September 12

September 12, 2018 Spot Test and Metadata – Soil B

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.

 

Category: Lucy Potts | LEAVE A COMMENT
September 12

Spot Test 9/12/18

Rationale: Now that we have collected soil metadata I will run a spot test to see if there is arthrobacter phage present in the soil taken from tree B. This way we can start putting together data comparing the presence of arthrobacter in trees that have been sprayed with pesticides versus those which have not.

Procedure:

  1. Spun down tube of enriched sample at 3000g for 5 minutes
  2. Took a plate and found that it was contaminated so I grabbed another plate. I

    labeled one side enriched and the other side PB control.

  3. Used a syringe and a 22μL filter to filter out about 3mL of my enriched

    lysate.

  4. I then made my top agar by mixing 2.0mL of LB broth, 22.5μL of CaCl2, 0.5mL of arthrobacter, and 2.5mL of 2x TA in that order.
  5. I pipetted up and down for 30 seconds before pouring into my plate. I swirled around until the top agar covered the whole plate and let sit for 10 minutes.
  6. I then pipetted 10μL of my enriched lysate onto the point labeled for enriched lysate and 10μL of phage buffer on the control side. I then let it sit for 15 minutes.
  7. I placed the plate without inverting it in the incubator to develop over the weekend.

Observations:

Plaque assay turned out negative.

Next Steps:
Next class I will conduct a plaque assay and using the results of these two assays I will compare against the other tree’s sample from Cameron park and the tree samples from the Baylor campus. My table can start compiling a spreadsheet of our results, so we can start analyzing whether or not the presence of pesticides is affecting the presence of arthrobacter phage.

Category: Sriram Avirneni | LEAVE A COMMENT
September 12

9.10.18 Soil Metadata and Washing

9.10.18 Soil Metadata and Washing

Rationale)

To wash Soil Sample B using a modified procedure in order to achieve a lysate that can then be enriched and/or left as a direct isolate. We will also collect metadata on Soil Sample B, including pH, percent water, and percent sand, silt, clay. We will also collect data on the leaves I collected from the tree I gathered soil from.

Procedures)

  1. Setup an aseptic zone by wiping down the work surface with CiDecon and 70% ethanol. Light an ethanol flame as well.
  2. Retrieve the vial labeled “NMN 9.5.18 Soil B” and using a 10mL serological pipette under aseptic conditions transfer 10mL of LB broth into the vial. We also filled the other Group 3 vials at the same time, using the same pipette all under aseptic conditions.
  3. Cap “NMN 9.5.18 Soil B” and shake for 15 minutes, afterward take the mass of the vial, 17.743g.
  4. After this, it was determined that water needed to be added to bring it closer to the mass of another vial in order for it to be centrifuged properly, roughly 5 drops of water added using a pipette to bring the mass to 17.855g.
  5. Centrifuge “NMN 9.5.18 Soil B” at 10,000g for 5 minutes.
  6. While “NMN 9.5.18 Soil B” is centrifuging, retrieve a weigh dish and record the empty mass, 2.45g. Label the dish as NMN %H2O and fill with 3.72g of Soil B from the bag, “NMN, Soil B+Leaf, 9.10.18”, containing the excess Soil B, place the weigh boat under the fume hood to dry for 48hrs.
  7. Retrieve a falcon tube and fill with 4mL of Soil B, do to the tube not being able to be labeled it was stored in the middle back section of the test tube rack for the purpose of identification.
  8. Add enough DI water to the falcon tube that the meniscus reaches 14mL, add 3 drops of soil dispersion liquid to the falcon tube and shake for 30 seconds, place the tube in the middle back of the test tube rack and let it sit for 48hrs.
  9. Retrieve the now centrifuged “NMN 9.5.18 Soil B” and a .22 micron filter, set the filter up under the fume hood and turn on the vacuum. Using a pipette transfer the supernatant from “NMN 9.5.18 Soil B” to the top of the filter apparatus.
  10. After all the supernatant has filtered through remove the filter apparatus from the attached 50mL conical vial and quickly cap the vial. Label the 50mL vial “NMN 9.10.18 Soil B Enriched Lysate” noting that only about 7.5mL of lysate was obtained. Dispose of the filter and accompanying discarded materials into the biohazard bag.
  11. Add .5mL of Arthrobacteria to “NMN 9.10.18 Soil B Enriched Lysate” under aseptic conditions.
  12. Retrieve the leaf gathered from the tree the soil was collected from in the bag labeled “NMN Soil B+Leaf 9.10.18”, compare the leaf to the leaf type sheet and determine the leaf to be an oblanceolate with an obtuse tip.
  13. Add about 2mL of the Soil B-water mixture from the falcon tube to a pH vial, filling the rest of the vial with DI water, cover with your finger and shake for 10 seconds.
  14. Take about 1 inch of pH paper and place it into the pH tube for about 30 seconds, remove the paper after this time and compare it with the pH paper comparison diagram, recording the pH is 6.
  15. Wash out the tube with DI after putting the contents of the vial and the pH strip in the biohazard bag.
  16. As we were finished relatively early, Shepard agreed to clean up the workspace as he still had work to do in order to finish the required assignments.

