Results:

Since the plates had no signs of contamination or plaques, it is safe to assume that no phage was present in the plaque picked on Wednesday (10/24). The pictures below show these plates.

Rationale:

Another plaque will be chosen and a plaque assay will be run on it to continue the purification process.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into a microcentrifuge tube labeled “KEA 10/26 10⁰.”
2. Used a micropipette tip to touch a plaque from the “KEA 10/22 10⁰-1 PA” plate and then swirled the tip in the “KEA 10/26 10⁰” microcentrifuge tube.
3. The “KEA 10/24 10⁰” microcentrifuge tube was vortexed.
4. 25 µL of 10⁰were added to a test tube which already had 0.5 mL of Arthrobacter.
5. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a conical vial.
6. Transferred and mixed 4.5 mL of the TA mixture from the conical vial into the test tube.
7. The test tube was poured onto a plate and the rest of the TA mixture was poured onto a different plate.
8. These plates were placed in the incubator at room temperature.

Observations:

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

Next Steps:

If there is contamination, a plaque assay with be run again with the same dilutions. If there are plaques, a third passage will be performed. If there are no plaques, a different plaque will be chosen.