15 October 2018 ✷ Enrichment and Metadata, soil 5

Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was filtered and enriched with LB broth in order to aid the phage isolation process.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• An empty weighboat was massed and then filled with “wet” soil and massed. The weighboat and soil were left to dry overnight under the fume hood.
• 4 mL of soil was added to a falcon tube and mixed with 8 mL of DI water and 3 drops of soil dispersion fluid and shaken for 30 seconds. The mixture was left to settle overnight.
• 10 mL of LB broth was added to 2 mL of soil and vortexed for 10 minutes. The mixture was then centrifuged at 3,000g for 10 minutes. The liquid that rose to the top was filtered through a 22 micron syringe filter into 2 vials: one for a direct lysate (~1 mL, refrigerated) and one for an enriched lysate (~5 mL, added 0.5 mL arthro and stored in shaking incubator).
• A small amount of soil was added to a pH test tube and mixed with DI water and shaken for 10 seconds. The pH was taken after allowing the mixture to settle for 45 seconds.

Observations, results, data

pH: 6.0

Mass of weigh boat: 2.03 g

Mass of wet soil + weigh boat: 6.83 g

Mass of wet soil: 4.80 g

Mass of dry soil + weigh boat: 5.98 g

Mass of dry soil: 3.95 g

Mass of water: 0.85 g

mass of water/mass of wet soil*100: 14.2%

% sand, silt, clay:

0.5 mL clay = 12.5%

0.5 mL silt = 12.5%

3.0 mL sand  = 75%

Interpretations, conclusion, next steps

A plaque assay will be conducted in the next lab in order to test for the presence of plaques.

By the fifth attempt at isolating a phage, the human error involved has been greatly reduced. Additionally, each group member tested a different sample, so the test for phage needs to be done with as little human error (+ contamination) as possible in order to guarantee a reliable result.