Results from previous lab:

• Plates were collected from Spot Test conducted on 8/27. No spot clearings were found on the plate indicating there is not a presence of bacteriophage in the soil sample. The Control plate was clear, indicating the Top Agar was not contaminated.

Objective:

• Plaque Assay will be conducted to confirm the results of the Spot Test from 8/27. The previous Spot Test was negative, but the Plaque Assay could come out positive since the phage has had more time to replicate. The Plaque Assay will provide more concrete answers.

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. 10 microliters of filtered enriched lysate (obtained on 8/27) was added to a the containing 0.5 mL of Arthrobacter. Tube was set aside to give potential phage and bacteria time to interact.
3. Then a 50 mL conical tube was obtained. 8 mL of LB broth, 90 microliters of CaCl2, and 10 mL of 2x Top Agar were added using pipettes. Mixture was swirled to ensure all components were evenly distributed. This created the Overlay Mixture without bacteria.
4. 1 mL of Overlay without bacteria was added to a plate that was shared with groups 2, 3, and 4. This was the Control plate. It was set aside to harden for 15 minutes.
5. Then, 4.5 mL of Overlay Mixture was piped into tube containing 0.5 mL of Arthro and 10 microliters of CaCl2. Mixture was swirled.
6. Overlay was then plated, let sit for 15 minutes, and incubated. Both the Overlay with bacteria and Overlay without bacteria (control) were incubated. Plates were left to be checked during next lab day (9/5).

Observations:

• Plate from Spot Test (8/27) was a yellow color and had a few air bubbles. This could have been mistaken as phage clearing spots, but after being viewed many times it was considered a negative plate.
• It can be difficult to get precise measurements while pipetting because contamination is a concern and you have to move quickly.
• Overlay Top Agar was the same color as the Spot Test.

Results:

• Spot Test results are negative.

• Results of Plaque Assay will be obtained next time in lab since experiment is not complete.

Conclusion:

• Since the Spot Test results were negative, it is likely that the soil sample Soil A does not contain any bacteriophage. The Plaque Assay will provide further answers as to whether or not the sample contains phage.

Next Steps:

• If the Plaque Assay comes back positive with the presence of phage, then the phage will be picked and processed. If the result is negative, then another soil sample will be obtained from a different tree in a different area of campus.