Rationale)

To collect another soil sample, and to collect metadata from the sample in addition to washing the soil sample in order to produce a direct and enriched lysate that can be used in plaque assays and spot tests in an effort to identify a plaque.

Results from 9.14.18)

My plaque assay tested negative for the presence of plaques when checked, our top agar control was contaminated as indicated by small bacterial colonies appearing throughout the top agar.

Questions)

1. The reason had the most well-defined plaque was likely due to his lysate likely being a higher titer, or concentration of phage compared to the other lysates in his group, which likely had a lower titer, or concentration of phage in their lysate, which explains why they would have a less defined plaque than a plaque created with Justin’s higher phage concentration  lysate.
2. 4.018 microliters of 10^0 lysate are needed to web the plate.

Procedures)

1. Sat up an aseptic zone by wiping down the work area and lighting an ethanol flame.
2. Collected a soil sample and a leaf from the tree it was near, I also recorded metadata for the soil, I stored the soil and leaf in a bag labeled “NMN 9.17.18  Soil C+Leaf Earle Hall”.
3. Added 2mL of the soil from the bag to a 15 mL vial and labeled it “NMN 9.17.18 Soil C”
4. Added enough LB broth to the vial to fill it to the 12.5mL mark.
5. The vial was massed and shaken for 15 minutes. Mass was 20.128.
6. Added enough DI water to the vial to reach 21.028g, in order for it to be centrifuged properly.
7. Labeled a weigh tray and label “NMN Soil C %H2O”, added 4g of Soil C and placed under the vent hood for 48 hours. The weigh dish weighed 2.36g empty.
8. Added 4mL of Soil C to a falcon tube and 10mL of water, added 3 drops of soil dispersion fluid and shook to mix. Let settle for 48 hours. The tube has “ss” on it.
9. Centrifuged “NMN 9.17.18 Soil C”
10. Filtered the supernatant of the centrifuged vial reserving 10mL for an enriched lysate in a 50mL conical vial labeled, “NMN 9.17.18 Soil C Lysate Enriched”, and .5mL for a direct isolation in a micro test tube labeled, “NMN 9.17.18 Direct Isolation”.
11. Added .5mL of arthro to the vial containing the lysate designated for enrichment, placed the vial on the shaking table for 48 hours.
12. Stored lysates and soil samples in the fridge and wiped down the table.

Data/Observations)

I observed that the supernatant was far clearer than previous supernatants I had filtered, however, the amount of time and the g’s that the vial was subjected to are unknown due to lack of information on the centrifuge. I also observed that the tree from which we gathered the soil was very healthy and relatively recently planted, additionally the soil was primarily brought in topsoil with only a little of what appeared to be native soil.

Conclusion/Next Steps)

Based on our negative results from our plaque assay conducted on Friday we proceeded to collect and wash another soil sample in order to produce a direct and enriched lysate. Our next step will be to conduct a plaque assay and spot test using the lysates produced in this procedure in an effort to find and isolate a phage via the formation of plaques in either or both tests.