Rationale: Perform plaque assays to see if phage are present from two soil samples.

Procedure: 

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone.

Soil A, Soil B, and 10^0 serial dilution. 

Both plaque assays from 10/08/18 failed (soil A and B). The same procedure was done 10/08/18 was done today, except 10^0 dilution that was performed 9/14/18 was used to perform a third plaque assay. Since the results form 9/14/18 were successful, this plaque assay should have positive results. The formula below was used to make the solution for 4 plates (three for the experiment and one positive control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

Observations: 

Control from the experiment 10/8/18 was positive, and the two plaque assays came back negative. Instead of adding 10 microliters of lysate into 500 microliters of Arthrobacter, 100 microliters of lysate was added to the Arthrobacter. This was done to amplify the phage so that plaques will be visible.

 Plaque Assay Soil A

 Plaque Assay Soil B

Conclusions:

Perform plaques assays on 10/12/18 whether or not phage is present. If phage is present, pick the plaque to create a serial dilution. If the experiment comes back to be negative, then plaque assays will be performed, and the contamination will be solved if possible.

Rationale: Perform a plaque assay to show that the plaques are indeed plaques. Passage of the plaques from plaque picking to phage buffer.

Procedure:

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. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate. Then got an ethanol burner, and the aseptic zone was created. Used the 10^0 lysate that was made on Friday 9/28/18 since this passed the third round of purification. Added the 10^0 lysate to the 90 microliter PB into the 10^-1 solution, and pipetted/mixed well through the microcentrifuge. Labeled this solution as the 10^-1 solution on the microcentrifuge. Got one more microcentrifuge caps, labeled one cap 10^-2. Added 90 microliters of PB to the 10^-2 solution. Added 10 microliters of the 10^-1 solution to the 10^-2 solution. All microcentrifuge caps had the lysate solution, then added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2). Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.

The formula below was used to make the solution for 9 plates (three for Michael and Justin, two for Cooper, and one for the control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro

Added the 2XTA last to the 50mL vials. shook the vial, and quickly pipetted the solution onto each vial containing the Artho/lysate solution. Sat each plate for about 15mins to the solution solidify. The remaining solution that was left in the 50mL vial was used for the control. Added TA and poured that solution onto the last plate, which all the plates sat to solidify for 15 minutes.

Observations:

The last three experiments performed in the lab by group 4, the control had been negative. The class as a whole had also been experiencing contaminations in their controls, which affect the outcome of the experiments. The plaque assay procedure was not hard since the experiment has been done several times. With the experiment performed Wednesday 9/26/18, the 2XTA split. Why did this split? Group 4 thinks the reason why the 2XTA split was that after the pouring of the plates, the plates sat <15 minutes.

Next Steps:

Prepare for titer calculations, and to determine whether or not the third round of purification passes. Count the number of plaques present, and determine how much lysate will be needed to completely web a plate.

Rationale: The last plaque assays have all failed since it failed the third round of purification, and group four’s control was once again contaminated. Preformed a plaque assay to pick from the original plate that the first plaque was picked, and passed. Picked soil from 2nd street

Procedure:

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.

Soil Metadata

• Soil pH
  • added about 3mL of Soil B into a 15mL vial.
  • filled the rest of the vial with DI water.
  • shook the vial for about 10 seconds and then the vial sat for 2 minutes.
  • placed pH paper.
  • recorded the results.
  • pH is 6.5
  • 
• Percent(%) Water
  • got a petri dish and labeled it ML 10/3/18.
  • recorded the weight of the dish itself.
    • 2.39g
  • added soil on the dish.
  • recorded the weight of the soil.
    • 13.82g
  • 
  • Placed this in the hooded cabinet.
• Sand Silt Clay
  • added 4mL of Soil B into a 50mL vial tube.
  • added 12mL of DI water.
  • added three drops of the dispersion liquid.
  • shook for 30 seconds.
  • placed in the hooded cabinet
  • 

Plaque Picking

• retrieved the successful plaque assay that was performed on 9/12/18.
• added 100 microliters of PB into a centrifuge cap.
• picked a plaque from the plate.
• Filtered the plaque through the 100 microliters of PB.
• Grabbed a 15mL vial and added 2mL LB broth.
• added 22.5 microliters of calcium chloride into the 15mL vial.
• added 10 microliters of lysate into 500 microliters of Arthrobacter.
• added the lysate/Arthrobacter into the 15mL vial.
• added 2.5mL of 2XTA.
• mixed the solution, and quickly poured the solution onto a plate.

