Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is a presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.  The new sample would provide a larger data size for the research, so besides the washing and Enrichment, the sample’s metadata is also very important for more insight into our research question.

Soil Washing and Enrichment for Sample (9.17.18):

Material:

• Two 15 ml Conical Tube
• One 50 ml Conical Tube
• LB Broth
• Syringe Filter
• Vortex machine
• Centrifuge
• Micropipette
• Serological pipette

Procedure:

1. Set up an Aseptic zone
2. Placed sample Soil (9.17.18) to the 2 ml mark in the 15ml Conical Tube.
3. Poured LB Broth to the 12 ml mark in the 15 ml Conical Tube (15 ml Conical Tube Soil (9.17.18) weight: 19.23 g)
4. Vortexed for 15 min to mobilize the soil.
5. Centrifuged at 3000 G for 10 min.
6. Filtered the supernatant from the 15 ml Conical Tube Soil 9.5.18 with syringe filter to a new 50 ml conical tube to 10ml mark and placed the rest in a new 15 ml conical tube. ( 50 ml conical tube labeled (9.17.18) Enrich, 15 ml conical tube labeled (9.17.18) Direct)
7. Added 0.5 ml Arthrobacter into the 50 ml conical tube with the filtered supernatant and place on the shaker.
8. Direct Isolation tube was stored in the fridge.

Soil Metadata for (9.17.18):

Soil Composition:

1. Added soil to the 4 ml mark on the falcon tube.
2. Added water to the 12 ml mark.
3. Added 3 drops of Soil Dispersion Liquid
4. Wait for 48 hrs to see the results

% Water:

1. Weigh the empty weigh boat: 2.40 g
2. Weigh the weigh boat with wet soil: 7.40 g
3. Wait for 48 Hrs for Soil to dry and check results

Soil pH:

1. Added a small amount of soil to the pH vial
2. Added water to 80% full
3. Shook the vial for 45 sec and waited for 2 min
4. put in a pH paper(should not exceed 1 inch)
5. Waited for 45 sec and compared the color with the pH scale to determine the pH of the soil
6. The pH level for Soil Sample 9.17.18 is  pH 6.0

Observations, Results & Data:

Plaque Assay for Sample (9.5.18):

• The Control again showed Contamination like the last run, white dots of colonies spreading on the plate.
• The Plaque plate showed negative results.

Interpretations & Conclusions:

Plaque Assay for Sample (9.5.18):

• The LB broth used for both Plaque Assays for Sample (9.5.18) is contaminated, which explains the reason why the control plate was still contaminated after being more cautious during the preparation of top agar solution.
• The LB broth and 2x Top Agar both must be double checked before use in case of a contamination of the bottle causing the inaccuracy of the experiments.
• The tests for Sample (9.5.18) are temporarily suspended to test the new sample (9.17.18) due to the fact that no traces of plaques were identified on both runs.

Next Step:

In the next lab, I will be running Plaque Assay for Sample (9.17,.18)