Rationale:

To pick a new plaque, dilute it using phage buffer, and create plaque assays

Procedure:

• First, wipe the table with CiDecon and Ethanol and set up an aseptic zone to prevent the spread of contamination.
• Arrange three small tubes in order, from 10^0 to 10^-2. Fill each tube with 90 µL of phage buffer.
• From a plate with a high concentration of plaques, locate a plaque and pick it.
• Place the picked plaque in the 10^0 tube of phage buffer.
• Transfer 10 µL of lysate from the 10^0 dilution tube to the 10^-1 dilution tube.
• After, transfer 10 µL of 10^-1 diluted lysate to the 10^-2 dilution tube.
• After performing the serial dilution, 2 mL of LB Broth and 22.5 µL of CaCl2 were combined together for as many plaque assays being made.
• Each dilution was combined with 0.5 mL of Arthrobacter and left alone for 10 minutes to infect.
• After infection, combine the Arthrobacter and dilution to the LB Broth and CaCl2 mixture.
• Add 2.5 mL of 2X Top Agar to the solution then pour over the assigned plate.
• Allow for the plate to solidify for 15 minutes before placing it invertedly in the incubator.

Results and Analysis:

There are no plaques in both dilutions. This may be due to the many mistakes that were made when conducting the dilutions and creating the plaque assays. Due to the errors, the entire procedure was conducted again with a new picked plaque.

Conclusion:

After setting up the correct measures to prevent contamination, three small tubes (10^0, 10^-1, and 10^-2) were filled with 90 µL each. From the plaque assay, pick a plaque and place in the 10^0 tube. From that tube, transfer 10 µL to another tube, creating the 10^-1 dilution. From the 10^-1 dilution, transfer 10 µL to the 10^-2 tube to create the dilution. Next, add each dilution to their respective 0.5 mL of Arthrobacter and allow it to infect for 10 minutes. While infection, combine LB broth and CaCl2. After 10 minutes, combine the lysate with the culture and add 2X Top Agar. Pour onto their respective plates and allow to sit for 15 minutes to solidify. Then place plate inverted in the incubator.

Future Plans:

In the future, dilutions will continue if there are plaques present on the plate. If not, another plaque will be picked.