Results:

No contamination was found on the plates, and plaques had formed. The “KEA 11/12 10⁰10µL” plate appeared to be the most webbed out of the plaque assays performed on 11/12. The pictures below show these plates.

Rationale:

In all efforts to receive a high titer, the “KEA 11/12 10⁰10µL” plate will be flooded, serial dilutions will be carried out to the 10^-8, and each dilution will be spotted.

Procedure:

1. Once an aseptic zone was established, 8 mL of phage buffer was poured onto “KEA 11/12 10⁰10µL” plate.
2. The plate was shaken on an incubator for five hours.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter which was added to the “KEA 11/12 FS lysate 10⁰” conical vial.
4. 10 µL of “KEA 11/13 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was vortexed.
5. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a conical vial.
6. 5 mL of the Top Agar (TA) mixture from the conical vial was poured onto a control plate.
7. 5 mL of Arthrobacter was combined with the remaining TA mixture and was poured onto the “KEA 11/13 Spot Test” plate.
8. Once the spot plate had solidified, 10 µL of 10⁰to 10^-8of the “KEA 11/12 FS lysate” and 10 µL of phage buffer were spotted.
9. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl₂needed for 2 plates.

• Step 4 was repeated with different dilutions to create dilutions out to 10^-8.
• When checking to see if the spots had solidified, it was noted that the spots moved. The spots did not collide with each other; however, the spots were no longer in a circle shape.

Next Steps:

Calculate the titer from the spot test plate.