Rationale: Isolate bacteriophage through both a Spot Test and Plaque Assay., and to collect more metadata soil sample B.

Procedures:

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
3. We then got an ethanol burner, and our aseptic zone was created.

Spot Test and Plaque Assay

Spot Test

• The enriched lysate was made on 9/10/18.
• Measured the mass of the enriched lysate. Mass of enriched lysate: 20.47 g.
•  The enriched lysate was spun in the centrifuge for 5 minutes at 3000 x g.
• The enriched lysate was filtered and put into a small cap.
• Divided one plate into four quadrants
  • I – ML Spot Test
  • II – CJJ Spot Test
  • III – JLL Spot Test
  • Buffer
• Added 2mL LB broth into a 50mL vial.
• Added 22.5 microliters of M Calcium Chloride in the 50mL vial.
• Added 0.5mL OF Artho PA Phage lysate
• Added 2.5 mL TA into the 50mL vial.
• Poured our solution onto the one plate that is divided into four quadrants.
• Sat the petri dish aside to let TA solidify (1o minutes).
• Added 0.5 microliters directed (filtered) lysate on top of the TA.
• Added the buffer onto the last quadrant, our negative control.
• After all the quadrants were filled with a spot, we placed the plate in the incubator.

Plaque Assay

• Got four different petri dishes, one for each lab partner, and the fourth one being our control buffer.
• In each dish we added:
  • Added 2mL LB broth into a 50mL vial.
  • Added 22.5 microliters of M Calcium Chloride in the 50mL vial.
  • Added 0.5mL OF Artho PA Phage lysate
  • Added 2.5 mL TA into the 50mL vial.\
    • Note this times 4 since we had to fill 4 petri dishes.
• We then separated the solutions into 15mL vials and we added our 0.5 microliters of direct lysate into the 15mL vial.
• Poured the Solution onto my petri dish labeled ML 9/12/18 Plaque Assay.
• Let the solution sit for 15 min and placed in the incubator.
• Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
• Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.

Soil Metadata

• Percent water of the soil was calculated to be 11%.
  • Mass Dry= 10.8
  • Mass Wet= 12.09
  • 10.8/12.09=.89
  • 1-.89=.11=11%
    • Side note: Lots of debris which could have an effect on the % water.

• The mass of the soil was recorded and left to sit under a vented hood in the lab (this was done 9/10/18). This allowed the procedure above to be done today in lab.
• % Soil, Clay, and Silt was then calculated
• 50mL vial was left under a vent hood from Monday to let the soil disperse.
• The vial was observed and these are the results.
  • 1.5mL Sand
  • .5mL Clay
  • 2mL Silt

Observations:

• The soil had a lot of debris in both the % water mass and the vial shown above which was used to measure % soil.
• Results from the metadata:
  • 11% Water
  • 1.5mL Sand
  • .5mL Clay
  • 2mL Silt
  • 6.5pH

Conclusions/Next Steps:

The overall spot test and plaque assays procedures were easy since we knew what to expect. We were better prepared versus last time. Next steps: to see if our Bacteriophage has been isolated, and if so, we will try to amplify it.