Rationale

Conduct a spot test with the enriched isolation produced on 8/22/18 in hopes of producing a plaque.

Procedure

1. Use the aseptic technique to clean your lab area. Using gloves, spray CiDecon on the lab table and wipe dry. Then wipe the table with 70% EtOH and let the excess evaporate.
2. Remove the 50 mL tube from the fridge containing the enriched isolation made on 8/22/18.
3. While working near an ethanol flame, use a syringe to suction 2 mL of the enriched isolation up and attach a 0.22 μm filter to the end of the syringe. Push down on the syringe to filter the enriched isolation into a micro test tube labeled “EAG 8/27/18 Filtered Enriched Isolation.”
4. Label a plate “EAG 8/27/18 Spot Test” and divide the plate into three quadrants labeled “direct”, “enriched”, and “negative control.”
5. Label a 50 mL conical tube “EAG, NMN, SS 8/27/18 Control Top Agar for Spot Test.”
6. Begin making the control top agar for the experiment by first adding 4.5 mLs of LB broth by using a 5 mL pipet attached to a suction device to the 50 mL conical tube.
7. Add 42.45 μL of calcium chloride into the conical tube using a micropipette.
8.  Quickly pipet 5.0 mL of 2X top agar into the 50 mL conical tube and swirl. Then pour the contents in the 5o mL conical tube labeled “EAG, NMN, SS 8/27/18 Control Top Agar for Spot Test” into a plate labeled “EAG, NMN, SS 8/27/18 Top Agar Control.” Let the plate sit for 15 minutes.
9. While the plate is sitting, get another 50 mL conical tube and label it “EAG 8/27/18 Spot Test Agar.”
10. In the 50 mL conical tube labeled “EAG 8/27/18 Spot Test Agar” pipet 4.5 mL of LB broth into the container.
11. Using a micropipette, pipet 45 μL of calcium chloride into the 50 mL conical tube.
12. Ask Lathan to add 0.5 mL of Arthrobacter into the tube. Then use a 10 mL pipet and a suction device to add 5 mL of 2X top agar to the 50 mL conical tube. Shake to invert.
13. Pour the contents of the 50 mL conical tube labeled “EAG 8/27/18 Spot Test Agar” into the plate labeled “EAG 8/27/18 Spot Test.” Let the plate sit for 10 minutes.
14. After 10 minutes, use a micropipette to pipet 5 μL of phage buffer into the negative control section on the plate. Pipet 5 μL of the direct isolation made on 8/22/18 onto the direct section of the plate. Pipet 5 μL of the filtered enriched isolation onto the enriched section of the plate.
15. Let the plate sit for approximately 8 minutes and then place into the incubator for 48 hours.

Observation

While pouring the 50 mL conical tube labeled “EAG 8/27/18 Spot Test Agar” into the plate, air bubbles formed on the plate. This may be confused with possible plaques after 48 hours of incubation.

Conclusion and Next Steps

In the future, the results of the spot test will be observed and a plaque assay will be conducted with the enriched and direct isolation to confirm the presence of plaques.