Results:

Many plaques formed on the “KEA 10/22 10⁰-1 PA” plate. The “KEA 10/22 10⁰-0 PA” plate was contaminated. One small plaque appeared on “KEA 10/22 10⁰-2” plate. The shared control plate was contaminated. The pictures below show these plates.

Rationale:

To continue the purification process, a plaque will be chosen from the “KEA 10/22 10⁰-1 PA” plate. Two plaque assays will be performed with this chosen plaque, with one using 10⁰and the other 10^-1.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into microcentrifuge tube “KEA 10/24 10⁰.”
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 10/24 10⁰” microcentrifuge tube.
3. The “KEA 10/24 10⁰” microcentrifuge tube was vortexed.
4. 10 µL of “KEA 10/24 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was then vortexed.
5. 10 µL of 10⁰and 10^-1were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
6. 6 mL of LB Broth, 67.5 µL of CaCl₂, and 7.5 mL of 2X TA were combined into a conical vial.
7. Transferred and mixed 4.5 mL of the TA mixture from the conical vial into each test tube.
8. Each test tube was poured onto their correlating plate.
9. 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 3 plates.

• The plaque circled bellowed was picked to make “KEA 10/24 10⁰” plaque, phage buffer mixture. The plaques on this plate were not as distinct as the plaques found on the “KEA 10/12/18 Soil E PA” plate.

• Air bubbles formed when pouring the “KEA 10/24 10^-1PA” plate.
• Strange contamination spots formed on the shared control plate as shown below.

Next Steps:

If there is contamination, a plaque assay will 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.