Rationale)

To wash Soil D again in order to produce an enriched lysate not created with contaminated arthro, I will also collect soil metadata.

Procedures)

1. Setup an aseptic zone
2. Added 2mL of Soil D to a 15mL vial labeled “NMN Soil D+ 9.26.18”
3. Added 10mL of LB broth to the vial, shook for 10 minutes. Took the mass.
4. Centrifuged for 15 minutes
5. Retrieved a weigh boat and labeled “NMN %H2O”, took the empty mass, then added 4g of Soil D and placed under the vent hood.
6. Got a falcon tube and added 4mL of Soil, filled to 12.5mL with DI water, and added 3 drops of soil dispersion liquid; covered and shook until mixed, let the tube settle for 48 hours.
7. Added 1-2mL of falcon tube liquid to a pH vial, shook to combine, tested pH. pH=5.5
8. Used a .22 micron syringe filter to filter the supernatant into a 50mL conical vial labeled “NMN 9.26.18 Soil D Enriched Lysate”
9. Added .5mL of arthro to the vial
10. Placed the vial on the shaker table for 48 hours.
11. Stored the bag with Soil D in the walk-in refrigerator.

Observations/Data)

The mass of the vial “NMN Soil D+ 9.26.18” was 18.042g. The empty mass of the weigh tray was 2.331g. The mass of Soil D in the tray was 4g. The pH of the soil was 5.5. I observed that the LB broth we used was clear and not contaminated, additionally, the arthro I used was also non-contaminated. The Falcon tube I used had a red asterisk on it.

Conclusions/Next Steps)

I can conclude that the soil from which I collected is acidic in nature. The next steps will be to conduct a plaque assay and spot test with the enriched lysate I produced today and the direct isolation I produced Monday in the effort to isolate a phage from Soil D, which can then be analyzed later.