Rationale: To isolate possible bacteriophages through the process of soil washing and enrichment. The soil metadata will also be found and recorded.

Description of Procedures:

1. The work space was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. 8mL of LB broth was added to the 15mL tube containing 2 mL of soil B, for a total of 10 mL. The tube was labeled LIP 9-10-18 Soil B.
3. The tube was then shaken for 10 minutes, and massed. The mass was found to be 18.212 grams.
4. While the tubes were being shaken, the some of the metadata was collected. The procedure to calculate the percent water was started. The weigh boat was found to be 2.36 g, and the weigh boat with the soil was found to be 7.27 g. The weigh boat was labeled LIP 9-10-18 Soil Dry, and was placed under the vented hood where it will sit for 48 hours to allow the water to evaporate.
5. The tube filled with LB broth and soil was put in the centrifuge for 5 minutes and spun at 10,000 x g. It was paired with a tube of similar mass (within 0.05g).
6. After the tube was spun in the centrifuge, the supernatant was filtered using a 2mL syringe and a 0.22 um filter. Approximately 7mL of lysate was collected in a 50 mL tube. There was not enough to create a direct lysate. 0.5mL of arthrobacter was added to the lysate to create the enriched lysate. The tube was labeled LIP 9-10-18 Enriched (Soil B).The tube will be shaken at 28 degrees C until Wednesday (approximately 48 hours).
7. The pH of the soil was then found. A small amount of soil was put into a pH vial and DI water was added to fill the vial. The vial was then shaken for 10 seconds and allowed to sit for 10 minutes. The pH paper was then put into the vial for 45 seconds and the paper was then compared to the pH color scale. The pH was found to be 6.0.
8. The table was then cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Data/Results:

• Observations:
  • The filtering process was difficult and took a long time.
  • There were no more large tubes for determining percent silt, sand, and clay, so this procedure will be completed Wednesday for more metadata.
  • The cap of the pH vial had a small amount of soil stuck to the top, which could affect the results of the pH test.
• Results:
• pH: 6.0
• 7.5 mL of enriched lysate was made (0.5 of this is arthrobacter)
• The weigh boat mass: 2.36 g
• Weigh boat with soil mass: 7.27 g

Interpretations/Conclusions/Next steps:

The procedure for enrichement was completed, but there was not enough to create a direct lysate. The soil metadata was not complete and will be completed Wednesday. The percent water will also be finished Wednesday after the water evaporates for 48 hours. The next step will be to finish metadata and perform a spot test and a plaque assay with the enriched lysate.

Rationale:

The purpose of this lab was to collect a soil sample and determine if there are arthrobacter phages present. The results of the plaque assay were negative, and the top agar control was contaminated. Due to this, new soil needs to be collected. The procedures done before will be repeated on Soil B to test for the presence of arthrobacter bacteriophages. The group question for groups 7 and 8 was to determine if the presence of phage differed between the soils around Red and White Oak trees.

Description of Procedure:

1. Group 8 (my group) was assigned to collect soil from the base of a Red Oak tree, and Group 7 was assigned to collect soil from a White Oak.
2. Soil was obtained from around a Red Oak near the Baylor Law School and McLane Stadium.
3. The survey was completed. The circumference of the tree, canopy width, and height of the tree were recorded for meta data. It was noted that the tree had no visible damage. The tree also had a large population of fire ants living around it. Leaves were also collected.
4. The soil sample was put into a bag, and 2 ml were put into a 15 ml tube. Both the bag and the tube were labeled LIP 9-5-18 Soil B.
5. The soil was taken back to the lab and will be stored in the refrigerator until Monday, 9-10-18 to preserve it in its current condition.

Observations/Data/Results:

• Observations:
  • The red oak tree had good, moist soil.
  • There were fire ants on and around the tree
  • The tree was close to McLane Stadium and the Baylor Law School.
• Data:
  • Tree height: 12 m
  • Tree Circumference: 96 cm
  • More data submitted through survey, as well as pictures.

Interpretations/Next Steps/Conclusions:
The collection of Soil B was complete. It will be stored until Monday, 9-10-18 in the refrigeration. The next step will be to wash the soil. We will also be adding 0.5 mL of Arthro, and incubating for 48 hours at 28 degrees Celsius. The collection procedure went more smoothly the second time than the first time.

