Objective:

• Plaque assay will be conducted to test for the presence of Arthorbacter phage in soil sample “Soil B” and help to answer the experimental question.

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. Enriched lysate that was collected in the previous lab (9/10) was gathered and centrifuged @ 3000 g for 5 minutes. This was done to pellet the Arthrobacter that had multiplied from being in contact with the LB broth for 48 hours.
3. Then a syringe and filter were used to filter 1 mL of the enriched lysate into a microcentrifuge tube.
4. 10 microliters of the filtered enriched lysate was added to 0.5 mL of Arthrobacter in a test tube. The mixture was left for 15 minutes while the Overlay Agar was made. This was done to ensure the possible phage were able to interact with the Arthrobacter before being added to the Overlay tube.
5. To make the Overlay Agar, a 50 mL tube was obtained. The agar was created for 3 soil samples in addition to a control sample. This was done to ensure the same base of Overlay Agar was used and no contamination occurred. 8 mL of LB broth, 90 microliters of CaCl2, and 10 mL 2x Top Agar were added to the tube.
6. After swirling the Overlay Agar in the 50 mL tube, 5 mL of the mixture was piped into a new 50 mL tube that would then receive the Arthrobacter and lysate mixture that was created earlier. The mixture was then poured onto a petri dish label “Soil B Plaque Assay” and allowed to solidify for 15 minutes.
7. Next, 5 mL of the Overlay Agar stock was plated and labeled “Control Group” to ensure their was not any contamination for Group 1. The plate was left to solidify for 15 minutes.
8. Both plates were incubated and left to be checked during next lab day (9/17)

Metadata=

1. The “wet soil” plate that was made during the previous lab (9/10) was collected and massed. It was found to be 10.15 g and labeled as “dry soil”. The difference in mass between the “wet soil” and “dry soil” was found to be 1.07 g. After calculations, it was found that soil sample Soil B was 26.6% water.
   1. Calculations:
      1. wet soil= 4.03 g      dry soil = 2.96 g
      2. 4.03 g – 2.96 g = 1.07 g
      3. (1.07 g / 4.03 g) x 100 = 26.6% water
2. In order to know the ratio between sand, silt, and clay in the Soil B sample, 10 mL of the soil was put in a 50 mL falcon tube. The tube was filled to the top with water and 3 drops of Soil Dispersion Liquid. The tube was hand-shaken for 2 minutes then left to sit for 48 hours to separate.

Results:

• It reviewing the metadata experiments that were conducted on 9/10, it was found that Soil B was 26.6% water.
• The plaque assay was not completed, so there are no results. Results will be recorded during next lab (9/17)

Next Steps:

• During the next lab, the plaque assays will be examined for clearings in the Arthrobacter lawn. If there are clearings, then there is the presence of Arthrobacter phage in Soil B and the phage will be picked for further testing. If no clearings are found, then a spot test will be conducted to confirm negative results. Results of the plaque assay/spot test will help to answer the experimental question.