Results:

The plaque assay run on Wednesday (9/26) was negative as shown in the pictures below.

Rationale:

With the newly collected soil from Monday (10/1), soil metadata will be collected for future correlations. The soil D will be filtered and enriched so the lysate will be ready for a plaque assay on Friday.

Procedure:

1. Once an aseptic zone was established, 2 mL of soil D and 10 mL of LB Broth were transferred into a conical vial which was shaken for 15 minutes the vial.
2. About 3 grams of soil D was poured out into a weigh boat labeled “KEA 10/1 soil D” and left under the flume hood to dry out.
3. A falcon tube labeled “KEA 10/3 soil D” was filled with 10 mL of soil D, 20 mL of de-ionized (DI) water, and three drops of texture dispersion liquid.
4. The “KEA 10/3 soil D” falcon tube was shaken for 30 seconds and then left under the flume hood for the next 48 hours.
5. A small amount of soil D was placed in a pH vial and the rest was filled with DI water.
6. The pH vial was shaken for 10 seconds and then settled for 2 minutes.
7. Used pH paper to determine the pH of the soil.
8. The conical vial was centrifuged at 3,000g for 10 minutes.
9. The supernatant liquid from the conical vial went through a 0.22 μL top vacuum filter into a conical vial labeled “KEA 10/3 soil D enrich.”
10. The “KEA 10/3 soil D enrich” conical vial was placed in the incubator at room temperature for the next 48 hours.

Observations:

• Soil D was collected under a Chinkapin Oak out by McLane Stadium. It was recorded that the tree had a circumference of 41 cm and a canopy circumference of 142 cm. The height was approximately 4.6 m. The following pictures of the tree were taken.

• The weigh boat weighed 3.40 grams. The weigh boat with wet soil weighed 6.30 grams.
• The conical vial weighed 17.77 grams before water was added to make the mass within 0.05 grams of another vial. With the addition of water, the conical vial final mass weighed 17.99 grams.
• The pH of the soil was determined to be 6.0 as shown in the picture below.

• 10 mL of the supernatant filtered out leaving only enough filtered lysate to do an enriched sample.

Next Steps:

On Friday during open lab, a plaque assay will be run to determine whether or not there are bacteriophages that target Arthrobacter in soil sample D. Also, the metadata will be completed.