Previous Results:

• The results of the metadata of soil composition were collected. The layers of the soil were measured by recording the markings on the tube. The total amount of soil was 16 mL with 59% silt, 37% clay, and 4% sand.
• Plaque assay from last lab was positive and had numerous clearings, too many to count so dilutions will be necessary to know concentrations.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a series of serial dilutions and plate the diluted lysate

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming 10^0 dilution
3. 10 microL of 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (9/19) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were positive and there are phage in soil sample B
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (9/19) the plates with the serial dilutions will be examined for phage. Another series of dilutions will be done to ensure only one strain of phage is being tested. The same steps will be conducted.

Objective:

• Plaque assay will be conducted to test for the presence of Arthorbacter phage in soil sample “Soil B” and help to answer the experimental question.

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. Enriched lysate that was collected in the previous lab (9/10) was gathered and centrifuged @ 3000 g for 5 minutes. This was done to pellet the Arthrobacter that had multiplied from being in contact with the LB broth for 48 hours.
3. Then a syringe and filter were used to filter 1 mL of the enriched lysate into a microcentrifuge tube.
4. 10 microliters of the filtered enriched lysate was added to 0.5 mL of Arthrobacter in a test tube. The mixture was left for 15 minutes while the Overlay Agar was made. This was done to ensure the possible phage were able to interact with the Arthrobacter before being added to the Overlay tube.
5. To make the Overlay Agar, a 50 mL tube was obtained. The agar was created for 3 soil samples in addition to a control sample. This was done to ensure the same base of Overlay Agar was used and no contamination occurred. 8 mL of LB broth, 90 microliters of CaCl2, and 10 mL 2x Top Agar were added to the tube.
6. After swirling the Overlay Agar in the 50 mL tube, 5 mL of the mixture was piped into a new 50 mL tube that would then receive the Arthrobacter and lysate mixture that was created earlier. The mixture was then poured onto a petri dish label “Soil B Plaque Assay” and allowed to solidify for 15 minutes.
7. Next, 5 mL of the Overlay Agar stock was plated and labeled “Control Group” to ensure their was not any contamination for Group 1. The plate was left to solidify for 15 minutes.
8. Both plates were incubated and left to be checked during next lab day (9/17)

Metadata=

1. The “wet soil” plate that was made during the previous lab (9/10) was collected and massed. It was found to be 10.15 g and labeled as “dry soil”. The difference in mass between the “wet soil” and “dry soil” was found to be 1.07 g. After calculations, it was found that soil sample Soil B was 26.6% water.
   1. Calculations:
      1. wet soil= 4.03 g      dry soil = 2.96 g
      2. 4.03 g – 2.96 g = 1.07 g
      3. (1.07 g / 4.03 g) x 100 = 26.6% water
2. In order to know the ratio between sand, silt, and clay in the Soil B sample, 10 mL of the soil was put in a 50 mL falcon tube. The tube was filled to the top with water and 3 drops of Soil Dispersion Liquid. The tube was hand-shaken for 2 minutes then left to sit for 48 hours to separate.

Results:

• It reviewing the metadata experiments that were conducted on 9/10, it was found that Soil B was 26.6% water.
• The plaque assay was not completed, so there are no results. Results will be recorded during next lab (9/17)

Next Steps:

• During the next lab, the plaque assays will be examined for clearings in the Arthrobacter lawn. If there are clearings, then there is the presence of Arthrobacter phage in Soil B and the phage will be picked for further testing. If no clearings are found, then a spot test will be conducted to confirm negative results. Results of the plaque assay/spot test will help to answer the experimental question.

Objective:

• Wash the soil sample (Soil B) to remove unnecessary material, such as dirt, rocks, and bacteria.
• Isolate possible bacteriophage found in the soil, in lysate.
• Collect metadata on soil sample “Soil B” to obtain more information about the environment the soil was collected in and have further data on the preferred environment of Arthrobacter phage.

Procedure:

Washing=

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. 15 mL tube was obtained and filled to 2 mL with Soil B and filled to 12 mL with LB broth.
3. The tube was hand shaken for 15 minutes to mix the soil and broth.
4. The tube was then massed (21.82 g) and centrifuged @ 10,000 g for 5 minutes.
5. After the centrifuge, the liquid found at the top of the soil in the tube was then filtered using a top filter and vacuum. This was to remove the bacteria from the remaining soil. 10 mL of filtrate was obtained from the filter in a 50 mL tube.
6. 0.5 mL of Arthrobacter was added to the lysate (10 mL) in the 50 mL tube. This created the enriched lysate.
7. The 50 mL tube was then placed in the fridge until the next lab (9/12).