Rationale: To perform a spot test to determine if there are any arthrobacter bacteriophages in the enriched lysate sample.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Procedure:

1. The first step in this procedure was to clean the lab station. This was done with CiDecon and 70% Ethanol. An aseptic zone was created with a 100% Ethanol burner being lit.
2. First the enriched lyste was filtered using a sterile 0.22 uL filter to filter the added arthrobacter out of the sample. 2 mL were removed from the enriched lysate, and 1.5 mL was filtered into a 1.5 mL micro centrifuge tube, using a sterile syringe. The tube was labeled EF, which stands for filtered sterilized enriched lysate.
3. 4 petri dishes were then obtained, one for each person in Group 4 and one for the Top Agar control. My individual plate was labeled LIP 8-27-18 SP1 (Spot Test 1), and three sections were drawn onto the bottom of the plate, one for the enriched, direct, and negative control spot tests. The Control plate was labeled Group 4 8-27-18 Control TA.
4. Next, the top agar was made in a 50 mL tube. The tube was labeled LIP 8-27-18 TA1 (Top Agar 1). The below calculation was done to determine the amount of CaCl2 needed.
5. 
6. The below calculation was done to determine the amount of CaCl2 needed for the control plate.
7. 
8. 4.5 mL of LB Broth was added first in an aseptic zone with a sterile pipette, to both the experimental and control tubes.
9. Next, 0.5 mL of arthrobacter was added to the experimental tube, but not the control tube.
10. 45 uL of CaCl2 were added to the experimental tube, while 42.75 uL of CaCl2 were added to the control tube.
11. Lastly, the 2x TA was added to both the control and experimental top agar solutions and gently mixed with the micro pipette.
12. The Top Agar solutions were then poured onto the correct labeled petri dishes.
13. The plates were then allowed to sit for 10 minutes until the Top Agar solidified.
14. After the plates set, 10 uL of the enriched and direct lysates, as well as the phage buffer for the negative control, were spotted into their correct areas on the petri dishes. Nothing was added to the control TA.
15. The liquids were then allowed to absorb into the agar for 15 minutes.
16. The plates were then incubated until Wednesday. This process was complete at 4:35.
17. The  lab station was cleaned before leaving. This was done with CiDecon and 70% Ethanol. All used materials were properly disposed of.

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Rationale: To visually confirm or deny the presence of plaques in the soil, after negative results on a spot test.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Procedure:

1. The workstation was cleaned with CiDecon and 70% Ethanol before the procedure was started. A 100% Ethanol burner was lit to form an aseptic zone.
2. To perform a plaque assay with the enriched lysate, the first step was to obtain 0.5 mL of arthrobacter. To this 10 uL of enriched lysate was added. This tube was labeled LIP 8-29-18 AEPA (Arthro Enriched Plaque Assay). This was allowed to sit for 15 minutes.
3. While the arthrobacter and enriched lysate were allowed to sit, The Top Agar solution was created. 8.0 mL of LB Broth was added to a 50 mL tube. The Top Agar for Group 4 was made in one tube, as well as the control top agar. This tube was labeled Group 4 8-29-18 TA Plaque Assay.
4. Next 90 uL of CaCl2 was added to the LB Broth. Because the solution contained 5 mL total, the amount of CaCl2 for each person was 22.5 mL, but since this TA was being used for three group members and a control plate, this number was multiplied by 4, to get 90 uL total.
5. 10 mL of 2X TA was then added to the LB Broth and CaCl2.
6. After, 4.5 mL of the TA solution was added to the tube containing arthrobacter and enriched lysate for each member of Group 4. It was mixed and then immediately poured on to a petri dish for each group member. My plate was labeled LIP 8-29-18 PA1 (Plaque Assay 1).
7. For the control TA plate, 1 mL of the TA solution was added to a petri dish. This control plate was shared by groups 3, 4, 7, and 8, so it was divided into 4 sections. Group 4’s 1 mL was put only into the quadrant labeled Group 4.
8. The plate was then allowed to sit for 10 minutes to solidify and was then stored in an incubator inverted until Friday, 8-31-18, at 2:00 pm, approximately 46 hours from the time it was finished. The time the procedure was complete was 3:40 pm.
9. The workstation was the cleaned with CiDecon and 70% Ethanol. All used materials were properly disposed of.

Observations/Results/Data:

Observations:

• The top agar solidified much faster with the plaque assay than with the spot test.
• Due to limited plates, 4 control top agar solutions were done on one plate for groups 3, 4, 7, and 8.
• There were some bubbles on the set plate when the procedure was completed.

