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 Metadata for (10.1.18):

Soil Composition:

1. Remove the supernatant in the falcon tube
2. Record the results

% Water:

1. Weigh the empty weigh boat
2. Weigh the weigh boat with wet soil
3. Wait for Soil to dry and check results

Plaque Assay for Soil (10.1.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (10.1.18) Lysate
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Spin down 1.2 ml Enriched lysate (3000g for 10 min) and filter with syringe filter(.22um).
3. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 10 min for infection.
4. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a tube
5. Add the infected lysate to the tube
6. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to the agar plate
7. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

Soil Metadata results for Sample (10.1.18):

1. Soil Composition:  2 ml Clay,  2 ml Silt,  1.5 ml Sand
2. Soil pH = 6.0

Interpretations & Conclusions:

Soil Metadata results for Sample (10.1.18):

Soil Composition: 36% Clay, 36% Silt, 28% Sand

Soil pH = 6.0

The soil belongs to clay on the soil category chart. Clay soil retains water very well, however, it also tends to deprive roots of oxygen so even though moist soil is an ideal environment for arthrobacter, the small particles in soil may hinder the cell respiration process of arthrobacters since they may have difficulty accessing oxygen.

Next Step:

If the Plaque Assay for Sample (10.1.18) shows positive results further purification process would be done, otherwise, new samples, multiwell enrichment or Plaque Assay re-run are all possible choices.