Metadata=

1. To find the percent water present in Soil B, a clean petri dish was obtain and massed. It was measured to be 7.19 g.
2. Then 4.03 g of Soil B was placed on the plate and left to dry out for 48 hours. Together, the soil and plate had a mass of 11.22 g. This total was recorded as the “wet soil” data.
3. The pH of the soil was also measured. Using a small vial, small amount of soil, and DI water, the mixture was shaken for 30 seconds. The vial was left to sit for 2 minutes to settle.
4. After settling, pH testing paper was placed in the vial for 5 seconds and the pH paper strip was compared to a pH key. It was determined that the pH of Soil B was 6.5.

Results:

• Since Soil B was a new sample being tested, there are no results to report from previous testing. Results from the “wet soil” metadata will be available in next lab (9/12).

Next Steps:

• In the next lab, a plaque assay will be completed using the enriched lysate that was made today. Additional metadata will also be collected, including the amounts of sand, silt, and clay present in the soil sample.

Objective:

• Collect a soil sample from a White Oak on campus to answer the question asked on Wednesday (9/5)

Procedure:

1. A plastic bag, a 15 mL tube, tape measure, and shovel were obtained to collect soil.
2. After walking around campus, a Bur Oak (White Oak) tree was chosen to be the site of soil collection.
3. The circumference of the tree was measured to be 73.5 cm, the circumference of the tree canopy was found to be 957.6 cm, and the height of the tree was about 959.6 cm. The circumference of the tree was calculated after finding the radius of the canopy (152.4 cm) and using the equation for the circumference of a circle. The height of the tree was found after taking a picture of the tree with the tape measure (152.4 cm) held vertically and comparing it to the height of the tree in the picture.
   1. Canopy Calculation:

      C = 2πr

      C = 2π(152.4 cm)

      C = 957.6 cm

4. Once data collection was complete and pictures were taken, a leaf was picked from the tree and stored in the plastic bag.
5. The soil sample was collected about 60 cm from the trunk of the tree using the shovel. The 15 mL tube was filled with 8 mL of dirt.
6. Both the plastic bag and tube were labeled “Soil B” and stored in the fridge until (9/10)

Observations:

• While collecting the soil is was noted that the soil was very moist, almost clay consistency, from the rain that was received the night before.
• The ground around the tree was mostly soil with very little grass or weeds.
• The tree was in a shady spot among other trees of similar age and size.
• Visually the tree was healthy. No roots were exposed on the ground, no markings on the bark, and the leaves were green (a few brown ones).

Next Steps:

• During the next lab the soil sample “Soil B” will be washed to obtain enriched and direct lysate. The lysates will then be tested for Arthrobacter phages using spot tests and plaque assays. Once the bacteria and lysates are plated, results will occur and there may be answers for the question being researched.

Objective:

• Create a testable question as a class that will be relevant for the entire semester.

Procedure:

• As a class we discussed the many different perspectives/ directions we could take to test the presence of Arthrobacter phage in the soil of Oak trees around campus.
• It was decided that tables will split up and test their own questions, 4 different questions
• Groups 1 and 2 formed a testable question

Question:

• Do White Oak trees that have been regulated with pesticides show a difference in the amount of phages than trees who have been isolated from pesticides?

Next Steps:

• Three samples of soil will be collected from 3 different White Oak trees in Cameron Park (9/7)
• Three samples of soil, from 3 different White Oak trees on campus (in an area sprayed with pesticides) will be collected on Friday (9/7)
• Soil samples will be labeled “Soil Sample B” and placed in the fridge until Monday (9/11)

Results from previous lab:

• Plates were collected from Spot Test conducted on 8/27. No spot clearings were found on the plate indicating there is not a presence of bacteriophage in the soil sample. The Control plate was clear, indicating the Top Agar was not contaminated.

Objective:

• Plaque Assay will be conducted to confirm the results of the Spot Test from 8/27. The previous Spot Test was negative, but the Plaque Assay could come out positive since the phage has had more time to replicate. The Plaque Assay will provide more concrete answers.

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. 10 microliters of filtered enriched lysate (obtained on 8/27) was added to a the containing 0.5 mL of Arthrobacter. Tube was set aside to give potential phage and bacteria time to interact.
3. Then a 50 mL conical tube was obtained. 8 mL of LB broth, 90 microliters of CaCl2, and 10 mL of 2x Top Agar were added using pipettes. Mixture was swirled to ensure all components were evenly distributed. This created the Overlay Mixture without bacteria.
4. 1 mL of Overlay without bacteria was added to a plate that was shared with groups 2, 3, and 4. This was the Control plate. It was set aside to harden for 15 minutes.
5. Then, 4.5 mL of Overlay Mixture was piped into tube containing 0.5 mL of Arthro and 10 microliters of CaCl2. Mixture was swirled.
6. Overlay was then plated, let sit for 15 minutes, and incubated. Both the Overlay with bacteria and Overlay without bacteria (control) were incubated. Plates were left to be checked during next lab day (9/5).

Observations:

• Plate from Spot Test (8/27) was a yellow color and had a few air bubbles. This could have been mistaken as phage clearing spots, but after being viewed many times it was considered a negative plate.
• It can be difficult to get precise measurements while pipetting because contamination is a concern and you have to move quickly.
• Overlay Top Agar was the same color as the Spot Test.

