Rationale: The purpose of this lab is to flood a webbed plate to determine the titer.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was created.
2. The webbed plate phage from soil C was flooded with 8 ml of phage buffer.
3. While covering the plate with parafilm, the plate was dropped and cracked. The top agar was transferred to a tube and covered with 7 ml of phage buffer, filling the tube to the 12 ml mark. The tube will be stored at 4 degrees Celsius until the next lab.
4. The workstation was cleaned using aseptic technique and all materials were properly stored and disposed of.

Observations:

• The procedure had to be altered due to a crack in the webbed plate.
• Red bacteria was found to be contaminating the plate.

Interpretations/Next Steps:

The procedure was complete. The next step will be to spin the tube in the centrifuge and filter the supernatant to create a new lysate.

Webbed Plate

Rationale: The purpose of this lab is to pick a plaque and conduct a plaque assay for soil C plaque purification, and to conduct a plaque assay for soil C repeat.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was created.
2. A plaque was picked from the plaque assay 1 and mixed with 100 ul of phage buffer and vortexed.
3. The enriched lysate from soil  C repeat was spun again in the centrifuge for 10 minutes at 5000 x g.
4. Percent soil calculations were conducted. 4 ml of soil were measured. Sand was found to be 25%, silt was found to be 50%, and clay was found to be 25% of the soil.
5. The percent water was then found for soil C repeat. 0.684 g were lost from the original 4.058 g, calculating a percent water of 16.86%.
6. Soil C repeat supernatant was filtered with a 0.22 um syringe filter.
7. 10 ul of soil c repeat lysate was mixed with 0.5 ml of arthrobacter and was allowed to sit for 10 minutes. 10 ul of the phage buffer containing the phage from soil C was also mixed with 0.5 ml of artrobacter and allowed to sit.
8. The top agar solution was made for 5 plates. 10 ml of LB broth, 112.5 ml of CaCl2, and 12.5 ml of 2x TA were added to a tube. 4.5 ml of this solution was then poured into the 0.5 ml of arthrobacter and immediately poured onto plates labeled LIP 10-8-18 PA 2, LIP EAG SS Control 10-8-18, and LIP SS EAG Soil C repeat.
9. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
10. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations/Results:

• Observations:
  • Plaque Assay 1 had plaques
  • Enriched lysate from soil C repeat was foggy, so it was respun and refiltered
  • Control plate had bubbles
  • Metadata for soil c repeat was very different from the original soil c metadata
• Results:
  • % Water- 16.86%
  • % Sand- 25%
  • % Silt- 50%
  • % Clay- 25%

Interpretations/Next Steps:
The procedures were complete for soil C repeat and the plaque from soil C. The next steps will be to complete a plaque assay for soil C unless it is webbed, and will then be flooded, and to check for plaques on soil C repeat.

Rationale: After two using soil C to make three enriched lysates and only finding plaques from one of them, the purpose of this lab is to collect new soil from the same tree as soil C and enrich this soil for future testing.

Description of Procedures:
Soil C was recollected from the same tree. 2 ml of soil and 10 ml of LB broth were added to a tube and shaken for 10 minutes. While it was being shaken, the percent water calculation for the soil was begun. The weigh boat was found to be 2.33 g. With the added soil it was found to be 6.388 g. The weigh boat was then stored under the vent hood and will remain there to allow water to evaporate until the next lab. Next, the test for the composition of the soil was started. 10 ml of soil and 20 ml of DI water were added to a tube with 3 drops of soil dispersion liquid. The tube was shaken for 30 seconds and then placed under the vent hood until the next lab. After shaking the tube, the tube was spun in the centrifuge at 5000 x g for 10 minutes. While spinning, the pH of the soil was found. Soil and DI water were added to a pH vial and allowed to sit for 10 minutes. When complete, the pH was found to be 5.5. After spinning in the centrifuge, the supernatant of the soil collection was filtered with a 0.22 um syringe filter. Approximately 7.5 ml of lysate were obtained. 0.5 ml of arthrobacter were added to this tube for enrichment. The tube was labeled LIP SS EAG Soil C Repeat 10-3-18. The tube will be shaken at room temperature until the next lab. The workstation was cleaned using aseptic technique and the materials were properly stored or disposed of.

Observations/Results:

• Observations:
  • Soil C repeat floated before being shaken.
  • Soil C repeat had a lower pH than the original Soil C.
• Results:
  • pH: 5.5
  • 7.5 ml of lysate obtained

Interpretations/Next Steps:

The procedure was complete. The next step will be to finish the soil metadata for Soil C repeat and to run a plaque assay with Soil C repeat.

