10.8.2018- Washing Soil Sample E

Rationale: Since a soil sample had been obtained, washing is necessary to prepare the sample for a spot test and plaque assay. Furthermore, since soil metadata procedures had been followed, results were obtained and processed. As confirmed on Wednesday, this began the analyzation of our final soil sample that was collected from campus.

Procedure:

1. Aseptic zone established
2. 2mL soil added to tube
3. 10mL LB Broth added to tube
4. Tube was shaken for 15 minutes to mix soil and LB Broth
5. Centrifuged for 15 minutes on high speed
6. Soil supernatant was filtered using a 3mL syringe and 22µm filter (added to tube labeled “HMB Enriched Soil E 10/8/18”)
7. 100µL of lysate was placed into tube labeled “HMB DI 10/8/18”
8. 0.5mL Arthrobacter was added to tube labeled “HMB Enriched Soil E 10/8/18”

Observations:

• The LB Broth used was made recently and showed no evidence of contamination; it was a clear solution with yellow color.
• It was far easier than normal to filter the supernatant (lysate) after centrifugation. This indicates that there were fewer soil particles that would be able to clog the filter, which would have made it far more difficult to filter the lysate.
• Filters were placed in a plastic wrap for the first time. While this is a very minute detail, it could have played a role in contamination seen in other procedures.
• Soil was consolidated into small balls rather than finer particles. This could have been due to refrigeration, as the soil sample had never been refrigerated in a conical tube before use until this procedure.

Results:

• % Water:
  • Weigh boat mass: 2.46g
  • Soil mass: 5.924g-2.46g= 3.464g/4.15g= 0.8347 percent soil
  • 1-0.8347= 0.1653 –> 16.53% Water
• Sand/Silt/Clay:
  • Sand: 2.5mL/4mL=62.5%
  • Silt: 1mL/4mL= 25%
  • Clay: 0.5mL/4mL=12.5%

Next Steps/Conclusions:

• Since the sample was successfully washed and processed, it will be possible to proceed with the testing process on Wednesday (Spot Test and Plaque Assay). Through this testing, it will be possible to see whether or not a phage is present in Soil Sample E. This will further contribute to the research question comparing old live oak trees compared to transplanted trees.

10.3.2018: Data Analyzation and New Sample

Rationale: Since a spot test and plaque assay were completed on Monday (10/3), it was necessary to obtain and examine results from those tests. However, since both tests showed negative results, it was necessary to discard the plates and begin the process of analyzing a new soil sample. Therefore, Soil Sample E was found and collected from Minglewood Bowl and metadata procedures were performed.

Data from Monday, October 1:

Both the spot test and plaque assay analyzations showed that no plaque had been caused by the samples derived from Soil Sample D. The only disruptions in the lawn growth of arthrobacter were caused by deformities and tears in the plate, and those deformities had been recorded prior to the completion of the test on the plate through dots of blue marker. However, it was also observed that control plates for both the spot test and plaque assay did not contain arthrobacter or any other form of contamination.

Top Row: Plaque assay, Spot Test

Bottom Row: Top Agar Controls

Procedure: Plates from 10/1/18 were analyzed. Soil was collected from a live oak in Minglewood Bowl near Brooks Residential Flats. Tree metadata form completed and submitted. 4mL of soil was placed in Falcon Tube. 8mL DI water was added to Falcon Tube along with 3 drops of soil dispersion liquid. Soil was shaken and vortexed for 30 seconds, then allowed to settle under fume hood to be left overnight. Added 4.15g of soil to a weigh boat (“HMB 10/3/2018”). Placed weigh boat under fume hood to be left overnight. Added roughly 10 drops of soil supernatant from Falcon Tube to pH tube. Filled the remainder of tube with DI water. Held pH paper in sample for 30 seconds to obtain a pH reading.

Observations:

• Soil sample was taken from an area without any grass covering the soil. Soil was still moist, and the sample was taken from approximately 2 inches below surface to avoid overly dry or caked mud.

Data from 10/3/2018:

• pH of sample was 6

Next Steps:

• The soil will need to be washed and prepared to test on Monday. Furthermore, the metadata procedures that were started today will also need to be observed on Monday, and the data will be collected then.

