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.  Since the last plaque assay yielded contamination on the control plate, plaque assay rerun would provide a more accurate result. Also, more samples were collected to expand the data size and provide more insight into whether the presence of Arthrobacter phages in the soil around red or white oaks has a correlation with the health condition of oak trees.

Plaque Assay for Soil (9.17.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (9.17.18) Lysate
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Added 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 10 min for infection.
3. Added 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) for the top agar solution.
4. Added the infected lysate to the solution
5. Added 2.5 ml of 2x Top Agar to the solution, pipette up and down then decant the solution to the agar plate
6. Waited for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

Sample (10.1.18):

•  White Oak
• Diameter: 277 cm
• Tree Height: 21.2 m
• Canopy: 985.52 cm

Plaque Assay 3(9.17.18):

The plaque assay control showed contamination on the right on the plate in the picture above, and the plaque assay yielded negative results. Further note, the LB broth used in the assay run showed signs of contamination.

Interpretations & Conclusions:

The soil collected for sample (10.1.18) showed a lot of clay-like nature. The tree is a lot greater in size comparing to previous samples,  and the soil has deviated from previous sample’s sandy nature to clay-like, so the sample’s results may be interesting.

The plaque assay being again contaminated is a concerning issue, since after multiple adjustments to no avail, the reason may be due to poor lab techniques or more fundamental reasons.

Next Step:

If the results of the Plaque Assay on (9.17.18) is negative, the main focus would be to wash and enrich sample (10.1.18) for Plaque Assay. If it yields positive results then further purification process would be done.