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.

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 23 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

For 9/21 Plaque Assay for sample (9.17.18) the control plate was contaminated, showing large areas of bacterial colonies, which made the results of the plaque assay invalid.  Furthermore, the plaque assay plate showed negative results with no plaques present on the plate.

On the side note, the LB Broth used on 9/21 also showed contamination, above is the comparison of an uncontaminated LB broth and the contaminated LB broth used on 9/21.

Interpretations & Conclusions:

There have been consecutive occurrences of contamination in the LB broth that group 5 uses. Although the ultimate reason for all contamination is unknown and all cases could be isolated incidents, the potential reason could be that the duration of pipetting fluid out of bottles or tubes were too long, which causes the particles in the air to have a significantly greater chance of infecting the broth.

Next Step:

If the Plaque Assay 2 for sample (9.17.18) result is negative, new soil samples or another plaque assay re-run would be the next step.