Results: The procedure was completed on time and the plates will be stored in an incubator inverted until Friday 8-31-18.

Interpretations/Next Steps/Conclusions:
The procedure was complete on time. It went smoothly and was done correctly. The plate will now be allowed to grow until 8-31-18 at 2:00 pm. If there are plaques, then they will be picked. If there are no plaques, a new soil sample will need to be found.

Rationale: To create a direct lysate sample for the experiment from the soil, without adding arthrobacter.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Procedure:

1. The sample of supernatant that had not yet been filtered was spun in a centrifuge for 5 minutes at approximately 4000 x g.
2. This sample was then put into a 0.22 um filter using a pipette and filtered. Approximately 7.35 mL of lysate was obtained from filtering the supernatant.
3. This lysate was then transferred into a 15 mL tube, not in an aseptic zone. This sample will be refrigerated until Monday and will eventually be plated.
4. The used materials were then properly disposed of and the work station was cleaned.

Observations/Results/Data:

Observations:

• After being spun in the centrifuge a second time, the filtering process was much quicker.
• The supernatant was cleared than it had been before the second spin.
• The transferring of the direct lysate to a smaller container was not done in an aseptic zone.
• Approximately 7.35 mL was obtained of direct lysate.

Results:

The procedure from Wednesday was completed, with the direct lysate sample having now been obtained. This sample is approximately 7.35 mL.

The sample will be stored in the refrigerator.

The tube was labeled LIP 8-24-18 Direct Lysate

Interpretations/Conclusions/Next Steps:

The procedure was now complete. The second spin in the centrifuge made it possible to quickly filter the rest of the supernatant and obtain the direct sample. The next step is to shake the enriched lysate until Monday. On Monday, the next step will be a spot test to determine if any arthrobacter bacteriophages have been found.

Rationale: To isolate the possible bacteriophages from the soil through the process of soil washing, and to collect lysate for enriched and direct samples.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Experimental Procedures:

1. In this procedure, aseptic technique was used to avoid contamination as much as possible. CiDecon and 70% Ethanol were used to clean the work space. A 100% Ethanol burner was used to form an aseptic zone.
2. The soil sample that was collected from the oak tree in Zone 4 was mixed with LB broth in a 50 mL tube. The tube contained approximately 13 mL of soil and was filled to the 35 mL mark with LB broth.
3. After adding the LB broth, the mass of the sample and the tube was found to be 53.332 grams. The tube was then shaken for 15 minutes to mix the mixture.
4. The class tubes were then paired with a tube that had a mass within +/- 0.1 g of the mass of their tube.
5. The tubes were then put into the centrifuge and spun at 3000 x g for 5 minutes.
6. After this process was complete, the supernatant was transferred into a 0.22 um filter using a pipette and filtered with a vacuum pump. Approximately 12.5 mL of lysate were obtained after filtering.
7. 0.5 mL of arthrobacter was then added to the lysate to create the enriched isolation, which will be shaken at room temperature until Monday, August 27. The rest of the supernatant was too thick and needed to be transferred to a 15 mL tube and put through the centrifuge again. This will be done on Friday, August 24.
8. The work station was then cleaned and all materials used were disposed of properly.

Observations/Results/Data: 

Observations:

• moist soil
• when shaken, bubbles formed
• soil mostly mixed with LB broth
• Created a milky color when mixed
• After spun in centrifuge:
  • Yellow supernatant on top
  • Pallet on bottom, brown color
  • bubbles are gone
  • some soil and sticks floating at the top
  • layered colors of soil at bottom.
• Filtering was slow due to items in the soil, causing the supernatant to require a second spin in the centrifuge.

Results:

The enriched sample was created. Approximately 12.5 mL were obtained of lysate. With the 0.5 mL of arthrobacter that was added to the sample, the enriched sample is 13.0 mL. The procedure to create both the enriched and direct lysate was not finished, but will be finished on Friday.

The sample will be stored in the laboratory until Monday at room temperature.

It is labeled: LIP 8-22-18 Enriched

Interpretations/Conclusions/Next Steps:

This procedure was not complete due to the sample not filtering completely.  In the future, trying to make sure there are less sticks and other material in the soil will allow this process to go more smoothly. The supernatant will need to be spun in the centrifuge again before collecting the direct lysate sample. The next step is to re-spin the supernatant and filter to create the direct lysate, which will be stored in the refrigerator.