Conclusions:

• Since the plaque assay and spot test for Soil Sample D did not show any plaques, it was concluded that Soil Sample D did not have any phage present.
• As noted earlier, the control plates did not display any evidence of contamination that was prevalently seen in the majority of the samples before this one. This shows that the recent change in Top Agar and LB Broth likely replaced contaminated samples that were causing systematic contamination of samples throughout the class. Therefore, the conclusion can be tentatively drawn that the contamination was caused by old and contaminated LB Broth and Top Agar.

10.1.2018- Spot Test and Plaque Assay Soil Sample D

Rationale: Lysate was obtained on Wednesday (9/26), but had yet to be tested from Soil Sample D. Therefore, it is necessary to determine whether or not a phage is present by using a spot test and plaque assay.

Procedure: Metadata results from Wednesday were obtained and recorded. 2mL Filter Sterilized Enriched Lysate (FSEL) created with syringe and 22µm filter. 10µL FSEL was added to 0.5mL Arthrobacter and was set aside for 15 minutes. 22.5µL CaCl2 was added to a control tube (“NMN HMB 10.1.18 Top Agar Control”) and experimental tube (“HMB Plaque Assay Soil D 10/1/18”). 2mL LB Broth added to both tubes. Arthrobacter with FSEL was added to experimental tube. 2.5mL Top Agar added to both tubes and poured onto plates. Plates were let sit for 48 hours. Obtain two new tubes (“HMB Spot Test Soil D 10/1/2108”) and (“HMB NMN 10.1.18 Top Agar Control-ST”). Add 2mL LB Broth to both tubes. 22.5µL CaCl2 was added to both tubes. 0.5mL Arthrobacter was added to the experimental tube. 2.5mL of Top Agar was added to both tubes, then the solutions were plated. Plates were labeled and let sit for 10 minutes. 10µL FSEL, Direct Isolate, and phage buffer were added to each respective section, then the plate was allowed to set for 15 minutes. Plate was incubated for 48 hours.

Observations:

• Metadata:
  • Soil Composition: 2.5mL Sand, 0.5mL Silt, and 0.5mL Clay were found in the sample. Results in 71.43% Sand, 14.29% Silt, and 14.29%
  • Percent Water: Sample’s mass when weigh boat was subtracted: 3.091g. Original mass of soil added: 4.126g-3.091g=1.035g/4.126g= 25.08% Water
• White particle was observed in direct isolate. It will be interesting to observe any possible effect that it has on the sample.
• Spots for spot test did not set as well as they normally have. This resulted in us not inverting our plates before overnight incubation.

Next Steps/Conclusion:

• Since the plates have been prepared, they will be able to be read and analyzed on Wednesday, October 3. If there are plaques, it will be possible to proceed with the plaque isolation process. However, if there are no plaques present, it will be necessary yet again to obtain a new soil sample, as there would not be any phages present in Soil Sample D.

9.26.2018 Rewashing Soil Sample D

Rationale: At the beginning of the lab time, it was revealed that many individuals had results that did not accurately reflect what they should have. This was attributed to the fact that the Arthrobacter used for all procedures on Monday was not Arthrobacter, which made it impossible for Arthrobacter-infecting phages to interact with them. Therefore, all procedures done on Monday that involved Arthrobacter had to be redone. Since the soil sample lysate had been enriched with a problematic bacterial sample, it was necessary to rewash and enrich the new lysate from the same soil sample.

Procedure (Metadata in Italics):

1. Established an aseptic zone.
2. Added 2mL of soil to tube (“HMB Soil Sample D 9/26/18”)
3. Added 10mL of LB Broth to tube
4. Shook for 10 minutes
5. Centrifuged sample for 15 minutes
6. Used syringe and 22 micron filter to filter supernatant of centrifuged sample. Placed into tube labeled “HMB Soil Sample D 9.24.18”.
7. Added 0.5mL of Arthrobacter to “HMB Enriched Soil D 9.26.18” tube to create enriched lysate.
8. Moved enriched lysate to shaker to be used on Friday 9/28.
9. Added 4.126g of soil to weigh boat and placed under hood to be analyzed on Friday.
10. 4mL of soil was added to a falcon tube. 8mL of DI water was added to the falcon tube along with 3 drops of soil dispersion liquid.
11. Shaken for 30 seconds for reexamination on Friday.
12. Added 0.5mL of soil supernatant from the falcon tube to the pH tube. Added DI water until the pH tube was full. Used pH paper to determine the pH was 5.5 after matching with chart. 

