Rationale)

To collect another soil sample and wash it, creating an enriched isolation that can be used to potentially isolate a phage. I will also collect metadata in regards to the soil sample I collected.

Results from Friday)

The results of the plaque assay were negative and the top agar control showed bacterial growth indicating contamination.

Procedures)

1. Setup an aseptic zone
2. Collected Soil D in a bag labeled “NMN 9.24.18 Soil D”
3. Added 2mL of Soil D to a 15mL vial labeled “NMN Soil D+ 9.24.18”
4. Added 10mL of LB broth to the vial, shook for 10 minutes. Took the mass.
5. Centrifuged for 10 minutes
6. Used a .22 micron syringe filter to filter the supernatant into a 50mL conical vial labeled “NMN 9.24.18 Soil D Enriched Lysate”, filtered .5mL into a micro test tube labeled “NMN 09.24.18 FDL”
7. Added .5mL of arthro to the “Enriched” vial
8. Placed the “Enriched” vial onto the shaker table for 48 hours.
9. Refrigerated the “FDL” tube for 48 hours.
10. Stored the bag with Soil D in the walk-in refrigerator.

Observations/Data)

The mass of the tube “NMN Soil D+ 9.24.18” was 18.336g. My plaque assay was negative with no plaques being formed and our top agar control showed bacterial colonies growing throughout. Once I stopped shaking my soil sample, sand began to settle out immediately.

Conclusions/Next Steps)

The next step will be to conduct a plaque assay and a spot test with the lysates produced from Soil D in the effort to isolate a phage from Soil D, in addition to collecting metadata on Soil D.