12 September 2018 ✷ Metadata Conclusion & Spot Test

Metadata (% silt, % sand, % clay, % water, and pH) was collected in order to further examine the links between tree type and phage presence. Today it will be finalized by recording final results. A spot test will be performed to indicate the presence of phage from the soil collection.

Procedure

• The enriched lysate was spun at 3000g for 5 minutes.
• The dried soil from Monday’s lab was massed and the percent water was calculated (see SEA Bears Day 5).
• I worked with one of my lab partners to mix the agar solutions for our plates and a control plate. We cleaned the workspace with CiDecon and 70% ethanol to promote an aseptic environment and then lit an alcohol burner. The control plate was made separately, but our two plates were made together, thus the volumes of the components were doubled. LB Broth was added first, then Calcium Chloride, followed by arthro to the spot test plates (not added to the control plate) and finally top agar. The volumes and concentrations of the components can be seen below.

• component	volume (control)	concentration	volume (spot test)	concentration
  2X Top Agar	2.5 mL	1X	5 mL	1X
  LB Broth	2 mL	–	4 mL	–
  1M Calcium Chloride	23 µL	4.5 M	45 µL	4.5 M
  Arthro	0 mL	–	0.5 mL	–

• The test plate was poured immedietally to prevent it from hardening in the conical vial; it was allowed to set for 10 minutes.
• The spot test plates were poured next from a serological pipette, which was also used to mix the mixture. Each plate was allowed to set for 10 minutes.
• The enriched sample was filtered through a 22 micron syringe filter and stored in a microcentrifuge tube. 5 µL of the filtered enriched sample was added to the designated space on the plate. 5 µL of phage buffer was added to the plate as a control, and 5 µL of the direct sample was added to the plate.
• The excess samples were stored in the refrigerator and the plates were placed in an incubator to sit until Monday.
• Because the % silt/sand/clay falcon tube was difficult to read, it was reshaken and repoured to attempt to get more accurate results for this metadata.

Observations/results/data

The “recipe” for a spot test plate requires 0.5 mL arthro for EACH plate, and we forgot to double this quantity when we mixed our plates. Thus, there may not be enough arthrobacter on the plate for the phage to infect and kill to make a noticeable plaque on the plate.

Results from the metadata collection are recorded on Day 5’s post.

Interpretations/Conclusion/Next Steps

The presence of arthrobacter in the plate is important for the plaques to form, and the limited amount in my plate may pose an issue. If results are inconclusive, I will run a plaque assay with my enriched sample in order to confirm the presence of a phage.