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.  And dilution of picked plaque lysate can help us calculate the titer of the phages, and successfully culture more phages for further analysis.

Plaque Assay for Sample (Gabe):

Materials:

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

Procedure:

1. Set up an Aseptic zone.
2. Filter lysate with syringe filter
3. Add 0.5 ml Arthrobacter and 100 ul 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 and repeat to another tube.
5. Add the infected lysate to their respective tubes.
6. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to an agar plate and repeat.
7. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

The control plate for the plaque assay on sample (Gabe) was contaminated, and the sample plate also displayed same type of contamination, the surface of the agar seemed to be covered by a coat of colonies.

Interpretations & Conclusions:

Due to the contamination on the plates, the sample would have to be re-run for amplification, since the results cannot be trusted.

Next Step:

The results of the new run would show if the lysate is enough to web the plate completely. If not then a new plate would be run to web a plate.