Objective:

• Conduct a Spot Test using the enriched lysate that was obtained on 8/22 to look for the presence of bacteriophages in the soil sample, Soil A.
• To correctly follow the recipe for Top Agar and plate it without the Agar slipping.

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. The Enriched lysate from 8/22 was obtained from fridge.
3. Using a sterile 3 mL syringe, 2 mL of lysate was taken up. The syringe was then connected to a filter and 1.5 mL of lysate was pushed through into a microcentrifuge tube. Tube was labeled “FEL” = Filtered Enriched Lysate. This was done to remove bacteria in the lysate, and to collect the possible bacteriophages.
4. Then a mixture was created for the Experimental Plate. In a 50 mL tube labeled “Top Agar,” 4.5 mL LB broth, 45 microliters of CaCl2, and 5 mL of Top Agar were added using a pipette. Lathan Lucas added 0.5 mL of Arthrobacteria.
   • Calculation of CaCl2:
     • C1V1 = C2V2
     • C1 = CaCl2 microcentrifuge concentration = 1 M
     • C2 = CaCl2 Predicted final concentration = 4.5 mM
     • V1 = unknown
     • V2 = Predicted volume = 10 mL
     • (1M)(V1) = (4.5 mM)(10 mL)
     • V1 = 45 microliters CaCl2
5. Step 4 was repeated in another 50 mL tube to create the Top Agar for the Control Group, but without the addition of 0.5 mL Arthrobacter.
6. Each 50 mL conical tube with the Top Agars were poured into separate petri dishes. Soultions were swirled until evenly distributed. Let sit for 15 min.
7. Once the Top Agars were hardened, the Experimental Group received 10 microliters of the filtered enriched lysate, 10 microliters of the direct lysate, and 10 microliters of a phage buffer. Each in separate, labeled areas on the plate.
8. Both the Control Group and Experimental Group plates were placed in the incubator.
9. Plates will be examined on Wednesday (8/29) for spot clearings indicating the presence of phage.

Observations:

• While filtering the enriched lysate through the filter and the syringe, resistance was felt, but filter was not popped.
• Both the Control Group and Experimental Group Top Agar had a golden- yellow color while being poured onto plates.
• Top Agar solidified pretty quickly. No slipping occurred.

Results:

• Test is not complete, therefore there are no results to report. Results possible on Wednesday (8/29).

Next Steps:

• On Wednesday (8/29), the spot test plates will be examined for clearings in the lawn of bacteria and the presence of phage. If clearings are found, a plaque assay will be conducted using the enriched lysate. If no clearings are found, 2 plaque assay will be conducted. One using the enriched lysate, and another using the direct lysate.