Data/Observations)

The pH of the soil was found to be 6.5, the mass of the empty weigh dish was 2.45g, and the mass of the undried Soil B that was added to the weigh dish was 3.72g. I also observed that my supernatant was much clearer compared to the previous time I washed soil, additionally the filtration process only produced 7.5mL of lysate instead of the desired 10mL, meaning a direct isolation was not kept and all the lysate was used in the enriched lysate. I also observed the live oak leaf I gathered to be an oblanceolate with an obtuse tip.

Conclusions/Next Steps)

The next steps will be to analyze and record the rest of the soil metadata on Wednesday after 48 hours have passed, including percent water and sand, silt, clay percentages. Additionally, we will also collect the enriched lysate that was left on the shaker table for the same 48 hours in order to utilize it in both a plaque analysis and a spot test, in the effort to isolate a phage from Soil Sample B.

 

 

 

Category: Nathan Newton | LEAVE A COMMENT
September 11

9/10/18 Soil Washing and Enrichment and Metadata Soil B

Rationale: Isolate possible bacteriophage through the process of soil washing and enrichment. The soil metadata will also be found and recorded.

Soil Washing 

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

  1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
  2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
  3. We then got an ethanol burner, and our aseptic zone was created.

Procedure:

  • 10mL LB broth was added to my 15mL vial, which contained 2mL of Soil B, giving a total volume of 12mL solution in my 15mL vial.
  • Started to shake my 15mL for 11 minutes.
  • Measured the mass of the 15mL vial which read 14.44g.
  • Found a partner that had .05+/- difference in mass for the centrifuge.
  • Spun the 15mL solution at 10,000g f0r 10 minutes.
  • Top filtered the solution after it was spun in the centrifuge, which came to about 7mL of my enriched lysate.
  • Then placed my 7mL solution into the Cabinet.

Observations: 

  • The soil wash was not hard at all compared to my first soil wash.
  • The only hard part was the filtering process since I had a lot of debris on the top of my enrichment.
  • 7mL of enriched lysate was made.

Soil Metadata (% Soil, Clay, and Silt. pH of Soil. % Water.)

While the Soil Wash tubes were being shaken, the soil metadata was collected.

Procedure %Water:

  • The procedure to calculate the percent water started with pouring soil B from the plastic bag to the petri dish.
  • Mass of the weight boat = 2.39g.
  • Mass of weight boat with soil = 14.48g.
  • The weight boat was then labeled ML 9/10/18 14.48g.
  • The weight boat was then placed under the vented hood, where it will sit for 48 hours to allow the water to evaporate.

Procedure for pH of the soil:

  • Poured a small amount of soil into a pH vial.
  • Filled rest of the vial with DI water.
  • Rested my hand over the top, and I shook the vial for about 45 seconds.
  • Put the short strip (<1 inch) pH paper into the vial for 45 seconds.
  • Quickly compared the pH paper to the pH scale (<1 min).
  • pH recorded

Procedure for Metadata:

  • Filled 4mL of soil B into a 50mL vial.
  • Added 8mL of DI water into the 50mL vial. Total 12mL solution.
  • Added three drops of soil dispersion liquid into the 50mL vial solution.
  • Shook the vial for 45 seconds, with my hand covering the top (no lids were available).
  • Labeled the vial ML Soil B 9/10/18
  • The vial was then placed under the vented hood, where it will sit for 48 hours to let the soil (clay, silt, and soil) completely separate.