Observations:

• Mass of tube while shaking was equal to 18.03g.
• At the end of filtering, a very small amount of white foam dripped from the filter into the lysate tube. It is unknown if the amount or composition will have significant effects in any way.
• As observed on Wednesday, the soil sample was very dark after adding LB Broth and sand had already begun to settle out before it was placed in the centrifuge.
• Mass of weigh boat: 2.325g.

Data Summary from Today: pH=5.5

Next Steps/Conclusions:

• The next step for processing this soil sample would be to perform a spot test and plaque assay. This procedure was intended to occur during this lab entry, but the delay due to the issue with Arthrobacter delayed the schedule of the lab. Therefore, the procedures will be completed on Monday.

9.24.18 Obtaining and Washing New Soil Sample

Results from Friday, 9/21: As suspected, the plaque assay revealed that there was no phage, indicating that the small clearings were likely caused by competition among bacterial groups or other sources. Therefore, Soil Sample C is officially negative. The control plate also was contaminated yet again, and similarly to Wednesday’s (9/19) sample, it is believed that the cause stems from a contaminated top agar or LB broth.

Rationale: Since the results from the test on Friday showed contamination on the control plate and only an extremely small mark on the plate, it was concluded that a new sample would be needed to restart the process. The washing procedure was also fit into the lab session after obtaining a new soil sample (Soil Sample D) to be ready to perform a plaque assay and spot test on Wednesday.

Procedure:

1. Establish an aseptic zone.
2. Collected new sample of soil from tree at Teal Residence Hall. Recorded metadata in survey.
3. Added 2mL of soil to tube (“HMB Soil Sample D 9/24/18”)
4. Added 10mL of LB Broth to tube
5. Shook for 10 minutes
6. Centrifuged sample for 10 minutes
7. Used syringe and 22μL filter to filter supernatant of centrifuged sample. Placed into tube labeled “HMB Soil Sample D 9.24.18”.
8. Moved 100μL of filtered lysate to microcentrifuge tube for a direct isolate.
9. Added 0.5mL of Arthrobacter to “HMB Soil Sample D 9.24.18” tube to create enriched lysate.
10. Moved enriched lysate to shaker to be used on Wednesday 9/26.
11. Moved direct isolate to fridge to be used on Wednesday 9/26.

Observations:

• Control Plate still contaminated using the last LB Broth and Top Agar combination. Will be determined if it is the cause when next plaque assay is completed, as new Top Agar and LB Broth solutions have been created.
• Soil was very dark and already began settling out sand particles before centrifugation. Through this, it can be predicted that there will be a higher proportion of sand than normal.
• Mass of tube with soil and LB Broth was equal to 18.376g as measured before being placed in the centrifuge.

Next Steps:

• The enriched and direct lysates will be used for a plaque assay and spot test. These tests will show accurately whether or not phages are present in the soil obtained. Furthermore, it will be necessary to take extra care when using the new Top Agar and LB Broth to avoid contaminating the fresh samples, as this will have adverse effects on the control plates certainly and potentially the experimental plates as well.

9.21.18 Possible Phage Analysis

Results from Wednesday, 9/19: Plaque Assay experimental plate shows one clearing that could have been caused by phage. Both the control plate predicted to show contamination and the control plate expected to not show contamination showed contamination. Spot test showed a very small clearing that appeared similar to the one found on the plaque assay.

Rationale: Results from Wednesday, 9/19 showed that there was a very small clearing on the Plaque Assay test completed. Therefore, to determine whether or not a phage was the cause of the clearing, a new plaque assay was completed to test the composition of the clearing.