Observations:

Plaque assays from Monday’s experiment fail, and the control was negative. Picked soil from a new location, washed the solution, and the metadata for the Soil sample B.

Next steps/conclusions:

Plaque assays and spot test with new lysate. Record the metadata results from the %water and %sand, clay, and silt. Check the plates from the last plaque assay to see if any results. If there are plaques, then continue to round two of passage.

Rationale: Perform a second third round of purification since the first third round failed, since the LB Broth was contaminated. The goal of the purification

Question: Why did the control fail for the last two experiments?

• The last two experiments, the control were both negative.
• One reason why this could have been negative since the same LB broth was used for both experiments. The eight groups in the class labeled there own LB broth so that the groups in the class could see which groups had the contaminated control. This experiment was done on the side, and the class quickly saw which groups had a contaminated LB broth.
• This led to the redo of the third round of purification.

Procedure:   

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.

• Picked Plaque from Plaque Assay, which was performed on 9/21/18.
• Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
• Got two more microcentrifuge caps, labeled one cap 10^-1 and the other solution 10^-2.
• Added 90 microliters of PB to both caps.
• Added 10 microliters of the 10^0 solution into the 10^-1 solution.
  • The 10^0 solution has the picked plaque. Transferred this to the 10^0 solution so that this could be added to the other two caps so that three Plaque Assays could be performed.
• Added 10 microliters of the 10^-1 solution to the 10^-2 solution.
• All microcentrifuge caps had plaques, added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2).
• Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
  • The formula below was used to make the solution for 9 plates (three for each of the three solutions and one for the control).
    • 20mL LB Booth (x9)
    • 22.5 microliters of Calcium Chloride (x9)
    • 25mL 2X TA (x9)
• Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
• Sat each plate for about 15mins to the solution solidify.
• The remaining solution that was left in the 50mL vial was used for our control.
  • Added TA and poured that solution onto the last plate.
    • Side note: the control solidified <15 minutes.

Observations:

The experiment performed 9/21/18, the control of the experiment was contaminated. This led to the uncertainty of the plaque assays, whether or not it had plaques or not. Because of the results, the experiment was done again since the LB broth was contaminated from the last experiment. Not everyone’s control was contaminated, but only a few groups.

Next steps/Conclusions:

On Wednesday, determine wheater the plaques are plaques and make a webbed plate. Determine if the plate has a high titer or a low titer simply by doing some calculations. If the results are negative, pick a plaque from the second round of purification, or simply pick new soil.

Rationale: Test to see if the bacteriophage is present by performing multiple plaque tests.

Question: Why did 10^0 serial dilution not have any plaque, yet both 10^-1 and 10^-2 have plaques?

• From group four, only Justin had successful plaques in all three serial dilutions. Cooper had only one successful serial dilution, and Michael had two successful serial dilutions.
• Rule out the possibility of contaminations since Group 4 used the same formula for the plating solution.
• Control was contaminated, but all groups had a contaminated solution,
• One reason why plaques are not showing up in the 10^o serial dilution is that the bacteria could be in a lytic cycle, whereas Justin’s bacteria is clearly in a lysogenic phase. The bacteria in both Michael’s and Cooper’s experiment could both be in the lytic cycle, meaning the bacteria are “temperate” and not as expressive as a lysogenic cycle.

Procedure:

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.

• Picked Plaque from Plaque Assay from the 10^-1, which was performed on 9/17/18.
  • 10^0 did not have any plaque.
  • 10^-1 had three plaques.
  • 10^-2 had one plaque.
    • Side note: Since 10^0 did not have plaque, this experiment performed on 9/19/18 was done to test whether or not phage is present by only performing two 10^0 plaque assays. No serial dilution was done since the purpose of this experiment was to simply see whether or not phage presence from the 10^o.
• Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
• Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
  • This formula was used to make our solution for 7 plates (two for each of the 10^0 solutions and one for the control).
    • 20mL LB Booth (x7)
    • 22.5 microliters of Calcium Chloride (x7)
    • 25mL 2X TA (x7)
• Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
• Sat each plate for about 15mins to the solution solidify.
• The remaining solution that was left in the 50mL vial was used for the control.
  • Added TA and poured that solution onto the last plate.

