Rationale:

Based off the results from the plates that had been spot tested on Monday (8/27), the experiment will proceed to perform a plaque assay to determine whether or not there are bacteriophages present in the soil sample that target arthrobacter.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Analyzed plates from Monday and decided to continue with a plaque assay.
3. Performed the following calculations to determine how much of the materials was required.

Calculations

*This calculation was performed after step 9, when the lab group recognized that only the original recipe amount was added.*

4. Obtained two plates (one for self and another one to serve as a control).
   • Labeled plate for self as “plaque assay enriched lysate Soil A.”
   • The control plate was split into four sections to be shared with four different groups due to a shortage of plates.

5. Through aseptic technique (over an EtOH (100%) flame), used a P200 micropipette to add 22.5 μL CaCl₂into a new 50 mL conical vial.
   • Labeled 50 mL conical vial “Team 2 TA 8/29/18.”

6. Used a cartwheel pipette with a 5 mL tip to add 2.0 mL of LB Broth to “Team 2 TA 8/29/18” conical vial.
7. Unfortunately, this vial spilled, so lab group started again with a new 50 mL conical vial.
   • Labeled 50 mL conical vial “Team 2 TA 8/29/18.”

13. Through aseptic technique (over an EtOH (100%) flame), used a P200 micropipette to add 22.5 mL CaCl₂into “Team 2 TA 8/29/18” conical vial.
14. Used a cartwheel pipette with a 5 mL tip to add 2.0 mL of LB Broth to “Team 2 TA 8/29/18” conical vial.
15. Used a P200 micropipette to add 67.5 μL CaCl₂into “Team 2 TA 8/29/18” conical vial to make a total of 90 μL CaCl₂.
16. Used a cartwheel pipette with a 10 mL tip to add 6 mL of LB Broth to “Team 2 TA 8/29/18” conical vial to make a total of 8 mL of LB Broth.
17. Used a cartwheel pipette with a 10 mL tip to add and mix 10 μL of enriched isolated lysate into a test tube with 0.5 mL arthrobacter in it.
    • Labeled test tube “KEA 8/29/18 Arthrobacter with Enriched Lysate.”
18. Allowed 15 minutes for the “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube to set.
19. Used a cartwheel pipette with a 10 mL tip to add and mix 1 mL of 2X TA into the “Team 2 TA 8/29/18” conical vial.
20. Used cartwheel pipette with a 10 mL tip to drop 1 mL of the Top Agar onto the control plate.
21. Used a cartwheel pipette with a 5 mL tip to add and mix 5 mL of 2X TA into the “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube.
22. Used a cartwheel pipette with a 5 mL tip to transfer over “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube contents onto “ERB 8/29/18 plaque assay Soil A” plate.
    • Crossed out “ERB” and labeled it with “KEA.”
23. Placed plates in incubator at room temperature.
24. Cleaned lab counter with CiDecan and EtOH (70%).

Results from the Spot Test of Soil A:

-On “Team 2 8/27/18 Soil A TA Control” plate, there were yellow spots indicating that the Top Agar was contaminated. The picture below shows this plate.

-On “KEA 8/27/18 Spot Test” plate, there were air bubbles that looked like plaques and yellow spots indicating contamination. The pictures below shows this plate.

Observations:

-When transferring over “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube contents, air bubbles formed and solidified on the plate. The picture below shows this plate.

Next Steps:

On Friday, check results of Plaque Assay. From these results, different things might happen. If it is contaminated, we might try running either a spot test or plaque assay with the same soil sample, or maybe filter the soil sample again. If it is negative, we will start all over with a new soil sample. If it is positive, we will celebrate and continue the experiment on the next lab day.