Results:

• Spot Test results are negative.

• Results of Plaque Assay will be obtained next time in lab since experiment is not complete.

Conclusion:

• Since the Spot Test results were negative, it is likely that the soil sample Soil A does not contain any bacteriophage. The Plaque Assay will provide further answers as to whether or not the sample contains phage.

Next Steps:

• If the Plaque Assay comes back positive with the presence of phage, then the phage will be picked and processed. If the result is negative, then another soil sample will be obtained from a different tree in a different area of campus.

Objective:

• Conduct a Spot Test using the enriched lysate that was obtained on 8/22 to look for the presence of bacteriophages in the soil sample, Soil A.
• To correctly follow the recipe for Top Agar and plate it without the Agar slipping.

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. The Enriched lysate from 8/22 was obtained from fridge.
3. Using a sterile 3 mL syringe, 2 mL of lysate was taken up. The syringe was then connected to a filter and 1.5 mL of lysate was pushed through into a microcentrifuge tube. Tube was labeled “FEL” = Filtered Enriched Lysate. This was done to remove bacteria in the lysate, and to collect the possible bacteriophages.
4. Then a mixture was created for the Experimental Plate. In a 50 mL tube labeled “Top Agar,” 4.5 mL LB broth, 45 microliters of CaCl2, and 5 mL of Top Agar were added using a pipette. Lathan Lucas added 0.5 mL of Arthrobacteria.
   • Calculation of CaCl2:
     • C1V1 = C2V2
     • C1 = CaCl2 microcentrifuge concentration = 1 M
     • C2 = CaCl2 Predicted final concentration = 4.5 mM
     • V1 = unknown
     • V2 = Predicted volume = 10 mL
     • (1M)(V1) = (4.5 mM)(10 mL)
     • V1 = 45 microliters CaCl2
5. Step 4 was repeated in another 50 mL tube to create the Top Agar for the Control Group, but without the addition of 0.5 mL Arthrobacter.
6. Each 50 mL conical tube with the Top Agars were poured into separate petri dishes. Soultions were swirled until evenly distributed. Let sit for 15 min.
7. Once the Top Agars were hardened, the Experimental Group received 10 microliters of the filtered enriched lysate, 10 microliters of the direct lysate, and 10 microliters of a phage buffer. Each in separate, labeled areas on the plate.
8. Both the Control Group and Experimental Group plates were placed in the incubator.
9. Plates will be examined on Wednesday (8/29) for spot clearings indicating the presence of phage.

Observations:

• While filtering the enriched lysate through the filter and the syringe, resistance was felt, but filter was not popped.
• Both the Control Group and Experimental Group Top Agar had a golden- yellow color while being poured onto plates.
• Top Agar solidified pretty quickly. No slipping occurred.

Results:

• Test is not complete, therefore there are no results to report. Results possible on Wednesday (8/29).

Next Steps:

• On Wednesday (8/29), the spot test plates will be examined for clearings in the lawn of bacteria and the presence of phage. If clearings are found, a plaque assay will be conducted using the enriched lysate. If no clearings are found, 2 plaque assay will be conducted. One using the enriched lysate, and another using the direct lysate.

Objective:

• Wash the soil sample (Soil A) to remove unnecessary material, such as dirt, rocks, and bacteria.
• Isolate possible bacteriophage found in the soil, in lysate.

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. 50 mL tube was obtained that contained the sample of Soil A up to the 12 mL mark. LB broth was then poured into tube up to 35 mL mark.
3. The mixture in the tube was shaken manually for 15 minutes.
4. After mixing, the tube was massed in order to find another tube with a similar mass (+/- 0.1g) that it could be placed with in a centrifuge. The 50 mL tube was found to be 55.215 g.
5. 50 mL tube containing mixture of Soil A and LB broth was centrifuged for 5 minutes.
6. Liquid found at the top of the soil in the tube was then filtered using a top filter and vacuum. This was to remove the bacteria from the remaining soil. 10 mL of filtrate was obtained from the filter in a 50 mL tube.
7. Half (5 mL) of the filtrate was put into a 15 mL conical vial and placed in the fridge. This was the direct lysate. Vial was labeled Direct Soil A.
8. 0.5 mL of Arthrobacter was added to the remaining lysate (5 mL) in the 50 mL tube. This created the enriched lysate. 
9. Enriched lysate was left to be shaken to be shaken until Monday (8/27).

Observations:

• After the soil sample was centrifuged the liquid was still murky. This may have lead to difficulty during the filtering,
• Filtering the lysate was difficult and took longer than other samples. Due to time, only 10 mL was obtained instead of all lysate that was available.

Results:

• No results to report since test has not been completed.

Next Steps:

• In the next lab, spot tests and plaque assays will be completed to see if bacteriophages are present in the soil sample, Soil A.