Rationale

Conduct a plaque assay to confirm the results from the previously conducted spot test. The formation of a plaque will indicate the possible presence of a phage.

Procedure

1. Use the aseptic technique to clean the area. With gloves on, spray CiDecon on the lab table and wipe dry. Then wipe 70% EtOH on the surface and let the excess evaporate.
2. Working near an ethanol flame, pipet 10 μL of the filtered enriched lysate created on 8/27/18 into a glass tube containing 0.5 mL of Arthrobacter. Wait 13 minutes.
3. While the Arthrobacter and the filtered enriched lysate are sitting in the glass tube, begin to make your top agar solution.
4. Using a 10 mL pipet and a suction device, pipet 8 mL of LB broth into a 50 mL conical tube labeled “EAG, NMN, SS 8/29/18 Top Agar for Plate Assay.”
5. Using a micropipette, pipet 90 μL of calcium chloride into the 50 mL conical tube.
6. Wait for the 13 minutes of the Arthrobacter and lysate sitting to be complete.
7. After the 13 minutes as passed, place 10 mL of top agar into the 50 mL conical tube.
8. Use a 10 mL pipet to dispense 5 mL of the solution in the 50 mL conical tube into the glass vial containing the Arthrobacter and lysate. Pour the contents of the vial onto a petri dish.
9. Swirl the contents in the petri dish around to distribute evenly.
10. Let the plate sit for 10 minutes before inverting and placing into the incubator for 48 hours.

Observation

There were possible plaques that appeared on the spot test conducted on 8/27/18, however they may be confused as air bubbles. When pouring the contents of the 50 mL conical vial onto the petri dish, the solution was no longer completely liquid and had assumed a slightly thicker texture.

Conclusion and Next Steps

The results of the plaque assay will be observed on Friday to determine if there are phages present in the soil collected.