10^0 Plaque Assays

Control

Observations:

• Group four had successful plaque assays from the experiment performed on 9/17/18.
• Justin had plaques on all three serial dilutions, whereas Cooper and Michael did not. Michael had plaques on his 10^-1 and 10^-2, and Cooper had only one plaque on his 10^-2 dilution.

 10^0 Plaque Assay

10^-1 Plaque Assay

10^-2 Plaque Assay

• control was contaminated.
  • Possible reason: LB broth contamination.

Next Steps/Conclusions:

On Monday, check all two plates to see if any plaques appear. If plaques do appear, perform the experiment again by picking the plaque of one of the plates. If no plaque, simply redo the experiment with the plaques assay that does have plaque (experiment performed on 9/12/18). Overall, the experiment was very easy since the experiment was done on Monday. The hard part was to determine where the plaques were, and whether if or not plaques were actually there, which they were.

Rationale: Pick Plaque from the plaque assay and to perform serial dilutions. Performed several plaque assays to isolate phage, and made a sample control to go along with our experiment.

Procedure: 

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.

• Picked Plaque from Plaque Assay, which was performed on 9/12/18.
• Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
• Got two more microcentrifuge caps, labeled one cap 10^-1 and the other solution 10^-2.
• Added 90 microliters of PB to both caps.
• Added 10 microliters of the 10^0 solution into the 10^-1 solution.
  • The 10^0 solution has the picked plaque. Transferred this to the 10^0 solution so that this could be added to the other two caps so that three Plaque Assays could be performed.
• Added 10 microliters of the 10^-1 solution to the 10^-2 solution.
• All microcentrifuge caps had plaques, added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2).
• Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
  • This formula was used to make our solution for 10 plates (three for each of the three solutions and one for the control).
    • 20mL LB Booth (x10)
    • 22.5 microliters of Calcium Chloride (x10)
    • 25mL 2X TA (x10)
• Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
• Sat each plate for about 15mins to the solution solidify.
• The remaining solution that was left in the 50mL vial was used for our control.
  • Added TA and poured that solution onto the last plate.
    • Side note: the control solidified <15 minutes.

Observations:

• Group 4 had plaques on all plaque assays, but only Justin had a plaque from his spot tests.
• On the plate of the plaque assay, it was very hard to identify a solid plaque but contained multiple little spots that could well be possible plaques.

 Plaque Assay with Plaque

• Spot Tests had zero plaque but had air bubbles. Justin had spots form group four.

Additional Questions:

Question 1. Group four all had plaques from their plaque assays, but only Justin had a plaque from his spot test.  This could have resulted from the soil since all three group members got soil from three different trees. Justin’s soil could have different phage from his group members, resulting in Justin’s better-defined plaques. The soil samples from group 4 at least have some similarities, given everyone in the group had a successful plaque assay.

Question 2. 

Lathan needs 4.01 microliters to web his plate (work is shown above).

Next Steps/Conclusions:

On Wednesday, check all three plates to see if any plaques appear. If plaques do appear, perform the experiment again by picking the plaque of all three plates. If no plaque, simply redo the experiment with the plaques assay that does have plaque. Overall, the experiment was very easy since Lathan’s lecture video basically covered everything that was needed to be known for this week lab. The hard part was to determine where the plaques were, and whether if or not plaques were actually there, which they were.

Rationale: Isolate bacteriophage through both a Spot Test and Plaque Assay., and to collect more metadata soil sample B.

Procedures:

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.

Spot Test and Plaque Assay

Spot Test

• The enriched lysate was made on 9/10/18.
• Measured the mass of the enriched lysate. Mass of enriched lysate: 20.47 g.
•  The enriched lysate was spun in the centrifuge for 5 minutes at 3000 x g.
• The enriched lysate was filtered and put into a small cap.
• Divided one plate into four quadrants
  • I – ML Spot Test
  • II – CJJ Spot Test
  • III – JLL Spot Test
  • Buffer
• Added 2mL LB broth into a 50mL vial.
• Added 22.5 microliters of M Calcium Chloride in the 50mL vial.
• Added 0.5mL OF Artho PA Phage lysate
• Added 2.5 mL TA into the 50mL vial.
• Poured our solution onto the one plate that is divided into four quadrants.
• Sat the petri dish aside to let TA solidify (1o minutes).
• Added 0.5 microliters directed (filtered) lysate on top of the TA.
• Added the buffer onto the last quadrant, our negative control.
• After all the quadrants were filled with a spot, we placed the plate in the incubator.

