9.26.2018 Rewashing Soil Sample D

Rationale: At the beginning of the lab time, it was revealed that many individuals had results that did not accurately reflect what they should have. This was attributed to the fact that the Arthrobacter used for all procedures on Monday was not Arthrobacter, which made it impossible for Arthrobacter-infecting phages to interact with them. Therefore, all procedures done on Monday that involved Arthrobacter had to be redone. Since the soil sample lysate had been enriched with a problematic bacterial sample, it was necessary to rewash and enrich the new lysate from the same soil sample.

Procedure (Metadata in Italics):

1. Established an aseptic zone.
2. Added 2mL of soil to tube (“HMB Soil Sample D 9/26/18”)
3. Added 10mL of LB Broth to tube
4. Shook for 10 minutes
5. Centrifuged sample for 15 minutes
6. Used syringe and 22 micron filter to filter supernatant of centrifuged sample. Placed into tube labeled “HMB Soil Sample D 9.24.18”.
7. Added 0.5mL of Arthrobacter to “HMB Enriched Soil D 9.26.18” tube to create enriched lysate.
8. Moved enriched lysate to shaker to be used on Friday 9/28.
9. Added 4.126g of soil to weigh boat and placed under hood to be analyzed on Friday.
10. 4mL of soil was added to a falcon tube. 8mL of DI water was added to the falcon tube along with 3 drops of soil dispersion liquid.
11. Shaken for 30 seconds for reexamination on Friday.
12. Added 0.5mL of soil supernatant from the falcon tube to the pH tube. Added DI water until the pH tube was full. Used pH paper to determine the pH was 5.5 after matching with chart. 

Observations:

• Mass of tube while shaking was equal to 18.03g.
• At the end of filtering, a very small amount of white foam dripped from the filter into the lysate tube. It is unknown if the amount or composition will have significant effects in any way.
• As observed on Wednesday, the soil sample was very dark after adding LB Broth and sand had already begun to settle out before it was placed in the centrifuge.
• Mass of weigh boat: 2.325g.

Data Summary from Today: pH=5.5

Next Steps/Conclusions:

• The next step for processing this soil sample would be to perform a spot test and plaque assay. This procedure was intended to occur during this lab entry, but the delay due to the issue with Arthrobacter delayed the schedule of the lab. Therefore, the procedures will be completed on Monday.