Rationale:

By setting up and running a spot test on the “KEA 8/24/18 enriched,” it will become apparent whether or not there are bacteriophages in the lysate that go after arthrobacter.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Used aseptic technique (over a lite EtOH (100%) flame) to filter with a 3 mL syringe with a 0.22 μm tip the enrichment isolation from the 50 mL conical vial labeled “KEA 8/24/18 enriched” into a microcentrifuge tube.
   • This microcentrifuge tube was labeled “KEA 8/27/18 FS lysate Soil A.” FS stands for filtered sterile.

3. Obtained two petri dishes (one for self and other to serve as a control Top Agar).
   • The petri dish for self was divided into thirds and sections were labeled: “Direct isolation Soil A,” “FS lysate Soil A,” and “Neg. Control.” The side of this petri dish was labeled “KEA 8/27/18 Spot Test.”
   • The petri dish that served as a control Top Agar was labeled “Team 2 8/27/18 Soil A TA Control.”

4. The following calculations were conducted before proceeding to the next steps.

Calculations:

Conversion factors:

1,000 μL = 1 mL

1 M =1,000 mM

Equation:

C₁V₁ = C₂V₂, where C is concentration and V is volume

For “KEA 8/27/18 Spot Test,”

(1 M) V₁= (4.5 mM) (10 mL)

(1,000 mM) V₁ = (4.5 mM) (10,000 μL)

V₁ = (4.5 mM) (10,000 μL)/(1,000 mM)

V₁= 45.0 μL

For “Team 2 8/27/18 Soil A TA Control,”

(1 M) V₁= (4.5 mM) (9.5 mL)

(1,000 mM) V₁ = (4.5 mM) (9,500 μL)

V₁ = (4.5 mM) (9,500 μL)/(1,000 mM)

V₁= 42.8 μL

5. Used a P200 micropipette to transfer 45 μL of 1 M CaCl₂into a new 50 mL conical vial.
   • This 50 mL conical vial was labeled “KEA 8/27/18 TA.”

6. Used a P200 micropipette to transfer 42.8 μL of 1 M CaCl₂into a new 50 mL conical vial.
   • This 50 mL conical vial was labeled “Team 2 8/27/18 TA.”

7. Used a cartwheel pipette with a 5 mL tip to transfer 4.5 mL of LB Broth into both “KEA 8/27/18 TA” and “Team 2 8/27/18 TA” conical vials.
8. Under a clean hood, added 0.5 mL of arthrobacter with a micropipette to “KEA 8/27/18 TA” conical vial.
9. Used a cartwheel pipette with 10 mL tip to transfer 5 mL of 2X TA to both “KEA 8/27/18 TA” and “Team 2 8/27/18 TA” conical vials.
10. Poured “KEA 8/27/18 TA” conical vial into “KEA 8/27/18 Spot Test” petri dish and “Team 2 8/27/18 TA” conical vial into “Team 2 8/27/18 Soil A TA Control” petri dish.
11. Allowed 10 minutes to pass for Top Agar to solidify in both petri dishes.
12. Used a P10 micropipette to add a 10 μL drop of direct isolation into the “Direct isolation Soil A” section on the “KEA 8/27/18 Spot Test” petri dish in an aseptic zone with EtOH (100%) flame.
13. With a fresh micropipette tip, used a P10 micropipette to add a 10 μL drop of FS lysate into the “FS lysate Soil A” section on the “KEA 8/27/18 Spot Test” petri dish in an aseptic zone with EtOH (100%) flame.
14. With a fresh micropipette tip, used a P10 micropipette to add a 10 μL drop of phage buffer into the “Neg. Control” section on the “KEA 8/27/18 Spot Test” petri dish in an aseptic zone with EtOH (100%) flame.
15. Placed the petri dishes in incubator at room temperature.
16. Cleaned lab counter with CiDecan and EtOH (70%).

Observations:

• When the Top Agar solidified, air bubbles formed. In the “KEA 8/27/18 Spot Test” plate, there were many bubbles in the “FS lysate Soil A” section and a few in the “Direct isolation Soil A” section. The picture below displays these air bubbles.

• When the 10 μL drop of FS lysate was placed with the P10 micropipette on the “KEA 8/27/18 Spot Test” plate, the drop hydroplaned over to be on top on the line separating the “FS lysate Soil A” and “Direct isolation Soil A” sections.

Next Steps:

On Wednesday, the spot test will be analyzed to see whether or not it contained a bacteriophage that specifically targets arthrobacter. If the plates are positive, the experiment will continue with a plaque assay. If the plates test negative, the experiment might either try a plaque assay or decide to start all over with a new sample of soil.