Results:

The Top Agar control was not contaminated. However, the strange patterns similar to the patterns that appeared from the previous plaque assay occurred again as shown in the picture below.

Rationale:

Another plaque assay will be run with soil D along with both TA control and Arthrobacter control plates. This will reveal whether or not these unusual patterns are plaques or just contamination. Soil E will be filtered and enriched, so if soil D has no phage, soil E will be ready to run a plaque assay during open lab.

Procedure:

1. Once an aseptic zone was established, 10 μL of “KEA 10/3 FS enriched” was added and mixed into a test tube which already had 0.5 mL of Arthrobacter.
2. 10 μL of phage buffer was added and mixed into a test tube which already had 0.5 mL of Arthrobacter.
3. 2 mL of soil E and 10 mL of LB Broth were transferred into a conical vial which was shaken for 15 minutes.
4. The conical vial was centrifuged at 3,000g for 10 minutes.
5. 10 mL of LB Broth, 112.5 μL of 1 M CaCl₂, and 12.5 mL of 2X TA were combined into a conical vial.
6. Transferred 4.5 mL of the Top Agar mixture from the conical vial into both test tubes.
7. The test tube mixtures were poured into their correlating plates.
8. The supernatant liquid from the centrifuged conical vial went through a 0.22 μL top vacuum filter into a conical vial labeled “KEA 10/10/18 soil E enrich.”
9. The “KEA 10/10/18 soil E enrich” conical vial was placed in the incubator at room temperature for the next 48 hours.
10. The “KEA 10/10/18 PA” and “SA LEF KEA 10/10/18 Arthrobacter” plates were placed in the incubator at room temperature.

Observations:

• Since the control TA plate from 10/8 appeared to have no contamination, it was hypothesized that the strange unusual pattern might be a result from either the Arthrobacter being used or the way the TA mixture was poured onto the plates.
• The following calculations were performed to determine enough LB Broth, TA, and CaCl₂needed for 5 plates.

• The conical vial weighed 21.71 grams before it was centrifuged.
• The centrifuged conical vial had a pellet that had only two distinct layers, a reddish-cinnamon brown layer on top of a milk chocolate brown layer. The vial is showed below.

• The tip of the serological pipette was accidentally used twice. To prevent any contamination, the TA mixture was tossed and a new TA mixture was made.

Next Steps:

In open lab, a plaque assay will be run with soil E if there is contamination or no plaques that result from the plaque assay from soil D. If the plaque assay is positive, a plaque will be picked.