Rationale: After the spot test turned up negative, so I will run a plaque assay to double check my spot test result.

Procedure:

1. Pipetted 10μL of lysate into 0.5mL of arthrobacter. Then began making top

   agar.

2. Using aseptic technique and fresh pipettes each time I put 8mL of LB broth

   in a 50mL tube, then 90μL of CaCl2, then 10mL of 2X TA.

3. Accidentally spilled some of a lab mates TA with their arthrobacter and

   lysate onto plate so started over.

4. This time only needed enough for 2 plates so mixed 4mL of LB broth, 45μL

   of CaCl2, and 5mL of 2x TA and poured 4.5mL into a control plate and poured the other 4.5mL into my arthrobacter and lysate mixture. I pipetted up and down to mix them then poured onto a plate.

5. Inverted and incubated for 48 hours.

Observations:
My control turned out contaminated, and the plaque assay had no plaques. This contamination could be from lack of aseptic technique, so we cleaned our pipette tips and made sure to mark our own LB broth bottle. Group 4 all had plaques in their plaque assay and Justin’s the most well-defined plaque.
Claire had a lot of plaques in her plaque assay, and her tree was from Morrison. Image on the left is plaque assay and Image on the right is control.

Interpretations and Next Steps:
Lathan checked a purified lysate by doing a plaque assay (10μL of lysate) of a 10-3 lysate. He counted 14 plaques. With an average plaque diameter of 1mm he would need 4μL of 100 lysate to web a plate with a diameter of 75mm. Arthrobacter has been known to have an affinity for a compound in pesticides so the findings thus far would support the positive correlation between pesticides and the presence of arthrobacter phage, as where there are bacteria there are phages. The next steps would be to collect more samples from different trees in Cameron park. This time I will be gathering samples from living Oak saplings. I will also be gram staining my control contaminant and the arthrobacter lawn from my plaque assay to see if the contaminant was arthrobacter or some other bacteria that may have come from the surroundings.