10 September 2018 ✷ Metadata & Enrichment

Metadata (% silt, % sand, % clay, % water, and pH) was collected in order to further examine the links between tree type and phage presence. While it isn’t imperative to the research process, the metadata may provide some interesting connections between phage presence in different groups/locations. Additionally, the soil was cleaned and enriched to help isolate any phage that may have been present in the original sample.

Procedure

• First, the workspace was cleaned with CiDecon and 70% ethanol and then an alcohol burner was lit to promote an aseptic work environment.
• 2 mL of soil was added to a 15 mL conical vial and then 10 mL of LB Broth was added to the same vial (reaching 12 mL line on tube). The vial was then shaken for 5 minutes, vortexed for 5 minutes, and shaken by hand again for a final 5 minutes. The vial was massed at the end of the 15 minutes and found to be 19.329 g.
• The vial was taken to be centrifuged at 10,000g for 10 minutes. In the time the Dr. Adair had my sample, I worked on the metadata.

% Water

1. I found the mass of a dry, empty weigh boat to be 2.31 g. I added some of my soil sample to the boat and found the mass of the soil + weigh boat to be 8.62 g. The soil/weigh boat was placed uncovered under the fume hood in the lab to dry until Wednesday, when it will hopefully be dried out and massed again.

% sand, % silt, % clay

1. 4 mL of dirt was added to a falcon vial and the vial was then filled to the 12 mL line with deionized (DI) water. 3 drops of soil dispersion liquid was added, the vial was covered, and the mixture was shaken by hand for 30 seconds. The supernatant was removed and stored in a separate 50 mL conical vial. Both were stored at room temperature under the fume hood.

pH

1. A small amount of soil was added to a pH tube and then the vial was filled with DI water, closed, and shaken for 10 seconds. A pH indicator strip was inserted for 30 seconds and read immediately after. The strip was a light green consistent with a pH of 6.5 (slightly acidic).

•  Once the sample was separated via centrifuge, 3 mL was removed with a syringe and filtered through a 22 micron filter attached to the end of the syringe. This was 3 times; each time the filter was removed to collect more lysate, the ends of it were cleaned with 70% ethanol. The intent was to have 10 mL lysate in a 50 mL conical vial and 1 mL in a 15 mL conical vial, but it ended up being 8 mL in the larger vial and 0.75 mL in the smaller. The smaller vial became the direct lysate and it was stored in the refrigerator.
•  0.5 mL of arthrobacter was added to the larger vial and stored in a shaking incubator at 28 degrees Celsius until Wednesday.

Observations, Results, Data

Pictured above is a leaf collected from the tree we collected soil from and an analysis of the traits of the leaf. This is more metadata that can be utilized in future steps of answering our scientific question.

interpretations/conclusion/next steps

The % sand, silt, clay separation didn’t work as well as hoped and the test will be redone next time to get better, more usable results.

Enriching the soil sample will hopefully lead to the discovery of a phage because the increased amounts of nutrients from LB broth will allow the arthrobacter to flourish, and because the phage feeds on bacteria, increased amounts of phage should be present.

The next lab period will consist of a spot test and conclusion of the metadata collection in order to isolate a phage and analyze the conditions it was found in (i.e. wetness from water percentage).