Observations/Results:

  • This metadata procedure was not hard. The only hard part was not to get any debris into any of the tests, which my soil B had a lot of debris. This could have an effect on all of my tests.
  • pH of soil: 6.5
  • Mass of weight boat: 2.39g
  • Mass of weight boat with soil: 14.48g

Next steps/Conclusions:

Prepare for a Spot Test/Plaque Assay and the continuation of Soil metadata results. Find the mass of water from the weight boat by doing a simple calculation. Find the composition of soil by observing the results in the 50mL tube. The next steps will be done 9/12/18. The spot test should not be hard, and hopefully, the simple calculations are not done incorrectly.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
September 11

09/10/2018- Washing And Enrichment of Soil B

09/10/2018

Washing And Enrichment of Soil B

Research Question:

Does the presence of arthrobacter appear more dominant in the soil of one oak species than the others? Is there a correlation between the presence of Arthrobacter Phage and the presence of oak wilt fungus?

Objective:

  • Prepare direct and enriched lysate for soil sample B
  • Collect metadata for soil sample B ( %water, pH, and sand silt clay percentages)

Materials Required:

50 ml conical vials, 15 ml conical vials, pH vials, pH paper, serological pipettes, Cidecon, 70% ethanol, LB broth, Soil Sample B, Falcon tubes, DI water, soil dispersion liquid, scooper, syringe, syringe tip filter (22 microns), shaking incubator, weighing boat

Procedure

  1.  set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%)( wipe on the desk and let it evaporate) after clearing the table.
  2.  lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

Washing and Enrichment:

  1. retrieved Soil Sample B from the refrigerator.
  2. retrieved the 15 ml conical vial with 2 ml of soil in it.
  3. retrieved the LB broth
  4. using a serological pipette, transferred LB broth to the 15 ml conical vial until it reached the 11 ml mark.
  5. closed the 15  ml conical vial and shook it by hand for 12 minutes
  6. after  12 minutes, find the mass of the vial ( 19.31g), and find another lab partner with a tube of mass within 0.05g of your tube mass.
  7. take the vials to the centrifuge and put them in for 10 minutes at an acceleration  10000 times g.
  8. take the vial back to the aseptic zone.
  9. using a sterile syringe and a syringe tip filter (22 microns), transfer 10 ml of filtered lysate to a 50 ml conical vial. ( take care to use the aseptic zone to avoid contamination of lysate.
  10. after you acquire 10 ml of filtered lysate, add 0.5 ml of arthrobacter to the vial. this is the enriched sample.
  11. place this tube, loosening the cap a little bit , into the shaking incubator for 48 hours
  12. filter the rest of the lysate into another 15 ml conical vial ( 1/2 ml for my sample). this will be your direct isolation sample
  13. store direct isolate in the fridge

% Water

  1.  weigh the weighing boat on the scale ( g)
  2. pour some soil onto the boat and weigh the weight of the boat plus the wet soil
  3. take the difference of the final weight ( boat and soil ) and initial weight (boat) to find the weight of the soil.
  4. put the soil outside for 48 hours to allow the water to evaporate.
  5. weigh the soil again.
  6. take the difference of the weight of the wet soil and dry soil to find weight of the water in the soil ( the water that evaporated)
  7.  calculate the percent of water in the soil by using     percent water= (weight of water/ weight of wet soil) x 100

Sand, Silt, Clay

  1. take 50 ml conical vial and fill it with soil sample to the 10 ml mark
  2. add DI water to the vial until it reaches the 30 ml mark.
  3. add soil dispersion liquid to the vial.
  4. shake vial vigorously for 30 seconds
  5. let the vial rest for 48 hours.

pH

  1. add a small amount of sand to the pH vial
  2. add water so that the level reaches the top of the vial
  3. shake vial for 10 seconds and then let is rest for 120 seconds
  4.  after 120 seconds, dip 1 inch of the pH paper in the vial for 45 seconds
  5. immediately compare the color of the strip to the scale to acquire pH

 

Analysis and Data:

there is no data that can yet be calculated. that will be done in pre lab calculation for the lab on 09/12/2018.

the following was collected for today

Mass of wet soil = 6.370 g

pH of soil= 6

the aseptic zone was properly maintained and there were no apparent events that may have caused contamination.

