Rationale: To visually confirm or deny the presence of plaques in the soil, after negative results on a spot test.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Procedure:

1. The workstation was cleaned with CiDecon and 70% Ethanol before the procedure was started. A 100% Ethanol burner was lit to form an aseptic zone.
2. To perform a plaque assay with the enriched lysate, the first step was to obtain 0.5 mL of arthrobacter. To this 10 uL of enriched lysate was added. This tube was labeled LIP 8-29-18 AEPA (Arthro Enriched Plaque Assay). This was allowed to sit for 15 minutes.
3. While the arthrobacter and enriched lysate were allowed to sit, The Top Agar solution was created. 8.0 mL of LB Broth was added to a 50 mL tube. The Top Agar for Group 4 was made in one tube, as well as the control top agar. This tube was labeled Group 4 8-29-18 TA Plaque Assay.
4. Next 90 uL of CaCl2 was added to the LB Broth. Because the solution contained 5 mL total, the amount of CaCl2 for each person was 22.5 mL, but since this TA was being used for three group members and a control plate, this number was multiplied by 4, to get 90 uL total.
5. 10 mL of 2X TA was then added to the LB Broth and CaCl2.
6. After, 4.5 mL of the TA solution was added to the tube containing arthrobacter and enriched lysate for each member of Group 4. It was mixed and then immediately poured on to a petri dish for each group member. My plate was labeled LIP 8-29-18 PA1 (Plaque Assay 1).
7. For the control TA plate, 1 mL of the TA solution was added to a petri dish. This control plate was shared by groups 3, 4, 7, and 8, so it was divided into 4 sections. Group 4’s 1 mL was put only into the quadrant labeled Group 4.
8. The plate was then allowed to sit for 10 minutes to solidify and was then stored in an incubator inverted until Friday, 8-31-18, at 2:00 pm, approximately 46 hours from the time it was finished. The time the procedure was complete was 3:40 pm.
9. The workstation was the cleaned with CiDecon and 70% Ethanol. All used materials were properly disposed of.

Observations/Results/Data:

Observations:

• The top agar solidified much faster with the plaque assay than with the spot test.
• Due to limited plates, 4 control top agar solutions were done on one plate for groups 3, 4, 7, and 8.
• There were some bubbles on the set plate when the procedure was completed.

Results: The procedure was completed on time and the plates will be stored in an incubator inverted until Friday 8-31-18.

Interpretations/Next Steps/Conclusions:
The procedure was complete on time. It went smoothly and was done correctly. The plate will now be allowed to grow until 8-31-18 at 2:00 pm. If there are plaques, then they will be picked. If there are no plaques, a new soil sample will need to be found.