Procedure:

1. Aseptic zone established.
2. Plates were removed from incubator and analyzed.
3. 100μL of Phage Buffer was placed in microcentrifuge tube (“HMB PPL”)
4. Pipette tip was used to take up plaque from plate, added to phage buffer and mixed.
5. 2mL LB Broth added to control and experimental tubes
6. 22.5μL CaCl2 added to control and experimental tubes
7. 10μL of Phage Buffer and Possible phage solution added to Arthrobacter. Let sit for 10 minutes.
8. Added 2.5mL top agar was added to control tube and plated mixture after swishing about.
9. 0.5mL Arthro and possible phage solution added to experimental tube
10. Added 2.5mL 2X Top Agar to experimental tube. Swished. Plated overlay. Let sit for 15 minutes before incubating until Monday 9/24.

Observations:

• Both control plates that were used showed signs of contamination. One of the control plates was expected to show contamination, but the other should not have. This likely would have been from contaminated 2X Top Agar solution, which was not changed between the two samples.
• A small clearing was observed on the plaque assay. It appeared to be turbid, and its size was quite small. These factors made it uncertain whether or not a plaque was present because there was not an obvious presence.

Next Steps:

• The next step for this experiment is to observe the results on Monday and follow the next proper step in picking and diluting (if plaques are present) or obtain a new soil sample (if no plaques are present on the plaque assay).

Conclusions:

• Systematic contamination of control plates is likely occurring from repeated use of contaminated LB Broth and Top Agar. Many of the control plates completed in group 3 in the past weeks have been contaminated in addition to other incidents of contamination across the laboratory , so it is not unrealistic to conclude that it is likely that there is a common source.
• There likely will not be a plaque on the plate on Monday. While it is possible that a phage caused the small clearing or plaque, it is strange that there was not any other sign of phage. Furthermore, the contaminated control plate introduces a situation where bacterial competition could have caused the clearing. Therefore, it was necessary to test with another plaque assay to determine whether or not phage was present.

9.19.18 Spot Test and Plaque Assay for Soil Sample C

Rationale: A Spot Test and Plaque Assay will be performed to analyze the composition of the lysates from Soil Sample C. The two tests will reveal if a phage is present through the presence or absence of a plaque on the bacterial lawn.

Procedure: (Metadata steps in italics)

1. Cleaned lab bench and lit burner for aseptic zone.
2. Obtained conical tubes and labeled “HMB Plaque Assay Soil C 9/19/18” and “Top Agar Plaque Assay Control 9/19/18”
3. Added 2mL of LB broth to both tubes
4. Added 25μL of CaCl2 to both tubes.
5. Used syringe and filter to create at least 10μL of Filter Sterilized Enriched Lysate. Placed in microcentrifuge tube labeled “HMB FSEL 9/19/18”.
6. It was observed that the LB Broth used in steps 3 and 4 was cloudy. This was thought to be due to contamination, so the control tube was kept and tested to confirm this thought while the other two tubes were remade (redid steps 3-4) with clear LB Broth.
7. Added 10μL of Filter Sterilized Enriched Lysate to 0.5mL Arthrobacter. Let sit for 15 minutes.
8. Obtained 3 new plates and labeled them “HMB Plaque Assay Soil C 9/19/18”, “Top Agar Contamination Control”, and “Top Agar Control 9/19/18”.
9. Added 0.5mL Arthrobacter with Filter Sterilized Enriched Lysate to conical tube labeled “HMB Plaque Assay Soil C 9/19/18”
10. 2.5mL of 2X Top Agar was added to all 3 tubes and was swished to mix.
11. Poured Top Agar solutions on to respective plates. Let sit for 15 minutes before incubating.
12. Obtained new 50mL conical tube labeled “TA Control for Spot Test HMB NMN 9/19/18” and “TA for Spot Test 9/19/18 HMB NMN”.
13. 2mL of LB Broth and 22.5μL of CaCl2 were added to Conical Tubes
14. 0.5mL of Arthrobacter added to experimental vial
15. 2.5mL of Top Agar was added to both Conical Tubes.
16. Conical Tubes were swished, then poured onto their respective plates.
17. At this point, it was observed that after letting the “HMB Plaque Assay Soil C 9/19/18” plate sit for 15 minutes that the Top Agar had broken and slid around the plate. Therefore, a new plate was created using steps 3-4, 7, 9-11 in that order. It was also given a new 15 minutes, then placed in incubator.
18. Weigh boat containing soil from Monday was reweighed and found to be 5.689g. 5.689g-2.43= 3.259g. 3.259g/4=0.815. 1-0.815= 0.185 –> 18.5% H2O.
19. Reexamined Soil Separation: 6mL of soil was separated. Measurements were found to be 2mL of sand (33.3%), 1mL of silt (16.7%), and 3mL of clay (50%). Soil was properly disposed of and tubes were cleaned.
20. pH tube and paper were obtained. Small amount of supernatant from soil separation sample was placed in bottom of pH tube and the rest of the tube was filled with Deionized Water. pH paper was placed in sample for 45seconds, then examined. Found to be a pH of 6.5 — more basic than previous sample.