Rationale: After a successful plaque assay with plaques and a successful picking of a plaque, the purpose of this lab is to perform a plaque assay for purification of the plaques.

Description of Procedures:

The workstation was cleaned using aseptic technique to begin the lab and an ethanol burner was lit to create an aseptic zone. 10 ul of phage buffer was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes. While sitting, the top agar solution was created for two plates. 4 ml of LB broth and 45 ul of CaCl2 were added to a tube. 5 ml of 2x TA was added to the top agar solution after the 10 minutes were through. 4.5 ml of the top agar solution were added to the arthrobacter and then poured onto a plate labeled LIP 10-3-18 PA-1P. The rest of the top agar solution was poured onto a plate labeled Control 10-3-18 LIP. The plates were allowed to sit for 15 minutes and then inverted and stored in the incubator until the next lab. The workstation was then cleaned using aseptic technique and all materials were properly stored or disposed of.

Observations:

• The plate made with too much phage buffer had two small plaques.
• The control plate had small bubbles in the center.

Interpretations/Next Steps:

The procedure was completed correctly and the lab went smoothly. The next step will to complete a second plaque assay for purification. This will be done approximately two more times.

Rationale: After a successful plaque assay, the purpose of this experiment is to pick a plaque for purification and perform a plaque assay.

Description of Procedures:

The workstation was cleaned using aseptic technique before beginning the lab. An ethanol burner was lit to create an aseptic work zone as well. To begin, a plaque was chosen to be picked on the plate and labeled p1. The plaque was the picked with a pipette tip and swirled into  phage buffer. It was then vortexed to mix the tube thoroughly. 10 ul of phage buffer solution was then mixed into 0.5 ml of arthrobacter and allowed to sit for 10 minutes. While waiting, the top agar for the plaque assay was made for four plates. 8 ml of LB broth, 90 ul of CaCl2, and 10 ml of 2x Ta were put into a tube and pipetted up and down to mix. Immediately after to ensure that the top agar did not solidify, 4.5 ml of the TA solution was added to the arthrobacter and poured onto a plate labeled LIP 10-1-18 Picked Plaque 1- PA. 4.5 ml were also poured onto a control plate labeled SS EAG LIP 10-1-18 Control. The plates were then allowed to sit for 10 minutes before being stored in the incubator until the next lab. While waiting for the plates to solidify, it was realized that the correct amount of phage buffer was not aliquotted for this experiment. The picking process was then repeated with the same plaque and the correct 100 ul of phage buffer. It was labeled LP P1 and stored in the refrigerator until the next lab when a plaque assay will be performed. When finished, the workstation was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results:

• There were many plaques found on the last plaque assay
• Bubbles were observed on the control plate.
• The phage buffer was not aliquotted correctly.

Interpretations/Next Steps:

The procedure was completed incorrectly. The wrong amount of phage buffer was used, so the plate made is very diluted and will most likely have no plaques. The plaque was re-picked and stored in the refrigerator until the next lab. The next step will be to run a new plaque assay with the picked plaque.

Plaques found

Rationale: After finding contaminated results in the previous lab, the purpose of this lab is to run another plaque assay.

Description of Procedures:

1. The work station was cleaned using aseptic technique and an ethanol burner was lit.
2. A pellet was seen in the bottom of the enriched lysate, so the lysate was spun again in the centrifuge for 5 minutes at 10,000 x g.
3. While spinning, enough top agar solution was made for four plates. 90 ul of CaCl2 and 8.4 ml of LB Broth were added to a tube.
4. The lysate was then filtered using a 0.22 ul syringe filter.
5. Next, 10 ul of lysate was added to 0.4 ml of arthrobacter and was allowed to sit for 10 minutes.
6. 2x TA was then added to the top agar solution and pipetted up and down to mix. 4.5 ml of the solution was poured into the arthrobacter and then poured directly onto a plate labeled LIP 9-26-18 Plaque Assay. 4.5 ml of the solution was poured directly onto a plate labeled Control LIP EAG SS 9-26-18.
7. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator.
8. The work station was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results:

• Observations
  • The enriched solution had a pellet at the bottom and was a murky color, so it was re-spun and re-filtered.
  • There were a few bubbles towards the center of the experimental plate.

Interpretations/Next Steps/Conclusions:

The experiment was complete. The next step will be to check for plaques in the next lab. If there are plaques, they will be picked. If not, a spot test will be run.

Contaminated Plates:

Rationale: The purpose of this lab is to perform a plaque assay with Soil C to isolate possible plaques, and to collect soil metadata.

