9.10.2018- Soil Sample B: Metadata and Enrichment

Rationale: Collecting metadata will allow for additional comprehension of soil composition and data connections to be made at a later date. The washing and enrichment process will prepare Soil Sample B for future Spot Test and Plaque Assay Procedures by creating a lysate that contains Arthrobacter.

Procedure:

1. Lab bench was cleaned with CiDecon and 70% ethanol to establish a clean work surface.
2. Lit ethanol burner to establish aseptic area.
3. Obtained soil sample bag containing conical tube, excess soil, and leaf samples.
4. (ASEPTIC) Added 10mL of LB Broth to the 11.5mL mark on the 15mL conical tube labelled “HMB Soil Sample B”
5. Shook for 15 minutes using a combination of shaking and vortexing. While shaking, mass of tube was found to be 17.849g. At the end of 15 minutes, centrifuge was used to spin the tube at 10,000g for 5 minutes.
6. 3.145g of soil was placed in weigh boat labeled “HMB water” and placed under a fume hood
7. 4mL of soil sample from bag was added to the 50mL tube. Then, DI (deionized) water was added to the 12mL mark. 3 drops of soil dispersion liquid was added. Mixture was shaken for 30 seconds to mix, then left to sit for 48 hours.
8. Top filter package and supplies was obtained and taken to the fume hood. Then, it was set up.
9. Under hood, supernatant of tube that was in centrifuge and labeled “HMB Soil Sample B” was added to filter. Filtrate was dripped into 50mL Conical tube labeled “HMB Enriched Lysate 9/10/18 S.S.B.”
   1. Resulted in 8mL of filtrate.
10. (Aseptic) 0.5mL Arthrobacter was added to 50mL conical tube. Let sit in shaker for 48 hours.
11. Obtained pH tube. Added a small amount of soil supernatant. Filled pH tube with DI water on top of soil supernatant. Added pH strip to sample for approximately 40 seconds. When compared with pH scale, pH was concluded to be 6.
12. My individual lab components were put away and disposed of properly. Since Shepard had additional procedure to go, he offered to clean the bench when he was done to allow us to leave early. The only item left in my section was a test tube rack with the three soil samples settling out from step 7 along with a guide to leaf observations.

Data:

• pH of soil was 6.

Observations

• After shaking with LB Buffer, this soil sample was darker than the first sample completed.
• Again, after shaking the volume of the mixture had decreased from 12mL to roughly 9mL.
• Leaf observations: Leaf obtained had an obtuse tip, entire sides, and acute shape at base. Appeared to be an oblanceolate leaf. Finally, leafs on branches appeared to be organized in the Palmately compound fashion.
• pH paper changed the color of soil and DI water solution to a blueish green color.

Next Steps

• During the next laboratory time, metadata results that were unable to be obtained today will be recorded. Also, the enriched lysate created today will be used to perform a Spot Test. The Spot Test will reveal any presence of phages that could have been in Soil Sample B.