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. Add 0.5 ml Arthrobacter and 125 ul lysate to a Centrifuge tube 10 min for infection.
3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a tube and repeat to another tube.
4. Add the infected lysate to their respective tubes.
5. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to an agar plate and repeat.
6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

The control plate was found with contamination and about 60% of the sample plate was covered with plaques.

Interpretations & Conclusions:

Due to contamination on the control plate, the current plaque assay would use the same amount of sample, since the effects of the contamination on the phages and formation of plaques are unknown.

Next Step:

The new results would determine if we could flood the plate and collect the phages.