Description of Procedures:

1. The work station was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. 10 ul of lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
3. The top agar solution was then made with 8 ml of LB broth and 90 ul of CaCl2, to make enough top agar for four plates. The 2x TA was not yet added, because it would solidify before the arthrobacter had sat long enough.
4. Next, the % water was calculated. The dry soil was found to be 5.331 g. Therefore the mass lost was 0.519 g. With the original mass of 3.53, the % water was calculated to be 14.7%.
5. The composition of the soil was determined next. Out of the 7 ml of soil, 5.5 ml were sand, 1.0 ml was silt, and 0.5 ml was clay. The percent compositions are listed under data.
6. Next, 10 ml of 2x TA was added to the TA solution.
7. 4.5 ml of the top agar solution was added to each tube of arthrobacter, and this mixture was quickly poured onto a petri dish. The rest of the top agar solution was poured onto a control plate. The experimental plate was labeled LIP 9-24-18 Soil C, and the control was labeled Control LIP SS EAG 9-24-18.
8. The plates were allowed to sit for 15 minutes and then inverted and stored in the incubator until the next lab.
9. The work station was cleaned using aseptic technique and all materials were stored or properly disposed of.

Observations/Data/Results:

• Observation:
  • When the plates were poured, there were less bubbles than before. Only one was seen on the experimental plate.
  • The control plate had more bubbles.
• Data:
  • % Water- 14.7%
  • % Sand- 78.6%
  • % Silt- 14.28%
  • % Clay- 7.14%

Interpretations/Next Steps/Conclusions:

The plaque assay and soil metadata were completed for Soil C. The next step will be to check for plaques in the next lab. If plaques are found, they will need to be picked. If no plaques are found, a spot test will be performed to test Soil C once more.

Rationale: After a negative plaque assay and a contaminated negative control, the purpose of this lab is to collect new soil, wash and enrich it, and collect metadata for the soil.

Description of Procedures

1. Soil was collected from a red oak tree, as well as the tree’s dimensions.
2. The work space was cleaned using aseptic technique. An ethanol burner was lit to create an aseptic zone.
3. 2 ml of soil and 8 ml of LB broth were added to a tube. The tube was shaken for 10 minutes, and then massed. The mass was found to be 17.74 g.
4. The soil was spun in the centrifuge for 10 minutes at 5000 x g.
5. While spinning, the % water test was begun. The weigh boat was found to be 2.32 g, and the weigh boat with soil was found to be 5.85 g. This will sit in the vented hood until the next lab to allow the water to evaporate.
6. Next the % concentration test was started. 10 ml of soil was added to a tube along with 20 ml of DI water. 3 drops of soil dispersion liquid was also added. The tube was then shaken for 30 seconds. It will sit under the vented hood until the next lab to allow the soil to separate into its layers.
7. The pH was found next. A small amount of soil was added to the pH vial, and then it was filled with DI water. The tube was then shaken for 10 seconds and then allowed to sit for 10 minutes.
8. While sitting, the tube containing soil was filtered with a 0.22 um filter and syringe. Approximately 7.75 ml of enriched lysate was obtained. No direct lysate was made. 0.5 ml of arthrobacter was added to this tube. It was labeled LIP 8-19-18 Enriched- Soil C. The tube will be shaken at room temperature until the next lab.
9. Next the pH was found to be 6.0, using pH paper.
10. The work station was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results/Data:

• Observations:
  • The soil collected was moist and dark in color.
  • The soil was collected from a red oak tree, along with a leaf.
  • The filtration process was easier than with Soil B.
• Data:
  • Weigh boat- 2.32 g
  • Weigh boat and soil- 5.85 g
  • pH- 6.0
  • 7.75 ml of enriched lysate collected
  • Tree circumference- 60.5 cm
  • Tree canopy- 303 cm
  • Tree height- 800.5 cm

Red Oak Tree- Soil Source

Negative Plaque Assay

Contaminated Control Plate

Interpretations/Next Steps/Conclusions:

The soil collection, washing, and enrichment processes were complete. Of the metadata, the soil pH was found, and the procedures for % water and % soil concentration were started, and will be completed in the next lab. The next step will be to run a spot test or a plaque assay to look for plaques. Soil B yielded no plaques, which is why Soil C was collected. Also, the control plate for the plaque assay for Soil B was contaminated.

Rationale: To attempt to isolate a phage by performing a plaque assay, after a negative spot test. This procedure will also finish calculating the soil concentrations of sand, silt, and clay.

