Previous Results:

•  The plaque assays and control plate (9/24) were all found to be contaminated. After examining other plates, it was concluded that the stocks of LB broth and 2x Top Agar used during the last plating were contaminated. The previous procedure and plaque assays would have to be redone.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a third series of serial dilutions and plate the diluted lysates with phage

Procedure:

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 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. 10 microL of new 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (10/1)) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were inconclusive
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (10/1)) the plates with the third round of serial dilutions will be examined for phage. If positive, then the experiment will move on to the next steps of calculating a high titer to create a webbed plate.