Observations

• pH of Soil Sample C is more basic than Soil Sample B. It will be interesting to see if this has anything to do with the presence of a plaque as a possible secondary study to the overarching question currently posed.
• When Top Agar did not set, plate was shaken somewhat vigorously to attempt to prevent Top Agar from setting with bubbles present. This likely caused the plate to not set correctly, creating slipping of top agar. Important to note for next time.
• There was much more clay in Soil Sample C than found in previous samples.
• Cloudy LB Broth could be the result of contamination that has been previously observed throughout the lab.

Next Steps: The next component of the process is to reexamine the plates on Friday to determine whether or not there is a phage. If there is a phage, we will be able to move ahead in the process of picking a phage on Monday, but if not, I will obtain a new soil sample over the weekend.

Conclusions:

• Over-swirling Top Agar likely results in slipping and dividing of Top Agar.
• Cloudy LB Broth could have been the cause of contamination viewed on many of the plates.

9.17.18 Obtaining and Washing Soil Sample C

Questions posed by Lathan:

1. The reason why Justin’s plate contained phage in the spot test and the plaque assay and the rest of group four only displayed plaque in the plaque assay was due to the titer of their solutions. Justin’s likely had the highest titer, or concentration, of phage in the plated sample, which would have allowed the spot to clear a plaque on the lawn growth. A lower titer would not be able to display a plaque in the same manner, which would have explained why the rest of group four had the results they did.
2. 4.018μL

Rationale: Since previous soil samples (Soil Sample A and Soil Sample B) did not contain a plaque that contained phage, it became necessary to obtain a third sample and prepare it (by washing the soil and collecting metadata) for testing that will occur on Wednesday in the form of a Spot Test and Plaque Assay.

Data from Friday, September 14

The Plaque Assay performed on September 14 needed at least 48 hours to develop results, which were first available to be observed during this lab session. As predicted by the Spot Test, there were no plaques on the plate containing lysate from soil sample B. However, the negative control exhibited slight presence of small Arthrobacter colonies of unknown origin. These colonies could be due to an error in preparation, or they could be from general contamination in the incubator or otherwise. Furthermore, there are uneven areas present on the experimental plate that cause imperfections in the lawn growth. These breaks are likely caused by uneven top agar spreading rather than phage, so they have no bearing on result. Pictures below illustrate these occurrences.

Procedure: (Sections in italics are metadata related)

1. Cleaned lab bench. Lit ethanol burner to create aseptic zone.
2. Observed plates with results and observations listed above.
3. Obtained materials for soil collection and obtained soil from an Earle Hall tree. Placed soil in bag labeled “HMB Soil Sample C Earle Hall”. Conical Tube labeled “HMB Earle Hall Soil Sample C 9/17/18”.
4. 2mL of soil was added to Conical Tube.
5. 12mL LB Broth added to Conical Tube.
6. Mixture of LB Broth and soil was shaken for 15 minutes. During the 15 minute period, the mass was found to be 20.795g. To match Nathan’s sample for centrifuging, water was added to the tube to make it 21.028g.
7. Sample was centrifuged for 10 minutes to pellet soil and waste.
8. Metadata: Weigh boat was massed and found to be 2.43g. 4g of soil was added to weigh boat. After, the weigh boat labeled with a “2” and distinguished by a crack in the plastic was placed under the hood to be reexamined on Wednesday. 
9. 4mL of soil was added to a 50mL Falcon Tube.
10. 8mL of DI water was added to 50mL Falcon Tube.
11. 3 drops of Soil Dispersion Solution were added to Falcon Tube. Tube was shaken for 30 seconds to mix soil. Soil let sit until Wednesday.
12. Syringe and filter used to add 10mL of lysate to 50mL conical tube and about 1.5mL of lysate into microcentrifuge tube as a direct lysate (which was labeled “HMB D 9/17/18” and placed into the fridge for storage).
13. 0.5mL of Arthrobacter was added to 50mL conical tube to create enriched lysate. Labeled “HMB Enriched 9/17/18”.
14. Enriched lysate was stored in shaker to be recovered in 48 hours.
15. Lab station cleaned and materials were returned to places they were found.

