Results:

• The positive control run by the TA’s was contaminated meaning everything run on Monday using Arthrobacter was unreliable.
• The control plate run on Monday was contaminated. The “KEA 9/24/18 PA” plate looked negative.

Rationale:

The plaque assay will be run again with the enriched lysate from soil C to determine if there are bacteriophages in this soil sample that go after Arthrobacter.

Procedure:

1. Once an aseptic zone was established, 10 μL of enriched lysate was transferred into a test tube which already had 400 μL of Arthrobacter.
2. 4 mL of LB Broth, 90 μL of CaCl₂, and 10 mL of 2X TA was combined into a conical vial.
3. 5 mL of the conical vial mixture was added and mixed into the test tube with the enriched lysate and Arthrobacter.
4. The mixture from the test tube was poured into a plate labeled “KEA 9/26/18 PA.”
5. The “KEA 9/26/18 PA” plate was placed in incubator at room temperature.

Observations:

• The positive control plate did not have any plaques on it. Both Anite and Murph are very high titer phages. The plate appeared negative as shown in the picture below.

• The control plate from the plate assay ran on Monday had strange particles in it and other unusual features as shown in the pictures below.

• The “KEA 9/24/18 PA” plate appeared negative as shown below.

• The following calculations were performed to determine enough LB Broth, TA, and CaCl₂needed for 4 plates.

• When 2X TA was pipetted into the conical vial, it was observed that the 2X TA was contaminated since there were white hair-like strains floating in the mixture. This conical vial was thrown away because the 2X TA added was contaminated. Steps 5-7 were repeated, but a different bottle with 2X TA was used.

Next Steps:

Next time in lab, if the plaque assay is contaminated, another plaque assay will be run. If the plaque assay is negative, new soil will be collected. If the plaque assay is positive, the experiment will move on to do the purification process.