Rationale:

The purpose of today’s lab was to check on the metadata experiments that had been performed the week prior (water percentage and soil composition) and also perform a plaque assay with the newly enriched lysate to test for the presence of phage.

Materials:

• 2.0-mL LB Broth
• 2.5-mL 2X TA
• 22.5-μL Calcium Chloride
• 0.5-mL Arthrobacter
• 0.22 micron syringe filter
• Enriched Lysate

Procedure:

1. Established an aseptic zone.
2. Checked soil composition and weighed dry soil to calculate sand, silt, clay makeup of soil and water percentage of soil.
3. After metadata was completed, aliquoted 2.0-mL Lb broth to conical vial.
4. Filtered enriched lysate through 0.22 micron filter.
5. Added 22.5-μL of calcium chloride to LB Broth.
6. Combined 10-μL of filtered lysate with 0.5-mL of Arthobacter and left to infect for approximately 20 minutes.
7. After 20 minutes had passed, added 2.5-mL 2X TA to broth, quickly added lysate mixture, and plated immediately.
8. Let solidify and left to incubate for 48 hours.

Observations/Results/Data:

• The soil composition did not vary as drastically as last samples. It did contain 4 different layers of the soil, but the fourth dark top layer was just additional organic matter that was not poured out.  It roughly had 1.5-mL of sand, 1.0-mL of silt, and 0.5-mL of clay. Making it 50% sand, 33.33% silt, and 16.67% clay.

• 

  Soil Composition 09/24/18 Bottom Layer: Sand Dark Middle Layer: Silt Light Layer: Clay

• LB broth before use was not contaminated, no cloudiness was reported and there was no precipitate in the solution at the time of use.
• The mass of the dry soil was 6.21 grams after being left out all weekend to encourage water evaporation. Therefore making the mass of water 3.91 grams.

Analysis/Conclusions:

• Based off graphic below and the percentages of the soil composition obtained from the experiment, the best estimate for the soil type is Loam. Loam is a fertile soil with a roughly 40-20-20 mineral composition of sand-silt-clay, which is very close to the results gathered in the experiment.
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• By using the water percentage equation, the final calculated percentage of water in the soil was 24.80%. This is only 4% more water than the previous soil sample which was gardeners soil. Possibly this increase in water will be beneficial for the presence of phage.
  

  Work for calculating water percentage

   

Next Steps:

• Analyze the plaque assay results for the presence of phage. If there are plaques present, a serial dilution will be performed to get a high titer of phage. If no plaques are present, a second plaque assay will be performed.