Observations

• During the metadata section, the soil seemed to separate more evenly than the first analyzed sample.
• As filter was used more during the filtration step, it became more and more difficult to push the lysate through the syringe. However, the tension never let up, which confirmed that the filter was at no point broken or dysfunctional.

Next Steps: On Wednesday, the enriched lysate will be used to perform both a Spot Test and a Plaque Assay. This will allow the sample to be read on Friday, which will reveal whether or not Soil Sample C had a viable phage that was causing plaques. Furthermore, if the testing process returns with a negative result, a new sample will be able to be collected more efficiently than if the washing process was put off until Wednesday.

Conclusions:
Since the second soil sample did not have a phage, it was necessary to repeat the process to obtain a third. Hopefully, this sample will contain phage and new procedures learned regarding picking the plaques and creating a webbed plate can be practiced.

9.12.18: Metadata Collection and Spot Test

Rationale: Metadata tests that needed additional time to settle had been given enough, so results of the tests could be processed and analyzed. Then, a Spot Test could be performed with Enriched Isolation that was obtained on Monday, 9/10/18.

Metadata Results/Data:

• % Water: 2.390g measured after 48 hours of evaporation/3.145g initial soil added= 75.99% Soil. 1-0.7599= 24.01% Water
• After observing tube that contained soil being analyzed for percentages of sand, silt, and clay, it was found to contain 3.25mL of layered soil. The bottom layer was 1.5mL of sand, the middle layer was 1mL of silt, and the top layer appeared to be 0.75mL of clay. The corresponding percentages were: 46.15% Sand, 30.77% Silt, and 23.08% Clay. The category of soil this fits under is Sandy Clay Loam.

Procedure:

1. Lab bench was cleaned with Cidecon and 70% ethanol.
2. Ethanol burner was lit to establish an aseptic zone surrounding the flame.
3. Obtained mass of 50mL Conical tube labelled “HMB Enriched Lysate 9/10/18 S.S.B.”
4. Paired tube with another of similar mass and sent for centrifuging at 3000g for 5 minutes in order to pellet out Arthrobacter.
5. While the tube was being centrifuged, metadata results were collected.
   1. Weigh boat containing original soil sample B labelled “HMB Water” was re-massed and found to contain 2.390g of soil. The corresponding calculations resulted in discovering the % water of the soil was 24.01%. Weigh boat containing soil was disposed of under the instruction of Leo.
   2. The tube with soil in layers was taken up from lab bench and examined. After examination and documentation, the tube was cleaned and returned properly.        
6. LB Broth, plate, and 50mL conical tube were obtained. Plate was labelled with each of Group Three’s initials on one section of the plate and “Spot Test 9.12.18” on the bottom.
7. Placed 2mL of LB Broth in the 50mL conical tube.
8. 0.5mL of Arthrobacter was added to same 50mL conical tube.
9. 22.5 microliters of 1M CaCl2 was pipetted into same 50mL conical tube.
10. Finally, 2.5mL of 2x Top Agar solution was added and swished in same 50mL conical tube. After mixing, solution was rapidly poured onto plate to avoid cooling and hardening in tube rather than on plate.
11. Plate with freshly poured Top Agar was given 15 minutes to settle and harden.
12. While plate was setting, a 3mL syringe was obtained along with the enriched lysate from Monday (9/10/18).
13. 2mL of enriched lysate were drawn up into syringe. Filter was screwed onto end, and the 2mL were pushed through the filter and into a microcentrifuge tube.
14. 10 microliters of filtered sterilized enriched lysate was added to the section of the plate labelled “HB”.
15. Plate was allowed to set for 15 minutes before being placed into the incubator to be observed on Friday.

