Rationale: To collect a new soil sample and to also perform another plaque assay due to the contamination in the control

Procedure:

First, a new soil sample was collected from a white oak. Measurements (diameter, canopy height, and tree height) and leaves were taken and soil (2 feet from base). Next, a plaque assay was made due to the contamination in the control. 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:

The latest control (right) was negative and the plaque assay (left) was negative.

A reason for the contamination may be due to the carelessness in performing the experiment (not remaining close to the aseptic zone.)

The Top Agar became contaminated.

Tree Data:

Diameter: 277 cm

Height: 2118.36 cm

Shortest Side of Canopy: 721.36 cm

Longest Side of Canopy: 1249.68 cm

Average: 985.52 cm

The soil was obtained from the largest tree on the large area of grass by iHop.

A large clump of grass had to be removed to access the soil. The soil became accessible 1.5 inches from the surface. Rather than being small particles, the soil was composed of large chunks of soil mixed with small pieces of grass. It had a dark coloration and was dry.

The leaves at eye level were dead and were decaying in random areas on the leaves. However, as the branches get higher and higher, the leaves looked healthier. Not only were there leaves on the tree, but many big acorns also the tree and would fall once in a while.

Due to the age and location of the tree, it is clear that there was no garden soil added.

Conclusion:

Due to the contamination of the negative control and negative result on the plaque assay, a soil sample was collected from a Burr Oak near iHop. Data such as the diameter, height, average canopy length, soil composition, and leaves were taken. After collecting a sample, a plaque assay was created using the current lysate to make sure there are no plaques. First, lysate and Arthrobacter were combined and left alone for 10 minutes to infect. Then, LB broth (checked for contamination) was added along with the calcium chloride. After the allotted time, the lysate and Arthrobacter were added to the LB broth and calcium chloride. 2X Top Agar was then added to the solution and poured onto the agar plate. For 15 minutes, the plates remained near an aseptic zone to solidify. After, the plates were placed in an incubator.

Future Plans:

If there are plaques in the current lysate, a plaque will be picked, diluted using phage buffer, then used to create a plaque assay. If there are no plaques along with no contamination, the new soil sample will be washed and enriched. If there are no plaques and contamination on the control, a new plaque assay will be made with methods being carefully used to avoid contamination.