Rationale:

To wash Soil F and produce a direct and enriched lysate which can be used to test for the presence of phage in Soil F via a spot test, plaque assay, and PCR. Additionally, I will collect soil metadata for Soil F.

Results from Friday:

My plaque assay was negative and my top agar control for it was somewhat contaminated. My spot test was also negative, though it showed evidence of contamination, the top agar control for it was free of contamination.

Procedures:

1. Setup an aseptic zone.
2. Added 3mL of Soil F to a 15mL vial.
3. Added 10mL of broth to the same vial and vortexed for 15 minutes.
4. Centrifuged the vial for 15 minutes.
5. Used a .22µ vacuum filter to produce a lysate from the Soil F supernatant.
6. Retained 100µL of the lysate as a direct isolation and stored in the fridge.
7. Added .5mL of arthro to the main tube of lysate to create an enriched lysate.
8. Placed the enriched vial on a shaker for 48 hours.
9. Added 5.332g of Soil F to weigh tray for percent water
10. A soil composition test was set up using 8mL of Soil F, the tube was then topped off to 25mL mark with DI water and 3 drops of soil dispersion fluid.
11. Added 3mL of the soil composition water to test for the pH in a vial topped off with DI water.

Observations/Data:

The pH of the soil was 6. The Falcon tube I used had a red star on it. The mass of the empty weigh tray was 2.381g. The supernatant was very clear. The LB broth also appeared to be free of contamination.

Conclusions/Next-Steps:

From the soil metadata I collected I can conclude that Soil F was somewhat acidic at a pH of 6. The next step will be to use both the direct lysate and the enriched lysate to use PCR in order to test for the presence of phage in Soil F via the formation of plaques.