Results:

No contamination or plaques were observed from the plaque assay performed on 10/26 as shown below.

Rationale:

Since it does not appear a phage was picked previously, two plaques will be picked from the “KEA 10/22 10⁰-1 PA” plate.  To continue the purification process, plaque assays will be performed with a plaque, phage buffer mixture solution made from both the newly picked plaque along with the “KEA 10/22 10⁰-1” mixture.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into both microcentrifuge tubes “KEA 10/29 10⁰ A” and “KEA 10/29 10⁰ ”
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 10/29 10⁰ A” microcentrifuge tube.
3. Used a micropipette tip to touch a different plaque from the “KEA 10/22 10⁰-1 PA” plate and then swirled the tip in the “KEA 10/29 10⁰ B” microcentrifuge tube.
4. Both “KEA 10/29 10⁰ A” and “KEA 10/29 10⁰ B” microcentrifuge tubes were vortexed.
5. 25 µL of “KEA 10/22 10⁰-1”, “KEA 10/29 10⁰ A” , and “KEA 10/29 10⁰ B” mixtures were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
6. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
7. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
8. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “ML KEA 10/29 Control.”
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 4 plates.

• The plaques circled belowed were picked to make “KEA 10/29 10⁰ A” and “KEA 10/29 10⁰ B” plaque, phage buffer mixtures. To increase the chances a phage was picked, the plaques were picked under a light microscope.

• When pouring the TA mixture, the TA mixture started to solidify causing bubbles and lumps to form on the plates.

Next Steps:

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