Rationale:

Due to the limited amount of agar plates, a plaque assay for the enriched will be conducted rather than both enriched and direct.

Scientific Question:

Does the presence of Arthrobacterphage appear more dominant in one oak tree species than the others? If so, in this species is there a correlation between the presence of Arthrobacterphage and the presence of oak wilt fungus growth?

Procedure:

• To prevent contamination, wipe the table with CiDecon and ethanol and also set up an aseptic zone.
• Remove the spot test plate from the incubator to check for plaques.

Plaque

• Use a pipette to add the enriched lysate to 0.5 mL of Arthrobacterphage, label it Culture 1, and allow it to sit for 15 minutes.
• Add 8mL of LB Broth to a 50 mL conical vial.
• Add 90 uL of CaCl2 to the 50 mL conical vial using 10uL – 100 uL pipette.
• When the 15 minutes for “Culture 1” is almost over, add 10 mL 2X Top Agar to the solution of LB Broth and CaCl2 in the 50 mL vial.
• Shake the vial several times to get an even mixture of the solution.
• Using a pipette, add 4.5 mL of the top agar into a new vial.
• 1 mL of the top agar was added to the top agar control plate (shared by four tables.)
• Pour “Culture 1” onto the agar plate and label it “PA 1 (enriched).”

Plaque Assay 1

Results and Analysis:

• The materials need to create the top agar was multiplied by four.
• It was a little difficult to get an exact amount of LB Broth and Top Agar due to the difficulty of seeing the marks.

Conclusion and Future Plans:

• Due to the presence of plaque in my spot test which indicated the presence of phage, the plaque assay provided a way to further prove that there are phages within soil sample 1.
• In the future (9/5/18), I will complete the plaque assay for the direct isolation lysate if there are enough agar plates.

Rationale:

Conduct a spot test to see whether or not there are phages in either Direct Isolation or Enriched.

Procedure:

• To prevent contamination, wipe table with CiDecon and ethanol and set up an aseptic zone using an ethanol lamp.
• Take enriched lysate out of the refrigerator and bring it to the table.
• Prepare a microcentrifuge tube and 0.22 um filter as they will be in use later on in the procedure.
• In the aseptic zone, use a syringe and remove 0.2 mL from the tube and place it in the microcentrifuge tube. Label it “Enrich 2.”
• Take an agar plate and label it into three sections: Enrich, Direct Isolation, and Negative Control.
• To make the top agar, calculate how much Calcium Chloride is needed from one mole using the C1V1=C2V2 formula to get a concentration of 4.5 mM.
• Combine LB Broth, 42.75 uL of Calcium Chloride, and LB 2X Top Agar (last) to have a total volume of 9.5 mL.
• Shake the tube and pour over the negative control agar plate.
• Calculate the amount of Calcium Chloride needed to conduct the spot test.
• Combine LB Broth, 45 uL of CaCl2, 0.5 Arthrobacter, and at last, add LB 2X Top Agar.
• Pour the top agar into the agar plate for the spot test.
• Allow the top agar to solidify.
• Add 0.1 mL of phage buffer to the negative control using a pipette.
• Prepare a 0.22 um filter, a microcentrifuge tube, and a syringe to filter the direct isolation.
• Add 5 mL of Enrich and Direct Isolation into their designated areas in the agar plate.
• Allow the plate to sit to avoid mixing of the spots.

Spot Test

• After 10 minutes, place both plates in the incubator.
• Clean the table with CiDecon and ethanol and place all equipment in their proper places.

Results and Analysis

• For the spot test, we used C1V1=C2V2 to find the concentration needed for a 10 mL solution out of 1M of CaCl2.

C1V1=C2V2

(1 M)(V1)=(4.5 mM)(10 mL)

(1000 mM)(V1)=(4.5 mM)(10000 uL)

V1= ((4.5 mM)(10000 uL)) / (1000 mM)

V1= (45000 mM x uL) / 1000 mM

V1= 45 uL

• For the top agar control, we used the same formula to find the concentration needed for a 9.5 mL solution.

