9.10.18 Soil Metadata and Washing

Rationale)

To wash Soil Sample B using a modified procedure in order to achieve a lysate that can then be enriched and/or left as a direct isolate. We will also collect metadata on Soil Sample B, including pH, percent water, and percent sand, silt, clay. We will also collect data on the leaves I collected from the tree I gathered soil from.

Procedures)

1. Setup an aseptic zone by wiping down the work surface with CiDecon and 70% ethanol. Light an ethanol flame as well.
2. Retrieve the vial labeled “NMN 9.5.18 Soil B” and using a 10mL serological pipette under aseptic conditions transfer 10mL of LB broth into the vial. We also filled the other Group 3 vials at the same time, using the same pipette all under aseptic conditions.
3. Cap “NMN 9.5.18 Soil B” and shake for 15 minutes, afterward take the mass of the vial, 17.743g.
4. After this, it was determined that water needed to be added to bring it closer to the mass of another vial in order for it to be centrifuged properly, roughly 5 drops of water added using a pipette to bring the mass to 17.855g.
5. Centrifuge “NMN 9.5.18 Soil B” at 10,000g for 5 minutes.
6. While “NMN 9.5.18 Soil B” is centrifuging, retrieve a weigh dish and record the empty mass, 2.45g. Label the dish as NMN %H2O and fill with 3.72g of Soil B from the bag, “NMN, Soil B+Leaf, 9.10.18”, containing the excess Soil B, place the weigh boat under the fume hood to dry for 48hrs.
7. Retrieve a falcon tube and fill with 4mL of Soil B, do to the tube not being able to be labeled it was stored in the middle back section of the test tube rack for the purpose of identification.
8. Add enough DI water to the falcon tube that the meniscus reaches 14mL, add 3 drops of soil dispersion liquid to the falcon tube and shake for 30 seconds, place the tube in the middle back of the test tube rack and let it sit for 48hrs.
9. Retrieve the now centrifuged “NMN 9.5.18 Soil B” and a .22 micron filter, set the filter up under the fume hood and turn on the vacuum. Using a pipette transfer the supernatant from “NMN 9.5.18 Soil B” to the top of the filter apparatus.
10. After all the supernatant has filtered through remove the filter apparatus from the attached 50mL conical vial and quickly cap the vial. Label the 50mL vial “NMN 9.10.18 Soil B Enriched Lysate” noting that only about 7.5mL of lysate was obtained. Dispose of the filter and accompanying discarded materials into the biohazard bag.
11. Add .5mL of Arthrobacteria to “NMN 9.10.18 Soil B Enriched Lysate” under aseptic conditions.
12. Retrieve the leaf gathered from the tree the soil was collected from in the bag labeled “NMN Soil B+Leaf 9.10.18”, compare the leaf to the leaf type sheet and determine the leaf to be an oblanceolate with an obtuse tip.
13. Add about 2mL of the Soil B-water mixture from the falcon tube to a pH vial, filling the rest of the vial with DI water, cover with your finger and shake for 10 seconds.
14. Take about 1 inch of pH paper and place it into the pH tube for about 30 seconds, remove the paper after this time and compare it with the pH paper comparison diagram, recording the pH is 6.
15. Wash out the tube with DI after putting the contents of the vial and the pH strip in the biohazard bag.
16. As we were finished relatively early, Shepard agreed to clean up the workspace as he still had work to do in order to finish the required assignments.

Data/Observations)

The pH of the soil was found to be 6.5, the mass of the empty weigh dish was 2.45g, and the mass of the undried Soil B that was added to the weigh dish was 3.72g. I also observed that my supernatant was much clearer compared to the previous time I washed soil, additionally the filtration process only produced 7.5mL of lysate instead of the desired 10mL, meaning a direct isolation was not kept and all the lysate was used in the enriched lysate. I also observed the live oak leaf I gathered to be an oblanceolate with an obtuse tip.

Conclusions/Next Steps)

The next steps will be to analyze and record the rest of the soil metadata on Wednesday after 48 hours have passed, including percent water and sand, silt, clay percentages. Additionally, we will also collect the enriched lysate that was left on the shaker table for the same 48 hours in order to utilize it in both a plaque analysis and a spot test, in the effort to isolate a phage from Soil Sample B.