Results:

Contamination was found on the control plate.

Rationale:

In efforts to receive a high titer, plaque assays will be performed with 400 µL of “KEA 11/5 FS lysate 10⁰,”10 µL of 10⁰and 10 µL of 10^-2of the lysate created on 11/9, 10 µL of 10⁰and 10 µL of 10^-2of the lysate created from flooding and filtering “KEA 11/7 PA 10⁰ 125 µL” plate.

Procedure:

1. Once an aseptic zone was established, 6 mL of phage buffer was poured onto “KEA 11/7 PA 10⁰125 µL” plate.
2. The plate was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “KEA 11/12 FS lysate 10⁰.”
4. 10 µL of “KEA 11/12 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was vortexed.
5. 10 µL of “KEA 11/12 10^-1” was added with 90 µL of phage buffer to create 10^-2dilution which was vortexed.
6. 400 µL of “KEA 11/5 FS lysate 10⁰,”10 µL of 10⁰and 10 µL of 10^-2of the lysate created on 11/9, 10 µL of 10⁰and 10 µL of 10^-2of the lysate created on 11/12 were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
7. 12 mL of LB Broth, 135 µL of CaCl₂, and 15 mL of 2X TA were combined into a conical vial.
8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
9. 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 “KEA 11/12 Control.”
10. 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 6 plates.

• The contamination found on the control plate appeared as small yellow spots as shown below.

• Tiny, microscopic plaques appeared on the plates indicating possibly that the plates were over lysed. The plaques are so small that they do not even appear in photos as shown with the “KEA 11/9 10^-2 10µL” plate below.

Next Steps:

If there is contamination, the experiment will be repeated. The titer will be calculated. If it is a high titer, transmission electron microscopy will take place. If not, webbing calculations and webbing of plates will occur.