Observations:

• Soil in tube was not evenly layered. For example, the silt on one side appeared to encompass over 1mL of the total sample, but on the other side it made up closer to 0.5mL. To get values, averages of these were taken, leading to possible inaccuracies in final values.
• While the top agar was being formed, some residual bubbles remained on the outside of the agar. They were unable to be popped, so they remained around the ring. However, there was more than enough space on the plate to avoid them.
• When samples were applied to the plate during the initial spot test, they seemed to sit on the surface momentarily before settling into the agar. However, the same phenomena was not observed during this Spot Test. It will be interesting to see whether or not the results are different, but any discrepancies would not be able to be formally attributed to that due to the number of variables changed.

Next Steps:

• On Friday during open lab, the spot test sample will be analyzed. If there is a plaque, it will need to be picked and a Plaque Assay will be run to ensure that the results obtained were accurate and repeatable. If there is no plaque, another soil sample will need to be obtained and examined.

9.10.2018- Soil Sample B: Metadata and Enrichment

Rationale: Collecting metadata will allow for additional comprehension of soil composition and data connections to be made at a later date. The washing and enrichment process will prepare Soil Sample B for future Spot Test and Plaque Assay Procedures by creating a lysate that contains Arthrobacter.

Procedure:

1. Lab bench was cleaned with CiDecon and 70% ethanol to establish a clean work surface.
2. Lit ethanol burner to establish aseptic area.
3. Obtained soil sample bag containing conical tube, excess soil, and leaf samples.
4. (ASEPTIC) Added 10mL of LB Broth to the 11.5mL mark on the 15mL conical tube labelled “HMB Soil Sample B”
5. Shook for 15 minutes using a combination of shaking and vortexing. While shaking, mass of tube was found to be 17.849g. At the end of 15 minutes, centrifuge was used to spin the tube at 10,000g for 5 minutes.
6. 3.145g of soil was placed in weigh boat labeled “HMB water” and placed under a fume hood
7. 4mL of soil sample from bag was added to the 50mL tube. Then, DI (deionized) water was added to the 12mL mark. 3 drops of soil dispersion liquid was added. Mixture was shaken for 30 seconds to mix, then left to sit for 48 hours.
8. Top filter package and supplies was obtained and taken to the fume hood. Then, it was set up.
9. Under hood, supernatant of tube that was in centrifuge and labeled “HMB Soil Sample B” was added to filter. Filtrate was dripped into 50mL Conical tube labeled “HMB Enriched Lysate 9/10/18 S.S.B.”
   1. Resulted in 8mL of filtrate.
10. (Aseptic) 0.5mL Arthrobacter was added to 50mL conical tube. Let sit in shaker for 48 hours.
11. Obtained pH tube. Added a small amount of soil supernatant. Filled pH tube with DI water on top of soil supernatant. Added pH strip to sample for approximately 40 seconds. When compared with pH scale, pH was concluded to be 6.
12. My individual lab components were put away and disposed of properly. Since Shepard had additional procedure to go, he offered to clean the bench when he was done to allow us to leave early. The only item left in my section was a test tube rack with the three soil samples settling out from step 7 along with a guide to leaf observations.

Data:

• pH of soil was 6.

Observations

• After shaking with LB Buffer, this soil sample was darker than the first sample completed.
• Again, after shaking the volume of the mixture had decreased from 12mL to roughly 9mL.
• Leaf observations: Leaf obtained had an obtuse tip, entire sides, and acute shape at base. Appeared to be an oblanceolate leaf. Finally, leafs on branches appeared to be organized in the Palmately compound fashion.
• pH paper changed the color of soil and DI water solution to a blueish green color.

Next Steps

• During the next laboratory time, metadata results that were unable to be obtained today will be recorded. Also, the enriched lysate created today will be used to perform a Spot Test. The Spot Test will reveal any presence of phages that could have been in Soil Sample B.