Rationale:

Due to contamination on the negative control, another plaque assay will be made.

Procedure:

To prevent contamination, the tables along with many of the items used (test tube racks and pipettes) were cleaned with CiDecon and Ethanol and two aseptic zones were set up. 0.1 µL of lysate and 400 µl of Arthrobacter were mixed together and left alone for 10 minutes. Next, 2.1 mL of LB broth and 22.5 µL were mixed together to form the solution of what would eventually become the top agar. After allowing the lysate and Arthrobacter to sit for 10 minutes, 2.5 mL of 2X Top Agar and lysate and Arthrobacter were poured into the Top Agar solution then poured onto the plate. The plate then sat for 15 minutes to solidify then put into the incubator.

Results and Analysis:

Due to the contamination of the LB broth previously used, a new LB broth was used.

The contamination in the negative control made on 9/24/18 was due to the fact that the Arthrobacter was contaminated. Because of this contamination, new measurements were used to create the plaque assay such as the 400 µL of Arthrobacter rather than the 0.5 mL that is usually used.

Conclusion and Future Plans:

Due to the contamination of the negative control caused by the contamination of the Arthrobacter, a new plaque assay was created to check for the presence of plaques. Also, an important note for the creation of this plaque assay were the new measurements that were used to create the top agar. First, the lysate and Arthrobacter (400 µL) were combined and left alone. Then, LB broth (uncontaminated and 2.1 mL) and CaCl2 (22.5 µL) were mixed together and left alone for 10 minutes. After the lysate and Arthrobacter solution was combined with the top agar solution then the 2X Top Agar was added. The solution was poured on the plate and left alone for 10-15 minutes. The plates were then placed in the incubator.

If there are plaques present, a plaque will be picked and diluted. If there are no plaques and no contamination, new soil will be collected. If there are no plaques and contamination, all instruments and other items used will be cleaned and all components of the top agar solution will be checked for contamination.