Results:

As expected, the control plate was contaminated. However, it was apparent that a plaque was picked. Plaques formed on both the “KEA 10/29 10⁰‘10/22’” and the “KEA 10/29 10⁰A” plates. No plaques formed on the “KEA 10/29 10⁰B” plate.

Rationale:

Although the phage has only gone through two passages, the plaques formed are distinct enough to move past the purification stage and go onto the amplification. Titer calculations along with flooding a plate will take place to form enough lysate to form a high titer.

Procedure:

1. Drew quadrants and counted total number of plaques on “KEA 10/29 10⁰A” plate.
2. Calculated the average diameter of plaques, the titer, and the amount of lysate needed to web the plate.
3. Once an aseptic zone was established, 8 mL of phage buffer was poured onto “KEA 10/29 10⁰A” plate.
4. Placed parafilm and stored “KEA 10/29 10⁰A” plate at 8ºC.

Observations:

• The control plate had a strange, contaminated yellow dot. The following pictures show the results.

• 514 plaques were counted from the “KEA 10/29 10⁰A” plate as shown in the picture below.

• The following measurements were recorded from 10 random plaques to approximate the average diameter: 0.1, 0.1, 0.05, 0.12, 0.04, 0.07, 0.08, 0.1, 0.1, and 0.1 mm. The average diameter calculated was 0.82 mm.

Calculations:

Next Steps:

In open lab, the flooded plate’s lysate, phage buffer mixture will go through a 0.22 µm filter to isolate the phage. A plaque assay will also be run with the mixture to receive a high titer.