Rationale: To isolate the possible bacteriophages from the soil through the process of soil washing, and to collect lysate for enriched and direct samples.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Experimental Procedures:

1. In this procedure, aseptic technique was used to avoid contamination as much as possible. CiDecon and 70% Ethanol were used to clean the work space. A 100% Ethanol burner was used to form an aseptic zone.
2. The soil sample that was collected from the oak tree in Zone 4 was mixed with LB broth in a 50 mL tube. The tube contained approximately 13 mL of soil and was filled to the 35 mL mark with LB broth.
3. After adding the LB broth, the mass of the sample and the tube was found to be 53.332 grams. The tube was then shaken for 15 minutes to mix the mixture.
4. The class tubes were then paired with a tube that had a mass within +/- 0.1 g of the mass of their tube.
5. The tubes were then put into the centrifuge and spun at 3000 x g for 5 minutes.
6. After this process was complete, the supernatant was transferred into a 0.22 um filter using a pipette and filtered with a vacuum pump. Approximately 12.5 mL of lysate were obtained after filtering.
7. 0.5 mL of arthrobacter was then added to the lysate to create the enriched isolation, which will be shaken at room temperature until Monday, August 27. The rest of the supernatant was too thick and needed to be transferred to a 15 mL tube and put through the centrifuge again. This will be done on Friday, August 24.
8. The work station was then cleaned and all materials used were disposed of properly.

Observations/Results/Data: 

Observations:

• moist soil
• when shaken, bubbles formed
• soil mostly mixed with LB broth
• Created a milky color when mixed
• After spun in centrifuge:
  • Yellow supernatant on top
  • Pallet on bottom, brown color
  • bubbles are gone
  • some soil and sticks floating at the top
  • layered colors of soil at bottom.
• Filtering was slow due to items in the soil, causing the supernatant to require a second spin in the centrifuge.

Results:

The enriched sample was created. Approximately 12.5 mL were obtained of lysate. With the 0.5 mL of arthrobacter that was added to the sample, the enriched sample is 13.0 mL. The procedure to create both the enriched and direct lysate was not finished, but will be finished on Friday.

The sample will be stored in the laboratory until Monday at room temperature.

It is labeled: LIP 8-22-18 Enriched

Interpretations/Conclusions/Next Steps:

This procedure was not complete due to the sample not filtering completely.  In the future, trying to make sure there are less sticks and other material in the soil will allow this process to go more smoothly. The supernatant will need to be spun in the centrifuge again before collecting the direct lysate sample. The next step is to re-spin the supernatant and filter to create the direct lysate, which will be stored in the refrigerator.