Results:

The plaque assay ran on Friday (9/14) was contaminated. However, there was no liquid in the plate like the spot test ran on Wednesday (9/12). Both the “KEA PA 9/14/18 enrich lysate” and the “KEA PA 9/14/18 control” plates looked similar to each other. The plates appear to look similar to each other and have a weird texture might be because the Top Agar started to solidify before the mixture was placed into the plates. The pictures below show these plates.

Rationale:

In today’s lab, a plaque assay will be run with the same enriched lysate from soil B. From this plaque assay, the researcher hopes to receive a plate without contamination and be able to determine whether or not there are bacteriophages that specifically target Arthrobacter in the collected soil sample.

Procedure:

1. Started off by cleaning the counter with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Next, 10 μL of enriched lysate from the soil sample B was added into a test tube which already had 0.5 mL of Arthrobacter.
3. Then, the test tube was set aside for 15 minutes.
4. The original recipe was quadrupled as shown below.

Calculations

*This calculation was used later since the amount of mixture in the control plate ended up with an excess amount.*

5. Through the used a serological pipette to transfer 8 mL of LB Broth into a 50 mL conical vial.
   • This 50 mL conical vial was labeled “SA LEF KEA 9/17/18 TA control.”

6. Then, 90 μL CaCl₂ was transferredinto “SA LEF KEA 9/17/18 TA control” conical vial using a P200 micropipette.
7. Obtained plates and labeled them “KEA 9/17/18 PA” and “SA LEF KEA 9/17/18 control.”
8. Next, 10 mL of 2X TA was added and mixed into “SA LEF KEA 9/17/18 TA control” conical vial using a serological pipette.
9. Then, 9.5 mL of the mixture was placed into the “SA LEF KEA TA control” plate.
10. This left only enough for two more plates. The Arthrobacter with enriched lysate was poured and mixed into 5.0 mL of the TA mixture twice. Then, poured this into the corresponding plates.
11. The steps 5-8 were performed the same way except with double the original recipe, and 5 mL were poured into both the control and the enriched lysate plate.
12. Allowed 15 minutes for the plate to solidify.
13. The plates were inverted and placed in the incubator at 48ºC for the next 48 hours.
14. Cleaned lab counter with CiDecan and EtOH (70%).

Observations and Interpretations:

• On the plate assay from Friday (9/14), most of the air bubbles which were created when the mixture started to solidify had disappeared.

Things to consider…

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

Justin’s group performed the procedures correctly. The plaques they had were extremely spread out. Since a plaque assay covers a whole plate, there is a better chance of receiving a plaque when compare to a smaller area where the plaque is dropped.

2. Lathan checked a purified lysate by doing a plaque assay of 10^-3 He counted 14 plaques. How many μL of Lathan’s 10⁰lysate should he add to web a plate (75 mm in diameter) if his average plaque diameter is 1 mm?

Data:

• Serial Dilution: 10^-3
• Number of Plaques Found on Plate: 14
• Plate Diameter: 75 mm
• Average Plaque Diameter: 1 mm

Calculations:

            Lathan should add 40.2 μL of 10⁰lysate to web the plate.

Conclusions:

After participating in the lab for about a month, the researcher has gained more respect for individuals who work in labs such as for the Environmental Protection Agency (EPA) and Food Safety and Inspection Service (FSIS). Research can be frustrating since one might perform the same tasks a multitude of times and still have negative or contaminated results.

Next Steps:

On Wednesday, if the plaque assay is contaminated, the experiment will proceed by running another plaque assay. If the plaque assay is negative, the experiment will start all over with a new soil sample. If the plaque assay is positive, the experiment will proceed to perform purification processes.