Plaque Assay

• Got four different petri dishes, one for each lab partner, and the fourth one being our control buffer.
• In each dish we added:
  • Added 2mL LB broth into a 50mL vial.
  • Added 22.5 microliters of M Calcium Chloride in the 50mL vial.
  • Added 0.5mL OF Artho PA Phage lysate
  • Added 2.5 mL TA into the 50mL vial.\
    • Note this times 4 since we had to fill 4 petri dishes.
• We then separated the solutions into 15mL vials and we added our 0.5 microliters of direct lysate into the 15mL vial.
• Poured the Solution onto my petri dish labeled ML 9/12/18 Plaque Assay.
• Let the solution sit for 15 min and placed in the incubator.
• Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
• Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.

Soil Metadata

• Percent water of the soil was calculated to be 11%.
  • Mass Dry= 10.8
  • Mass Wet= 12.09
  • 10.8/12.09=.89
  • 1-.89=.11=11%
    • Side note: Lots of debris which could have an effect on the % water.

• The mass of the soil was recorded and left to sit under a vented hood in the lab (this was done 9/10/18). This allowed the procedure above to be done today in lab.
• % Soil, Clay, and Silt was then calculated
• 50mL vial was left under a vent hood from Monday to let the soil disperse.
• The vial was observed and these are the results.
  • 1.5mL Sand
  • .5mL Clay
  • 2mL Silt

Observations:

• The soil had a lot of debris in both the % water mass and the vial shown above which was used to measure % soil.
• Results from the metadata:
  • 11% Water
  • 1.5mL Sand
  • .5mL Clay
  • 2mL Silt
  • 6.5pH

Conclusions/Next Steps:

The overall spot test and plaque assays procedures were easy since we knew what to expect. We were better prepared versus last time. Next steps: to see if our Bacteriophage has been isolated, and if so, we will try to amplify it.

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.

Scientific Question:

What is the difference in the amount of bacteriophage present between the soil surrounding newly planted trees versus the trees that are well established?

Rationale:

Under the premise of our newly defined scientific question, we collected soil samples from newly planted trees. We will compare the soil samples from age established oak trees to newly planted oak trees to see if bacteriophage are present in one or the other. We will eventually ascertain a lysate from this soil sample so we can test it through both spot tests and plaque assays for the presence of phage, to test our question.

Procedures:

• As a class, attempted to develop a question, but then as a table, we developed our own question.
• I then went out and collected a soil sample with my 15mL vial and a plastic bag so I could have extra soil. Collected the soil without touching the soil with my hands to rule out any possible contaminations from the beginning. I labeled the bag 9/5/18 ML Soil B.
• I then recorded the height measurements of the tree and recorded it onto Canvas, which I will finish entering the data gathered.
• Back in the lab, I transferred some of the soil in my 15mL vial into the bag since we only 2mL of soil in the vial. I then put everything into the plastic bag, my 15mL vial with 2mL of soil, soil B, and my three leaves.

Observations/Metadata:

The tree in which I gathered my soil samples were right in of the Baylor Science Building on 2nd Street. My trees height was roughly about 5m tall, and roughly 8m across. The trunk was roughly about 58cm, which indicates it is one of the new trees on campus. Observing the tree, trunk looked healthy, the leaves were green (only 3-10 leaves were brown), and the branches throughout the tree looked proportional. The tree looked healthy with no evidence of damage.

Conclusion/Next Step:

From the class conversation about our Scientific Question, we changed our initial point of view from a broad topic to a more narrow topic. We designed a question that was testable in our hands, and we went to gather soil. The sample that was collected will allow us to test if we have bacteriophage present in our soil/tree via spot tests and plaque assays. From this information, we will then be able to begin answering our many questions regarding the amount of bacteriophage present in recently planted trees versus trees that are well established.