Rationale: After a negative plaque assay and a contaminated negative control, the purpose of this lab is to collect new soil, wash and enrich it, and collect metadata for the soil.

Description of Procedures

1. Soil was collected from a red oak tree, as well as the tree’s dimensions.
2. The work space was cleaned using aseptic technique. An ethanol burner was lit to create an aseptic zone.
3. 2 ml of soil and 8 ml of LB broth were added to a tube. The tube was shaken for 10 minutes, and then massed. The mass was found to be 17.74 g.
4. The soil was spun in the centrifuge for 10 minutes at 5000 x g.
5. While spinning, the % water test was begun. The weigh boat was found to be 2.32 g, and the weigh boat with soil was found to be 5.85 g. This will sit in the vented hood until the next lab to allow the water to evaporate.
6. Next the % concentration test was started. 10 ml of soil was added to a tube along with 20 ml of DI water. 3 drops of soil dispersion liquid was also added. The tube was then shaken for 30 seconds. It will sit under the vented hood until the next lab to allow the soil to separate into its layers.
7. The pH was found next. A small amount of soil was added to the pH vial, and then it was filled with DI water. The tube was then shaken for 10 seconds and then allowed to sit for 10 minutes.
8. While sitting, the tube containing soil was filtered with a 0.22 um filter and syringe. Approximately 7.75 ml of enriched lysate was obtained. No direct lysate was made. 0.5 ml of arthrobacter was added to this tube. It was labeled LIP 8-19-18 Enriched- Soil C. The tube will be shaken at room temperature until the next lab.
9. Next the pH was found to be 6.0, using pH paper.
10. The work station was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results/Data:

• Observations:
  • The soil collected was moist and dark in color.
  • The soil was collected from a red oak tree, along with a leaf.
  • The filtration process was easier than with Soil B.
• Data:
  • Weigh boat- 2.32 g
  • Weigh boat and soil- 5.85 g
  • pH- 6.0
  • 7.75 ml of enriched lysate collected
  • Tree circumference- 60.5 cm
  • Tree canopy- 303 cm
  • Tree height- 800.5 cm

Red Oak Tree- Soil Source

Negative Plaque Assay

Contaminated Control Plate

Interpretations/Next Steps/Conclusions:

The soil collection, washing, and enrichment processes were complete. Of the metadata, the soil pH was found, and the procedures for % water and % soil concentration were started, and will be completed in the next lab. The next step will be to run a spot test or a plaque assay to look for plaques. Soil B yielded no plaques, which is why Soil C was collected. Also, the control plate for the plaque assay for Soil B was contaminated.