Description of Procedures:

1. The work space was cleaned using aseptic technique and a 100% ethanol burner was lit to create an aseptic zone.
2. 10 ul of enriched lysate was added to 0.5 ml of arthrobacter. This was allowed to sit for 10 minutes.
3. Next, the top agar solution was made. One tube was used to make four 5 ml plates. 8 ml of LB broth and 90 ul of CaCl2 were added to the tube.
4. 2x Top Agar was then added to the tube and pipetted up and down to mix the top agar solution.
5. 4.5 ml of the solution was added to each of the groups arthrobacter and phage mixture, and immediately poured onto petri dishes. The remaining 4.5 ml was poured onto a control top agar plate. The experimental plate was labeled LIP 9-17-18 Plaque Assay. The control was labeled EAG, SS, LIP 9-17-18 Control Plaque Assay. The plates were then allowed to sit for 10 minutes.
6. While the plates were sitting, the soil metadata collection was finished. There were 7 ml of soil total, and it was found that 5.5 ml were sand, 0.5 ml were silt, and 1.0 ml was clay. The percent sand found was 78.57%. The percent silt was 7.14%. The percent clay was 14.29%.
7. After 10 minutes, the plates were inverted and placed in the incubator for 48 hours.
8. The work station was cleaned using aseptic technique and the materials were properly disposed of.

Observations/Results/Data:

• Observations:
  • There were small bubbles on the control plate, and one larger one on the edge.
  • There were 1-2 small bubbles on the control plate.
  • The soil had many layers of silt, all small, but different colors.
• Data:
  • % Sand: 78.57%
  • %Silt: 7.14%
  • %Clay: 14.29%

Interpretations/Next Steps/Conclusions:

The procedures were complete. The soil composition was found and the plaque assay, both the control and experimental, were completed. The next step will be to wait 48 hours to allow the bacteria to grow and plaques to form. If there are plaques, they will be picked. If not, then new soil will need to be found and the procedures will be started again.

Additional Questions:
1. Group 4’s Plaques: The group members in Justin’s group did not all collect soil from the same tree. The soil was from three different trees in the same area. This could account for the differing results. The soil Justin collected could have different phages from his group members, allowing him to have more and better defined plaques. However, the soil was most likely at least similar, as all three members of group 4 had plaques.

2. Lathan will need 4.01 ul to web his plate. (Work shown below).

Rationale: To isolate bacteriopages through a spot test, and to collect more metadata for soil sample B.

Description of Procedures:

1. The workstation was cleaned using aseptic technique, and an aseptic zone was created.
2. The enriched lysate sample made on 9-10-18 was spun in the centrifuge for 5 minutes at 3000 x g. The mass before spinning was found to be 20.49g. The enriched sample was spun to form an arthro pellet, to help separate the arthrobacter from the lysate.
3. While spinning, the percent water of the soil was calculated. The mass of the dry soil with the weigh boat was found to be 6.896g. From this, the mass of the dry soil was found to be 4.54g. The original mass of the soil was 4.91g. Subtracting the original mass from the final calculates the mass lost to be 0.37g. Dividing the difference by the original mass and multiplying by 100 tells us that the percent water in the soil was 7.5%.
4. To set up the spot test, one plate was divided into four quadrants, one for each group member and one for the negative control. My quadrant was labeled LIP 9-12-18 ES (Enriched sample).
5. Next, approximately 1.5 ml of the enriched sample was filtered into a 15mL tube labeled LIP 9-12-18 Filtered EL (Enriched Lysate). A 0.22 ul syringe filter was used to filter the lysate.
6. To make the top agar, 2.0 ml of LB broth was pipetted into a 50 mL tube. 22.5 ul of CaCl2 was added to the LB broth, as well as 0.5 ml of arthrobacter. Next 2.5 ml of the top agar was added to the tube, and this solution was lightly mixed. It was then immediately poured on to the petri dish and allowed to sit for 10 minutes.
7. While the plate settled, the percent concentrations of sand, silt, and clay test was started. The vial was labeled LIP 9-12-18 % Soil C (Composition).
8. 10 ml of soil was added to the vial. The vial was then filled to the 30 ml mark with DI water.
9. After 10 minutes of sitting, the top agar was settled and the procedure for the spot test was completed. 5 ul of lysate was spotted into the correct quadrants for each group member. 5 ul of phage buffer was spotted into the negative control quadrant. This was then allowed to sit for another 10 minutes.
10. Three drops of dispersion liquid were added to the 50 ml vial containing the soil. The solution was shaken vigorously for 30 seconds. The tube was then put under the vent hood to allow it to sit until the next lab.
11. The workstation was cleaned and the materials were properly stored and disposed of.

Observations/Results/Data:

• Observations:
  • The TA had some bubbles when it was settled.
  • The soil clumped to the bottom of the vial of the percent composition tube while mixing.
• Results:
  • % H2O: 7.5%

Interpretations/Next Steps/Conclusion:
The procedure for the spot test was complete. In the next lab, the % composition of the soil will be determined. Also, the plate will then be checked for plaques.