Results:

Since the TA mixture started to solidified when placed on plate, it is hard to tell if the plates were contaminated or not as shown in the picture below.

Rationale:

It is not certain if a phage was picked previously, so plaque assays will be run with the 10⁰dilution from last time, a 10⁰dilution made from a newly-picked plaque from the “KEA 10/12/18 Soil E PA” plate, and a 10⁰dilution made from a newly-picked plaque from the “KEA 10/17 10⁰ PA” plate. From this, maybe one of the plates will have many plaques, and then a second passage can be performed.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into both microcentrifuge tubes “KEA 10/22 10⁰-1” and “KEA 10/22 10⁰-2.”
2. Used a micropipette tip to touch a different plaque from previous plaque assay from the “KEA 10/12/18 Soil E PA” plate and then swirled the tip in the “KEA 10/15 10⁰-1” microcentrifuge tube.
3. Used a micropipette tip to touch a plaque from the “KEA 10/17 10⁰ PA” plate and then swirled the tip in the “KEA 10/15 10⁰-2” microcentrifuge tube.
4. Both microcentrifuge tubes were vortexed.
5. 10 µL of 10⁰, 10⁰-1, and 10⁰-2 dilutions were 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 Top Agar 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 plates with the dilutions did have strange marks which most likely were air bubbles.
• The plaque circled bellowed was picked for the “KEA 10/15 10⁰-2” dilution.

• The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl₂needed for 3 plates.

• Made a shared control plate with a different group.
• Since the “KEA 10/12/18 Soil E PA” plate had been left in the incubator, not the fridge, it appears that another plaque had formed.

Next Steps:

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