Results:

The webbed plate was not completely webbed as shown below.

Rationale:

The “KEA 11/2 Web” plate will be flooded to collect lysate with a higher titer. Serial dilutions and plaque assays will be performed in order to make a plate from which the new titer can be calculated.

Procedure:

1. Once an aseptic zone was established, 4 mL of phage buffer was poured onto “KEA 11/2 Web” 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 11/5 FS lysate 10⁰.”
4. 10 µL of “KEA 11/5 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1
5. 10 µL of “KEA 11/5 10^-1” was added with 90 µL of phage buffer to create 10^-2
6. 10 µL of 10⁰, 10^-1, and 10^-2dilutions were added to correlated test tubes which already contained 0.5 mL of Arthrobacter.
7. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 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 11/5 Control.”
10. 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.

• When pouring the TA mixture, it started to solidify causing bubbles to form on the plates.
• The “KEA 11/5 PA 10^-2” plate did not solidify, and the TA mixture folded over on itself as shown below.

Next Steps:

If there is contamination, a plaque assay will be run again with the same dilutions. If not, the titer will be calculated. If the titer is not high, the plates will be flooded and a web plate will be made. If the titer is high, the experiment will go on to characterization procedures.