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 (9.5.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (9.5.18) Lysate
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 20 min for infection.
3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a 50 ml conical tube
4. Add the infected lysate to the 50 ml conical tube
5. Add 2.5 ml of 2x Top Agar to the 50 ml conical tube, pipette up and down then decant the solution to the agar plate
6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Soil Sample (9.17.18):

1. Location: 31.5450 N, -97.1187 W
2.  Species: Burr Oak
3. More information would be taken later this week.

Observations, Results & Data:

Plaque Assay (9.5.18):

• The control plate was contaminated with white dots of colonies.
• The Plaque Assay plate showed negative results.

Spot Test (9.5.18):

• The Stop Test showed some uneven solidification of top agar.
• The plate showed negative results.

Interpretations & Conclusions:

Considering this:

1. Group 4 all had plaques on their plaque assays. Justin had the most and well-defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?

• The possibles reasons for having the most and well-defined plaque on the plaque assay are:

1. His experimental techniques were more in the bacteriophage’s favor.
2. The phages and Arthrobacter were more active in his sample and his enrichment lysate.

• The possible reasons for being the only one having a plaque on the spot test:

1. The amount of sample he put down on the plate.
2. The phage activity on the plate.

1. Lathan checked a purified lysate by a plaque assay using 10 µL of a 10^-3 lysate. He got 14 plaques. How many µL of Lathan’s lysate should he add to web a plate (8 cm in diameter) if his average plaque diameter is 1 mm.

• 10^0 Lysate = 14(pfu) / 10(ul) * 10^3
• Plate(Area) / Plaque(Area) = [40(mm)]^2 / [0.5(mm)]^2
• Final quantity needed:   {[40(mm)]^2 / [0.5(mm)]^2 } / (10^0 Lysate) = 4.6 (ul)

Spot Test:

• The unequal solidification of the top agar might be caused by the mixing of the solution since the top agar was prepared in a 50 ml conical tube for the whole group so it could be possible that a part of the solution had started solidifying when it was decanted to the plate.

Plaque Assay:

• The reason causing the control plate to be contaminated might be the last time when the agar solution was being prepared it was too far away from the aseptic zone, lettiing microbes in the environment to contaminate the plate.

Next Step:

If the results of the Plaque Assay on (9.5.18) is negative, the main focus would be to wash and enrich sample (9.17.18) for Plaque Assay. If it yields positive results then further purification process would be done.