Rationale: Isolate possible bacteriophage through the process of soil washing and enrichment. The soil metadata will also be found and recorded.

Soil Washing 

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
3. We then got an ethanol burner, and our aseptic zone was created.

Procedure:

• 10mL LB broth was added to my 15mL vial, which contained 2mL of Soil B, giving a total volume of 12mL solution in my 15mL vial.
• Started to shake my 15mL for 11 minutes.
• Measured the mass of the 15mL vial which read 14.44g.
• Found a partner that had .05+/- difference in mass for the centrifuge.
• Spun the 15mL solution at 10,000g f0r 10 minutes.
• Top filtered the solution after it was spun in the centrifuge, which came to about 7mL of my enriched lysate.
• Then placed my 7mL solution into the Cabinet.

Observations: 

• The soil wash was not hard at all compared to my first soil wash.
• The only hard part was the filtering process since I had a lot of debris on the top of my enrichment.
• 7mL of enriched lysate was made.

Soil Metadata (% Soil, Clay, and Silt. pH of Soil. % Water.)

While the Soil Wash tubes were being shaken, the soil metadata was collected.

Procedure %Water:

• The procedure to calculate the percent water started with pouring soil B from the plastic bag to the petri dish.
• Mass of the weight boat = 2.39g.
• Mass of weight boat with soil = 14.48g.
• The weight boat was then labeled ML 9/10/18 14.48g.
• The weight boat was then placed under the vented hood, where it will sit for 48 hours to allow the water to evaporate.

Procedure for pH of the soil:

• Poured a small amount of soil into a pH vial.
• Filled rest of the vial with DI water.
• Rested my hand over the top, and I shook the vial for about 45 seconds.
• Put the short strip (<1 inch) pH paper into the vial for 45 seconds.
• Quickly compared the pH paper to the pH scale (<1 min).
• pH recorded

Procedure for Metadata:

• Filled 4mL of soil B into a 50mL vial.
• Added 8mL of DI water into the 50mL vial. Total 12mL solution.
• Added three drops of soil dispersion liquid into the 50mL vial solution.
• Shook the vial for 45 seconds, with my hand covering the top (no lids were available).
• Labeled the vial ML Soil B 9/10/18
• The vial was then placed under the vented hood, where it will sit for 48 hours to let the soil (clay, silt, and soil) completely separate.

Observations/Results:

• This metadata procedure was not hard. The only hard part was not to get any debris into any of the tests, which my soil B had a lot of debris. This could have an effect on all of my tests.
• pH of soil: 6.5
• Mass of weight boat: 2.39g
• Mass of weight boat with soil: 14.48g

Next steps/Conclusions:

Prepare for a Spot Test/Plaque Assay and the continuation of Soil metadata results. Find the mass of water from the weight boat by doing a simple calculation. Find the composition of soil by observing the results in the 50mL tube. The next steps will be done 9/12/18. The spot test should not be hard, and hopefully, the simple calculations are not done incorrectly.