Rationale: The plates ran last week turned out to not be webbed. They had two distinct plaque morphologies, so I will pick a plaque from each and run plaque assays to make sure this is the same phage making both types of plaque.

Procedure:

1. Filled 2 microcentrifuge tubes with 100µL of 1X Phage Buffer labeled one alpha and the other beta.
2. Picked a large plaque and placed in the tube labeled alpha. Pick a small small plaque and placed in the tube labeled beta.
3. Diluted each sample out to the 10^-4 dilution factor.
4. Enriched alpha dilutions in 0.5mL arthrobacter for 20 minutes.
5. Made top agar with 10mL of LB broth, 12.5mL of 1X TA, and 112.5µL of CaCl2.
6. Plated each mixture of alpha dilution and arthrobacter after pipetting in 4.5mL of top agar.
7. Repeated steps 4-6 with the beta dilutions.
8. Made a top agar control by plating 4.5mL of top agar.
9. Let sit then inverted and placed in the incubator.

Observations:

The marked plaque on the top right is the plaque picked for the alpha samples. The plaque picked on the bottom left is the plaque picked for the beta samples.

The results show that both alpha and beta lysates produced both large and  small plaques. The top agar was contaminated, so when the  first two dilutions of alpha lysed the plate the contamination was able to grow back in place of arthrobacter.

Conclusions  and Next Steps: This means that  there is just one type of phage in my plate making the radically different plaques. Since I have a plate closed to being webbed and a lysed plate I will flood both so I can have enough lysate to perform DNA extraction. I’m flooding the lysed plate because the plate with a lower dilution factor also lysed, but the plate with a higher dilution factor nearly webbed so there is most likely some amplification that occurred.