Results:

The plaque assay from Monday (9/17) was contaminated. Just like from the Spot Test ran on 9/12, there was a strange yellow ochre liquid in both the control and enriched plate. This amount was smaller than in the Spot Test. The pictures below show the plates.

Rationale:

Since there were no more positive plates, the experiment will start all over and try with new soil samples. By selecting white oak trees in a different area of campus, perhaps the sample will have a bacteriophage. Through filtration and enrichment of this sample, the sample will be ready to run a plaque assay to determine whether or not it contains a bacteriophage that specifically targets Arthrobacter.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Through aseptic technique (over an EtOH (100%) flame),10 mL of LB Broth were added with a serological pipette to the conical vial with 2 mL of soil C in it.
3. This conical vial was shaken for 15 minutes.
4. This conical vial was weighed and approximately 0.5 mL of water was added so the conical vial would be within 0.05 grams of another conical vial tube.
5. The conical vial was centrifuged at 5,000 g for 5 minutes.
6. With the use of a 0.22μL top vacuum filter, the supernatant of the centrifuged conical vial was filtered into a 50 mL conical vial which was labeled “KEA 9/19/18 Soil C enriched filtered lysate.”
7. Poured into “KEA 9/19/18 Soil C enriched filtered lysate” conical vial was 0.5 mL of Arthrobacter.
8. The“KEA 9/19/18 Soil C enriched filtered lysate” conical vial was placed in the incubator at room temperature for the next 48 hours.
9. The counter area was cleaned with CiDecan and EtOH (70%).

Observations, Metadata, and Interpretations:

• As far as the strange liquid on the plaque assay plates, it has been determined, from a gram-strain, that it is most likely from contamination in the LB Broth used.
• The tree selected for soil C was located outside Teal by East Village. There was mulch surrounding the tree. From the leaves, the tree was identified as a post-oak. The tree’s leaves contained a few brown patches. No broken branches or any other unusual signs. The pictures below show the leaves.

• The tree trunk had a circumference of 48.1 cm. The tree had a canopy of approximately 177.4 cm. The tree’s height was approximately 6.858 meters. The picture below was used to approximate the trees height.

  

• Before the conical vial was centrifuged, it weighed 18.42 g.

Next Steps:

In open lab on Friday, soil metadata will be collected from the soil C sample. On Monday, the remaining soil metadata will be collected and a plaque assay will be run with soil C.