Rationale: Redo plaque assay due to contamination in the negative control

Procedure:

First, the previous plaque assays were examined only to find the negative control to be filled with contamination. Due to the later understanding of the source of contamination (LB broth), the negative control was filled contamination along with a negative result for the plaque assay. The lab table was wiped down with CiDecon and Ethanol, after which an aseptic zone was set up to prevent contamination. Then, 0.5 mL of Arthrobacter and 1 µL were mixed together and left alone for 10 minutes. Next, LB broth (2 mL) and CaCl2 (22.5 µL) were combined. After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solution along with the lysate and Arthrobacter. The solution was then poured onto the plate and given enough time to solidify. After solidification, the plates were placed into the incubator and left there for 48 hours.

Results and Analysis:

Plaque Assay on the left and Negative Control on the right

The contamination was due to the contamination of the LB broth.

On the left is the LB broth was used on Friday to do the plaque assay while the LB broth on the left is an example of uncontaminated.

Conclusion:

Because of the contamination, another plaque assay was created. First, the tables were cleaned with CiDecon and ethanol along with an aseptic zone. Then, the lysate and Arthrobacter were mixed together to infect. The LB broth and CaCl2 were added together into a vial of which would become the top agar solution. After 1o minutes, the 2X top agar was added along with the lysate and Arthrobacter onto the plate. It was left alone to solidify then put into the incubator.

Future Plans:

The plaque assays will be checked for the presence for plaques. If there are no plaques with contamination, another plaque assay will be created. If there are no plaques and no contamination, then a new soil sample will be collected, If there are plaques,  a plaque will be picked and will be diluted using phage buffer. After diluting it, a plaque assay will be created to begin the process to get a high concentration of plaque.