C1V1=C2V2

(1 M)(V1)=(4.5 mM)(9.5 mL)

(1000 mM)(V1)=(4.5 mM)(9500 uL)

V1= ((4.5 mM)(9500 uL)) / (1000 mM)

V1= (42750 mM x uL) / 1000 mM

V1= 42.75 uL

• Between the time of cleaning the table and starting the experiment, my elbows were on the table and instruments were also placed on the table.
• The narrow end of the filter was touching the table and a little more than 0.2 mL from the tube was put into the microcentrifuge tube.
• After I added the enriched to the plate, two bubbles appeared in the middle but was moved to the side.

Conclusion and Future Plans

• Using both lysates (Direct and Enriched), a spot test was conducted to see if phages are present within the sample. Two separate sets of Top Agar were made: one was without the arthrobacter to serve as the Top Agar control which show if the top agar was contaminated and the other was for the spot test. The experiment was completed with few mistakes; none that cause drastic changes in the results.
• In the future (8/29/18), I plan on conducting the plague assay experiment to further prove the existence of phages in my sample.

Rationale:

Filter the Direct Isolation lysate

Procedure:

• Take the Direct Isolation out of the refrigerator and get a new vial.
• Fill the new vial with water with the same mass as the Direct Isolation.
• Place the tubes directly across from each other in the centrifuge and allow it to spin from 5-10 minutes.
• Take the Direct Isolation out of the centrifuge and place it in the test tube rack in the refrigerator.

Results and Analysis:

• Upon receiving my direct isolation, I noticed that the sample was already separated. Some minutes later, without me shaking the tube, the sample became evenly mixed again.

Conclusion

• I did not fulfill the purpose of this lab which was to filter the direct isolation. However, I did complete half of the procedure which was to put it in the centrifuge to separate the phage from the dirt and bacteria.

Future Plans

• In the future (8/28/28), I plan to finish filtering my Direct Isolation lysate. I also plan to conduct a spot test in which the filtered Direct Isolate will come in play.

Rationale:

Separate the phages and bacteria from an environmental sample with the purpose of obtaining phages for experimentation

Procedure:

• To prevent contamination, wipe the tables with CiDecon and ethanol (70%) and set up an aseptic zone by using an ethanol lamp
• Near the lamp, pour the LB Broth up to the 35 mL mark in the 50 mL conical vial containing the soil sample and then close the sample as quickly as possible
• Shake the sample vigorously for 15 minutes.
• After shaking for 15 minutes or more, place the sample in a centrifuge for five minutes
• Using the tube top vacuum filter under the vacuum hood, filter the supernatant. Use a bulb pipette to transfer the supernatant to the filter.
• Filter supernatant until reaching 20 mL of lysate.
• After reaching 20 mL, carefully unscrew the filter from the vial filled with lysate and quickly screw on the lid to prevent contamination
• Separate the lysate evenly; half of the sample will stay in the vial while the other half will go into a new one (15 mL conical vial).
• The lysate in the new vial, labeled “Direct Isolation”, will go into the fridge to be filtered later.
• Combine the other half of the lysate with 0.5 mL of Arthrobacter (the host) near the lamp and label it “Enriched 1.”
• Close the lid and place the lysate in a test tube rack to be placed in the refrigerator.
• Place all equipment back to their proper place and wipe the table with CiDecon and ethanol (70%).

Results and Analysis:

• Rather than placing the sample in the bag, I placed it in the vial and sealed it shut. Over the course of two days, the vial remained in my room with cold temperatures (68 F). During this time, soil particles and perspiration lined the walls.
• Due to the large spaces between the clusters of soil, I tapped the vial against the table to fill those spaces. In doing so, my soil sample went from 17 mL to 10 mL; 2 mL less than the minimum requirement. Due to this problem, I did not produce as much lysate as I should have to create my Direct Isolation and Enrichment.
• The rate at which the supernatant flowed through the filter was incredibly slow and I lightly tapped the vacuum filter against the table.
• After pouring the Arthrobacter in the lysate, I noticed that there was still some left in the vial.
• The soil sample combined with the LB Broth reached a mass of 51.4 g.
• The Direct Isolation had a mass of 13.8 g.

Conclusion and Future Plans:

• The procedure was carried with little mistakes but none that can cause much contamination. Despite having less soil than what was required, I have enough to carry on for further experimentation.
• In the future (Friday- 8/24/18), I will filter the Direct Isolation using the tube top vacuum filter.