Future:

calculate % water, %sand, %silt, and % clay.

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
September 11

Soil Metadata and Enrichment of Soil B (09/10/18)

Rationale:

By conducting different procedures, different soil metadata, such as percent water, percent clay, percent silt, percent sand, and pH, is gathered for later use to see if there is any correlation in the data. By filtering and enriching the sample, the sample will be prepared for a spot test and plaque assay which will reveal the presence of Arthrobacter phages.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Started enrichment process by filling a 15 mL conical vial with 2 mL of the soil.
    • Labeled the conical vial as “KEA 9/7/18 Soil B.”
  1. Through aseptic technique (over an EtOH (100%) flame), filled “KEA 9/7/18 Soil B” conical vial with LB Broth to the 12 mL mark using a serological pipette with a bulb with a 10 mL tip.
  2. Shook “KEA 9/7/18 Soil B” conical vial for 15 minutes.
  3. Weighed “KEA 9/7/18 Soil B” conical vial.
  4. Centrifuged “KEA 9/7/18 Soil B” conical vial at 10,000g for 10 minutes.
  5. Started the soil metadata procedures by filling a falcon tube with 10 mL of soil.
    • This falcon tube was labeled “KEA 9/10/18 Soil B.”
  1. Filled the “KEA 9/10/18 Soil B” falcon tube with de-ionized (DI) water to the 30 mL mark.
  2. Added three drops of texture dispersion liquid to “KEA 9/10/18 Soil B” falcon tube.
  3. Used a disposable latex glove to cover the “KEA 9/10/18 Soil B” falcon tube while shaking it for 30 seconds.
  4. The “KEA 9/10/18 Soil B” falcon tube was placed under a flume hood to sit for 48 hours.
  5. Weighed a petri dish.
    • Labeled petri dish “KEA 9/10/18 Soil B.”
  6. Placed and weighed approximately 3.5 grams of soil onto “KEA 9/10/18 Soil B” petri dish.
  7. Set “KEA 9/10/18 Soil B” petri dish under flume hood for 48 hours.
  8. To test the pH, added a small amount of soil to a small pH vial.
  9. Filled this small pH vial with DI water to the top.
  10. Closed thumb over top of small pH vial and shook for 10 seconds.
  11. Allowed 2 minutes for the small pH vial to settle.
  12. Placed a small piece of pH paper into the small pH vial and left it in for 45 seconds.
  13. Recorded the pH and washed out small pH vial with water.
  14. Back to the enrichment process, the supernatant was then pipetted into a 0.22 μm top vacuum filter to separate the lysate.
  15. The lysate was collected in a 50 mL conical vial.
    • Labeled conical vial “KEA 9/10/18 Soil B enrich.”
    • Only collected a little less than 10 mL of lysate so all was used for the direct enrichment. None was saved for isolation.
  1. Added 0.5 Arthrobacter to “KEA 9/10/18 Soil B enrich” conical vial.
  2. Placed “KEA 9/10/18 Soil B enrich” conical vial in incubator at 28 ºC for 48 hours.
  3. The lab counter was cleaned with CiDecan and EtOH (70%).

Observations/Data:

  • With the addition of the LB Broth, the conical vial weighed 19.33 grams.
  • The empty petri dish weighed 7.58 grams.
  • The petri dish with soil weighed 11.00 grams.
  • The pH paper revealed that the soil sample had a pH of 6.0 as shown in the picture below.

  • As the falcon tube settled, the sand became apparent as shown in the picture below.

  • The supernatant was a light shade of yellow. The pellet of Soil B was a lighter brown than the Soil A sample. The picture below shows the “KEA 9/7/18 Soil B” conical vial after it was centrifuged.

 

Next Steps:

On Wednesday, finish procedures and conduct calculations to obtain the soil metadata for the percent water, percent sand, percent silt, and percent clay. Also, the lysate in “KEA 9/10/18 Soil B enrich” conical vial will be used to conduct a spot test.

Category: Kathryn Adkins | LEAVE A COMMENT
September 11

September 10 2018 Soil Washing and Enrichment and Metadata – Soil B

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.

