6. Split up the remaining supernatant between two smaller tubes (both of which weighed

   11.7 grams). Spun those two tubes at >4000rpm for 5 minutes.

7. Filtered each new supernatant using a different filter of the same size. Ended with 13mL

   of filtered supernatant. Stored 3mL in a small tube labeled direct and stored in fridge. Stored remaining 10mL in larger tube and added 0.5mL of arthrobacter using aseptic technique, labeled enriched. Stored enriched sample in shaking incubator.

Observations:
Throughout a lot of this procedure there were possible times that my sample may have been contaminated. For example, while using the fume hood an external particulate may have fallen into my tube before I closed it. I had been coughing a lot that day in the lab and there is a high likelihood that some of the expelled fluids from my cough may have ended up in my sample before filtering. However, most likely they had been filtered out.

Interpretations and Next Steps:

The tree we observed definitely seemed to be sick. However, I can’t confirm whether or not it truly was oak wilt. Using the enriched lysate and direct lysate we will conduct a spot test and a plaque test to try to find phages within my sample that affect arthrobacter. Since my tree seemed sick it would be interesting to compare the results of my plaque assay and spot test against groups who seemed to have healthy trees. Maybe there will be a correlation in the presence of phages or not.