Rationale: Perform Plaque assays for both Soil A and B after washing soil sample B.

Procedure:

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone.

Spot Test 

A spot test was performed for both Michael’s and Cooper’s Soil sample B. Obtained a 50mL vial and used the formula below as the plates solution. After the solution was made within an aseptic zone, the solution was poured onto a plate, which sat for >15minutes. Then 10 microliters of lysate (Soil B) were added to the solidified solution, along with one spot for the negative control (Phage Buffer 10 microliters). Then the plates were placed in the incubator for 48 hours. The formula below was used to make the solution for 1 plate (one for the spot test, which was divided into three parts).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro

Plaque Assay

A plaque assay was performed for Michael’s two soil samples A and B. Since the phage was lost during the passage of phage, the lysate from the beginning of the lab (9/12/18) was obtained to perform a plaque assay, in hopes to passage phage. The formula below was used to make the solution for 4 plates (two for Michael’s plaque assays, one for Cooper, and one for the control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

This was all done within an aseptic zone. Pippeted 10 microliters of lysate into the 500 microliters of Artho. Pippeted 4.5mL of the 50mL vial solution into each tube of arthro + lysate, then quickly poured the solution onto plates. The plates sat for about 15 minutes, and then they were placed in the incubator for 48 hours.

Soil Metadata results:

% Water: 21%

% Sand: 1.5 mL

% Silt: 2mL

% Clay: .5mL

Observations:

The plaque assay that was performed on Wednesday 10/3/18 was contaminated. This was the last plaque available to pick, and the experiments of the results came back negative. This resulted in two plaque assays that were performed 10/8/18 (Soil A and Soil B). All of the controls in the lab came back negative once again.

Conclusions/Next Steps: 

Perform more plaque assays to purify plaques if plaques are present. The previous experiments results resulted in the start of the new soil sample b. Soil sample A lysate was used to perform another plaque assays since it has had positive results, but the passage of plaques have failed from contaminations or the picking of a bubble. Knowing that soil sample A will have positive results ensured that on Wednesday, 10/9/18, it will be passed.