Previous Results:

• During the last lab, while the lysate was take to do TEM, plaque assays were create by the lab group. They made 5 plates using Lysate #6 10^-2. The control plate did not have contamination. 2 plates had slipped and one plate looked as though Arthrobacter was not mixed in the overlay Agar. It was determined that the procedure should be repeated.

Objective:

• Create plaque assays using 10^-2 diluted Lysate #6 without slipping and contamination

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Lysate #6 was obtained and a 10^-2 dilution was remade. Microcentrifuge tubes labeled “10^-1” and “10^-2” each had 180 microliters phage buffer
3. 20 microliters of Lysate #6 was added to “10^-1” tube, then 20 microliters were taken from that tube and put into the “10^-2” tube
4. After mixing the tube, 20 microliters of 10^-2 lysate was added to 5 tubes containing 0.5 mL of Arthro and left to infect for 10 min
5. An Overlay Agar was mixed using 12 mL LB broth, 15 mL 2x TA, and 135 microliters CaCl2
6. 4.5 mL of Overlay Agar was added to each Arhtro tube, leaving 4.5 mL of Overlay in the tube for a control plate
7. The mixtures were then plated (6 plates: 5 experimental and 1 control) and left to harden for 15 min, then incubated for 48 hours

Results:

• The plaque assays were not completed, therefore there are no results to report
• One of the experimental plates experienced plate slipping, but was incubated with the other plates

Next Steps:

• During the next lab, the plates will be flooded, the lysate collected, and plaque assays created to try and calculate a high titer
• A TEM will be run again on Lysate #6 using another staining agent