Category: Lucy Potts | LEAVE A COMMENT
September 10

9.10.2018- Soil Sample B: Metadata and Enrichment

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.
Category: Henry Burns | LEAVE A COMMENT
September 10

Collecting Soil Metadata and Washing 9/10/18

Rationale: Today we are collecting metadata on the soil samples we collected last week in order to have data to minimize confounding variables in our findings, therefore increasing our reproducibility.

Procedure: Collecting Metadata:

  1. First, I prepared the percent water analysis. I weighed a petri dish by itself which was 7.57g. I then added some soil I collected from tree B, described in 9/5/18 notebook. The mass of the dish and soil was 12.51g.
  2. I placed the dish under the vent hood to let dry over the next 48 hours.
  3. Next, I prepared the soil, silt, clay analysis. I poured 10mL of the tree B soil

    into a falcon tube. I then filled it with DI water to the 30mL mark.

  4. I then added 3 drops of soil dispersion fluid then dropped my tube on the

    floor causing all the contents to spill.

  5. After taking proper precautions and cleaning up my area and cleaning out

    my falcon tube I repeated the last two steps.

  6. I shook the tube vigorously for a little less than a minute then I waited for a

    minute and placed it in a test tube rack to sit for 48 hours.

  7. Before starting my pH test I started my soil washing described below.
  8. I took a pH test vial and cap and added an extremely small amount of soil

    sample then filled the rest of the vial with water.

  9. I placed the cap on top and shook back and forth for about a minute then let

    sit until the soil settled.

10.I placed an inch-long piece of pH paper in the vial then analyzed the color

against the chart to find the pH.
11.I repeated the process to make sure my findings were correct and got the

same result. Soil Washing:

  1. Stored about 2mL of soil from tree B in a 15mL tube then filled with LB broth up to 12mL.
  2. Shook using a vortexer for about 14.5 minutes.
  3. Added 5 drops of DI water to get the right mass to match my partner within

    .1 grams.

  4. After spinning down my tube I filtered my supernatant using a 22μL filter

    and a vacuum to a 50mL tube.

5. I added 0.5mL of arthrobacter to the tube and placed the tube in a shaking incubator after labeling it “enriched”.

Observations:
The results of the soil, silt, and clay analysis were: 19.05% Clay, 2.381% Sand, and 78.57% Silt. The pH of the soil was about 8.0 or 8.5. Lastly, the weight after the 48 hours of drying was 12.21 grams. 4.94 grams of soil with moisture and 4.64 grams after drying therefore, a 6.47% moisture in the soil sample recorded.

Interpretation and Next Steps:
The soil was unusually basic and had a very low amount of moisture. This could either be a cause of the death of the tree or a result of the death of the tree. It is a good idea to keep this data in mind when I analyze my data further down the road, but for now I should proceed in performing a spot test and a plaque assay to search for the presence of phage in this soil.

Category: Sriram Avirneni | LEAVE A COMMENT
September 9

Plaque Assay Results and Soil Collection (Soil B)

Results:

On Friday, it was determined that my Plaque Assay for Soil A turned out negative without contamination.

Non-contaminated Control Plate

The negative plaque assay

Rationale:

Collect a new sample of soil, as well as come up with our scientific question

Scientific Question:

Each table section came up with their own question, and ours is, “Is there a difference in Arthrobacterphage in live oak trees at Baylor versus planted trees in developed areas?”

Procedure:

Our table groups (Groups 3 &4) came up with our scientific question, and then went out to collect soil (In a 15-mL vial and bag). My group (Group 4) was tasked with gathering soil from planted trees, so we went to Teal Residential College and looked at trees there. My tree is on the northern side, and closer to the building than the rest of the trees. I gathered soil two feet from the base of the tree, as well as took leaf samples for identification.

Observations:

  • My tree is not an oak tree, and I have yet to find out the specific type it is (I will have to look through a tree category book)
  • Although my tree had lots of brown leaves on it, most of it looked healthy and green
  • While digging through the soil, I found a grub of some sort

Next Steps:

We will be washing and filtering my soil on Monday, and will be creating a direct and enriched lysate

Category: Justin Yu | LEAVE A COMMENT