Objective:

• Conduct a third series of serial dilutions for the 10^0 plate to see if the contaminated results from (9/26) effected the previous results
• Create a webbed plate to prepare for plate flooding

Procedure: 3rd Purification of 10^0

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked from the 10^0 plate (created 9/24) by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
3. The dilution was poured into a tube of 0.5 mL Arthrobacter to allow phage to interact with bacteria
4. A mixture of Overlay Agar was made for the experimental and control plate using 4 mL of LB broth, 45 microL CaCl2, 5 mL 2xTA. The mixture was divided into 2 50 mL tubes
5. The mixture of Arthrobacter with the dilution was then added to the 50 mL tube of  Experimental Overlay Agar
6. The experimental tube and the control tube were plated and left to harden for 15 min
7. Plates were placed in incubator for 48 hours.

Procedure: Webbed Plate

1. The webbed plate was made under the same aseptic conditions
2. Using the results from the titer calculations (10/1), the left over 90 microL of 10^0 lysate (created 9/24 for 3rd purification) was poured into a tube of 0.5 mL Arthrobacter to allow phage to interact with bacteria
3. The Overlay Agar for the webbed plate was created using 1.9 mL LB broth, 22.5 CaCl2, 2.5 mL 2xTA. Mixed in 50 mL tube
4. The 0.5 mL of Arthrobacter and the dilution was added to the 50 mL tube
5. The plate was left to harden for 15 min, placed in incubator of 48 hours

Results:

• This step in the experiment is not complete, therefore there are no results to report

Next Steps:

• During the next lab (10/8), both the 3rd purification plate and the webbed plate will be examined for positive results. The control plate will be examined for contamination. If the webbed plate has positive results, then the plate will be flooded to collect the phage for further testing. If the webbed plate is not completely webbed, then titer calculations will be reviewed and modified.