Rationale: The purpose of this lab is to collect a high titer lysate and perform a spot titer test to calculate the titer, start the DNA extraction process, as well as perform TEM with the phage.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. The webbed plates were flooded with 8 ml of phage buffer and put in the refrigerator overnight.
3. Lysate was collected from the webbed plates and filtered using a 0.22 um top filter.
4. 35 ml was collected and separated into two tubes for two samples.
5. 10 ml of lysate was poured into a separate tube for DNA extraction. 40 ul of nuclease was added and the tube was inverted 10 times. 4 ml of PEG was then added, the tube was inverted once and then put in the incubator at 37 C for 25 minutes.
6. The tube was then allowed to sit at room temperature for 40 minutes.
7. While waiting, electron microscopy was performed. A TEM grid was made with 20 ul of lysate, two 20 ul dots of DI water, and a drop of uranyl acetate. The TEM grid was allowed to sit in the lysate for 5 minutes, and water for 2.5 minutes. It was then allowed to sit in the UA for 1 minute and then dried.
8. The image of the phage showed it to have an average head size of 58 nm and a tail of 160 nm.
9. A spot titer test was performed to calculate the titer of the lysate collected, with dilutions out to 10^-7. A 5 ml plate was made and inverted and stored in the incubator until the next lab. It was labeled LIP 11-28-18 STT (Spot Titer Test)
10. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations/Results:

• Head of phage: 58 nm
• Tail of Phage: 160 nm
• 35 ml of lysate collected
• Bubbles seen on the control plate.

Interpretations/Next Steps:
The procedure was complete for TEM and spot titer test, but the DNA extraction was not complete. The lysate was not spun in the centrifuge due to an error with the machine and will have to be performed in the next lab. The next step will be to finish DNA extraction and possibly run PCR.

Flooded Plates and Image of Phage: