• NOVEMBER 9TH, 2018
  • OBJECTIVE:
    • Create a lysate from the new soil sample collected 
  • PROCEDURE:
    • • Soil was filled to the 2mL mark of a test tube 
      • Then 10mL of LB broth was added to the tube 
      • It was shaken fro 15 minutes, before then bing placed into the centrifuge for 10 minutes where it was then spun at 3,000G 
      • Once the tube being centrifuged was done, a top filter was then used to filter out the supernatant 
      • Then .5mL of Arthrobactor was added to the filtered supernatant 
      • The tube was then left in the incubator 
  • RESULTS: 
    • No results to report 
  • CONCLUSION: 
    • No results to discuss
  • NEXT STEPS:
    • Run a plaque assay 
• NOVEMBER 12TH, 2018
  • OBJECTIVE:
    • To plate the plaque assay with no contamination 
  • PROCEDURE: 
    • Tables were cleaned, lamps were lit
    • A test tube was filled with: (2X TA was added last to prevent solidifying) 
      • 4mL LB broth
      • 45𝝁L  CaCl2
      • 5mL 2X TA
    • 15 𝝁L of lysate was pipetted into test tube containing arthro and was left to sit for 15 minutes 
    • After the 15 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the  lysate and arthro 
    • The test tubes were then poured into the plates, which were then given 10 minutes to solidify
    • Plates were then inverted and placed in the incubator 
  • RESULTS: 
    • As seen in Figure 21, the control and test plate were free of contaminants (control is top plate, bottom is test plate)
  • CONCLUSION: 
    • Its very difficult to view in Figure 21, but there were 5 small clear circles which are believed to be bacteriophage, and will be tested
  • NEXT STEPS: 
    • Pick plaques 
• NOVEMBER 14TH, 2018
  • OBJECTIVE: 
    • To pick what is believed to be plaques, and plate them without contamination 
  • PROCDURE:
    • Tables were cleaned and lamps were lit
    • The plaques were circled and labeled 
    • 5 micro centrifuge tubes were filled with 100 𝝁L of phage buffer 
      • A compound microscope was used to view the plaques, which were then “picked” by gently tapping the plaque with the end of a micro pipet 
      • The pipet is then placed into the micro centrifuge tube containing 100 𝝁L phage buffer 
      • The tube was then vortexed for 2 seconds 
    • The steps above were repeated for every plaque 
      • A test tube was filled with: (2X TA was added last to prevent solidifying) 
        • 12mL LB broth
        • 135𝝁L  CaCl2
        • 15mL 2X TA
      • 15 𝝁L of lysate was pipetted into test tube containing arthro and was left to sit for 15 minutes 
      • After the 15 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the lysate and arthro 
      • The test tubes were then poured into the plates, which were then given 10 minutes to solidify
      • Plates were then inverted and placed in the incubator
  • RESULTS: 
    • The results can be seen in Figure 22, 23, and 24
    • As it can be seen the control was contaminated, and samples P1, P2, and P5 were all negative for phage presence 
    • Samples P3 and P4 were positive for phage presence, as seen in Figure 23, and 24
  • CONCLUSION: 
    • Samples P4 and P3 both contain phage, while the other samples appear to be negative. The source of contamination on the control is unknown, and may be due to exposure to bacteria during plating. 
  • NEXT STEPS:
    • Begin serial dilution, then flood plate

• OCTOBER 29TH, 2018
  • OBJECTIVE:
    • To enrich new sample again and run soil metadata 
  • PROCEDURE:
    • Tables were cleaned and lamps lit
    • The samples that were incubated last time were contaminated 
    • The sample was discarded and a new enriched lysate was made:
      • Soil was filled to the 2mL mark of a test tube 
      • Then 10mL of LB broth was added to the tube 
      • It was shaken fro 15 minutes, before then bing placed into the centrifuge for 10 minutes where it was then spun at 3,000G 
      • Once the tube being centrifuged was done, a top filter was then used to filter out the supernatant 
      • Then .5mL of Arthrobactor was added to the filtered supernatant 
      • The tube was then left in the incubator 
  • RESULTS:
    • No results to report 
  • CONCLUSION:
    • No results to report 
  • NEXT STEPS: 
    • Run plaque assay
• OCTOBER 31ST, 2018
  • OBJECTIVE:
    • Plans were changed, and new groups were assigned, where group members will be adopting phages
    • Goal —> adopt new plaque and get high titer!
  • PROCEDURE: 
    • Group member  calculated the the amount to make a high titer 
    • High titer amount= 546mL
    • Plate was flooded with 8mL phage buffer and left over night 
  • RESULTS:
    • No results to report 
  • CONCLUSION:
    • No results to report 
  • NEXT STEPS: 
    • Plate high titer 

• OCTOBER 22ND, 2018
  • OBJECTIVE:
    • Conduct a gel electrophoresis to test if the PCR was positive 
  • PROCEDURE:
    • .8 grams of the agarose powdered was measured out
    • Then 40mL of 1X TAE was put into a flask
    • The agarose powder was then poured into the flask, the flask was then repeatedly swirled 
    • The flask when then placed in the microwave, and heated until it started to boil 
    • The flask was then taken out and swirled until it cooled, and was just warm (instead of burning hot)
    • Then 2𝝁L EtBr was added by a Teaching Assistant 
    • The solution was then poured into a gel apparatus, where a gel comb was then inserted 
    • Then 20 minutes was allowed for gel to cool
    • Gel comb was removed 
    • Then the box holding the gel was filled with 1X TBE to submerge to gel 
    • Next 10 𝝁L of each sample was loaded into the appropriate wells seen in the figure below: 
    • The lid was then placed on the box, and the power cords were connected to the power apparatus, and set at 100 volts (make sure positive end is on end away from wells)
    • This was run for 40 minutes, then gels were taken to MCB to be imaged by Teaching Assistant 
    • Imaging was done by cleaning the UV plate the with ethanol, placing the gel on it, then placing it in the BioRad Gel Doc EZ imaging machine 
  • RESULTS: 
    • The control gel results came out normal, or as expected 
    • The plate ran with all the samples was negative for any DNA presence 
  • CONCLUSION:
    • Since there was no DNA present in the gels, it can be determined that the soil samples obtained do not contain phage 
  • NEXT STEPS: 
    • Collect new sample, then wash sample to run another PCR
• OCTOBER 24TH, 2018
  • OBJECTIVE:
    • Wash new soil sample to prepare to run PCR
  • PROCEDURE:
    • Soil was filled to the 2mL mark of a test tube 
    • Then 10mL of LB broth was added to the tube 
    • It was shaken fro 15 minutes, before then bing placed into the centrifuge for 10 minutes where it was then spun at 3,000G 
    • Next a well plate was obtained, and 2mL of the supernatant was placed into each of the three wells 
    • The well plate was then heated to 55C for 5 minutes 
    • The sample was then cooled or 5 minutes
    • Then .5 𝝁L of arthro was added to each well 
    • The well plate was then incubated at 28C and were wired at 150 RPM 
  • RESULTS:
    • No Results to report 
  • CONCLUSION:
    • No Results to report 
  • NEXT STEPS: 
    • Run PCR

• OCTOBER 12TH, 2018
  • RESULTS: (RESULTS FROM 10/10 WERE VIEWED UNTIL 10/12)
    • The results showed in Figure 17 show a small amount of contamination on the arthro control, and TA control and the plaque assay were clear of any contamination
    • The plaque assay was negative for phage presence 
  • CONCLUSION:
    • The contamination seen on the arthro control is most likely from contamination from a airborne bacteria that ended up on the plate. Therefore we know the arthro was not contaminated, and it can be concluded that this soil sample does not contain phage. 
  • OBJECTIVE:
    • Enrich new soil sample 
  • PROCEDURE: 
    • The sample was retrieved from the incubator when it was noticed that the sample was pink due to contamination that can be seen in Figure 18
    • Then the sample was placed into the centrifuge for 10 minutes at 3,000G 
    • The bacteria then gathered at the bottom of the tube as seen in Figure 19 
  • RESULTS:
    • Due to contamination the sample could not be used 
  • CONCLUSION:
    • The sample was discarded due to the contamination having affecting the potential phage population, if there was phage present. 
  • NEXT STEPS:
    • Wash and enrich the sample soil sample
• OCTOBER 15TH, 2018
  • OBJECTIVE: 
    • To wash the soil sample and have no contamination 
  • PROCEDURE:
    • Soil was filled to the 2mL mark of a test tube 
    • Then 10mL of LB broth was added to the tube 
    • It was shaken fro 15 minutes, before then being placed into the centrifuge for 10 minutes where it was then spun at 3,000G 
    • One the tube being centrifuged was done, a syringe and .22 𝝁m sister was then used to filter out the supernatant 
      • • Then .5mL of Arthrobactor was added to the filtered supernatant 
        • The tube was then left in the incubator 
  • RESULTS: 
    • No results to report
  • CONCLUSION:
    • No results to discuss 
  • NEXT STEPS:
    • Conduct PCR
• OCTOBER 17TH, 2018
  • OBJECTIVE 
    • conduct PCR, and understand the fundaments behind it
  • PROCEDURE:
    • Lysate was spun in the centrifuge for 5 minutes at 3,000G 
    • 2mL of the spun lysate was put into a micro centrifuge tube and was boiled 
    • After the sample was boiled 
    • Four tubes each containing 12.5𝝁L  Taq + dNTPS were then labeled “1” “2” and “3” and “c” 
    • The tubes labeled 1,2, and 3 all had 1𝝁L of DNA added from each soil sample (samples 5L and 5S)
    • The tube labeled c (serving as the control) then had 1𝝁L of dd water added to it
    • 4 𝝁L of the corresponding primer was added to tubes 1,2, and 3 (ex. primer P1 was added to tubes 1) 
    • 4 𝝁L of P2 was added to the control tube 
    • Then 6.5 𝝁L of dd water was added to each tube 
    • The sample was then taken to be run through the PCR machine by T.A 
  • RESULTS:
    • Waiting on results
  • CONCLUSION
    • Waiting on results 

• • NEXT STEPS:
    • Conduct gel electrophoresis 

• OCTOBER 5TH, 2018
  • RESULTS: (META DATA RESULTS WERE NOT VIEWED UNTIL 10/5)
    • Soil meta data Results:
      • Sand: 65%
      • Silt: 25%
      • Clay: 10%
  • CONCLUSION: 
    • The soil composition has been determined, now the percent water must be calculated in order to have more data on the soil sample. 
  • OBJECTIVE: 
    • Plate plaque assay with no contamination 
  • PROCEDURE:
    • Tables were cleaned and lamps were lit
    • The sample was filtered using a syringe to push the solution through a .22𝝁m filter into a micro centrifuge tube
    • A weigh boat was massed and then had wet soil added which was then massed 
    • 10 𝝁L of the lysate was then pipetted into the Arthro, and was left to sit for 10 minutes 
    • During the 10 minutes a test tube was filled with: (TA is added last) 
      • 6mL LB broth
      • 7.5mL 2X TA 
      • 67.5 𝝁L CaCl2
    • 4.5mL of the 2X TA solution was added to the test tube with the lysate and Arthro 
    • Before the tubes were able to be poured into plates, the 2X TA solidified in the test tubes, so the tubes were held over an open flame to put it back into a liquid form
    • It was then poured onto plates and were inverted and placed into the incubator
  • RESULTS: 
    • Percent Water:
      • Plate: 7.575g
      • Wet soil + plate: 9.940g
      • Dry soil + plate: 9.420g 
      • % water: 5.23%
    • Plaque Assay Results:
      • As seen in Figure 15, the control plate has no contamination, while the plaque assay has been contaminated
  • CONCLUSION: 
    • It was discovered that the arthro used in the experiment was contaminated, hence why the control, which has no arthro on it, was not contaminated while the plaque assay, which contains arthro, was contaminated. This has rendered the results to be inconclusive. 
  • NEXT STEPS:
    • Conduct the plaque assay with the same soil sample, due to contamination rendering the results inconclusive 
• OCTOBER 8TH, 2018
  • OBJECTIVE: 
    • Conduct another plaque assay with the same sample 
  • PROCEDURE: 
    • Tables were cleaned, lamps were lit
    • A test tube was filled with: (2X TA was added last to prevent solidifying) 
      • 8mL LB broth
      • 90 𝝁L  CaCl2
      • 10mL 2X TA
    • 10 𝝁L of lysate was pipetted into test tube containing arthro and was left to sit for 10 minutes 
    • After the 10 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the  lysate and arthro 
    • The test tubes were then poured into the plates, which then solidified, and were then inverted and placed in the incubator 
  • RESULTS: 
    • As seen in Figure 16, the control is not contaminated while the plaque assay is contaminated 
  • CONCLUSION:
    • The source of the contamination is unknown, it could potentially be arthro or a contamination issue that is occurring during the pouring of the TA 2X solution on to the plate.
  • NEXT STEPS: 
    • A new soil sample will be collected, and a plaque assay will be conducted one more time with the same sample, and an arthro control will also be conducted to help determine the source of contamination 
• OCTOBER 10TH, 2018 
  • OBJECTIVE: 
    • Wash new soil sample, and conduct another plaque assay with the old sample and have no contamination 
  • PROCEDURE:
    • Tables were cleaned and lamps were lit
    • A test tube was filled to the 2mL mark with soil, which then had 10mL of LB broth added to it 
    • The tube was then shaken for 15 minutes
    • The tube was then centrifuged for 10 minutes at 3,000 G
    • During this time the 10 𝝁L  lysate (from the previous sample) was put into a test tube with arthro and was also left to sit for 10 minutes
    • A separate test tube containing arthro had 10 𝝁L of phage buffer added (this will serve as arthro control) 
    • A large test tube was then filled with: (2X TA was added last to prevent solidifying)
      • 10mL LB broth
      • 112.5 𝝁L  CaCl2
      • 12.5mL 2X TA
    • After the 10 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the  lysate and arthro, and the test tube with phage buffer and arthro 
    • The test tubes were then poured into the plates, which then solidified, and were then inverted and placed in the incubator 
    • After the soil sample was done being centrifuged, a top filter was used to filter the supernatant 
    • .45mL of arthro was then added to the filtered supernatant, which was then placed into the incubator
  • RESULTS:
    • Waiting for results 
  • CONCLUSION:
    • Waiting for results
  • NEST STEPS: 
    • Conduct a plaque assay with new sample 

• OCTOBER 1ST, 2018
  • RESULT ANALYSIS AND NEW SOIL COLLECTION
    • RESULTS: (RESULTS FROM 9/26 WERE NOT VIEWED UNTIL 10/1)
      • As seen in Figure 14 the control and plaque assay plate were both clear of contaminants
      • The plaque assay seen in the lower plate on the picture is negative for phage presence 
    • CONCLUSION: 
    • • The plaque assay was negative for phage presence therefore, it can be concluded that the soil sample does not contain phage. 
      • NEXT STEPS: 
      • A new soil sample will obtained and a new plaque assay will be conducted with the sample 
    • OBJECTIVE: 
      • Obtain a new soil sample
    • PROCEDURE: 
      • A new soil sample had to be collected, and a tree was located outside of Mclane stadium
      • A shovel was used to collect to dirt 2 feet away from the base of the tree, then the tree’s height, canopy diameter, and trunk circumference were measured 
      • Data for the tree will be entered into tree data survey 
    • RESULTS: 
      • No results to be recorded 
    • CONCLUSION:
      • Nothing to conclude 
    • NEXT STEPS: 
      • To wash new soil sample
• OCTOBER 3RD, 2018
  • SOIL WASHING AND FILTRATION
    • OBJECTIVE: 
      • To wash and filter soil to prepare for plaque assay 
    • PROCEDURE: 
      • Tables were cleaned and lamps were lit
      • A test tube was filled to the 2mL mark with soil, which then had 10mL of LB broth added to it 
      • The tube was then shaken for 15 minutes
      • The tube was then centrifuged for 10 minutes at 3,000 RPMs
      • A falcon tube was then filled with 10mL of soil, and was then filled to the 30mL mark with DI water
        • 3 drops of dispersion fluid were then added to the tube, and the tube was then shaken for 30 seconds, then was left to settle
      • Once the tube being centrifuged was done, a top filter was then used to filter out the supernatant 
        • Then .5mL of Arthrobactor was added to the filtered supernatant 
        • The tube was then left in the incubator 
  • RESULTS: 
    • Waiting for metadata results 
  • CONCLUSION:
    • Waiting for metadata results 
  • NEXT STEPS: 
    • Plate plaque assay 

• SEPTEMBER 21ST, 2018
  • SOIL FILTRATION
  • OBJECTIVE: 
    • Filter out new soil sample 
  • PROCEDURE: 
    • Tables were cleaned and lamps were lit 
    • 4 mL of soil was put into a test tube and filled to the 24mL mark with LB broth 
    • Tube was then shaken for 15 minutes 
    • Then a separate tube was filled with water to be its “mass buddy” and it was centrifuged for 7 minutes 
    • Next a weigh boat was massed and wet soil was added and massed 
      • The weigh boat its the soil was then left to dry in the fume hood
    • A falcon tube was then filled with 10 ml of soil and was then filled to the 20 ml with DI water
      • Then 3 drops of dispersion liquid was added to the tube, it was then shaken for 30 seconds and then left to sit over night
    • Then the tube that was then spun was retrieved and then had the supernatant filtered using a top filter 
    • 10mL of the supernatant was left in the test tube and 5mL was put into a different test tube 
    • The test tube containing the 10mL had .4mL of Arthro added to it (enriched sample)
    • Was then left to incubate 
    • The 5mL test tube was then put in the fridge (direct sample)
  • RESULTS: 
    • Soil Meta Data 
      • Sand 65%
      • Silt 15%
      • Clay 20%
  • Water percent
    • • Mass of plate: 7.57g 
      • Mass of wet soil: 13.01g
      • Mass of dry soil: 12.23g
      • Mass of water: .78g
      • 6% water
  • CONCLUSION:
    • Metadata was collected, will now conduct plaque assay next to test for phage presence 
  • NEXT STEP: 
    • Conduct a plaque assay 
• SEPTEMEBER 24TH, 2018 
  • PLATING PLAQUE ASSAY 
  • OBJECTIVE: 
    • Conduct a plaque assay with NO CONTAMINATION
  • PROCEDURE:
    • Tables were cleaned and lamps were lit
    • Direct and enriched samples were filtered out using a syringe and a .22 𝝁m filter 
    • Next, a large test tube was filled with:
      • 10 mL of LB
      • 12.5 mL of 2XTA 
      • 112.5 CaCl 
    • .10 𝝁L of lysate was then added to the containing .5mL of Arthro and was left for 15 minutes 
    • The tube contains the 2X TA solution was then placed in a hot water bath so it would not solidify 
    • Next 5mL of the TA solution was added to a plate, where 10 𝝁L of the direct were added to it 
    • The plate was swirled then was left to solidify
    • Another 5mL was pipetted onto a different plate to serve as control 
    • Next the tubes contain the lysate and Arthro had 5mL of the TA solution added to it
      • Solution was mixed then was poured onto plates 
    • Plates were left to solidify and were then inverted and placed into incubator 
  • RESULTS:
    • The results as seen in figure 13 were contaminated due to the Arthro sample being a different bacteria 
  • CONCLUION: 
    • The results seen have been deemed inconclusive due to laboratory issue with Arthro 
  • NEXT STEPS: 
    • Conduct another plaque assay 
• SEPTEMBER 26TH, 2018
  • PLATING ANOTHER PLAQUE ASSAY (SAME SAMPLE)
  • OBJECTIVE:
    • To conduct another plaque assay without contamination 
  • PROCEDURE:
    • Test tubes containing .4mL of Arturo had 10 𝝁L of lysate added to it, and was left to sit for 10 minutes
    • A large test tube was filled with 
      • 10mL of 2X TA 
      • 90 𝝁L of CaCl
      • 8.4 mL LB broth 
    • 5mL of the TA solution was poured onto a plate and served as the control 
    • Then 5mL of TA solution was put into the tubes contains the Arturo and lysate
      • The contents of the tubes were mixed then poured onto plates 
      • The plates then solidified for 10 minutes and were then inverted and placed in the incubator 
  • RESULTS: 
    • Waiting to view plates and obtain results
  • CONCLUSION:
    • Waiting on results 
  • NEXT STEPS: 
    • View results and determine if phage are present, if so dilutions will be carried out, and if not a new soil sample will be collected 

• SEPTEMBER 17TH 2018
  • RESULTS: (following results and conclusion are from 9/12/18 and were not viewed until 9/17/18)
    • Spot test, seen in figure 7, was cloudy, had a yellow liquid in the bottom of dish, and was contaminated due to it not being inverted 
    • The control, seen in figure 8, was cloudy from contamination, and also had yellow liquid  gathered in the bottom of the dish, was most likely contaminated from not being inverted 
  • CONCLUSION: 
    • As a result of the contamination the plates were cloudy and had no signs of plaques being present. A plaque assay will be conducted next to determine if there are plaques or not. One group member did have plaques present, and her sample was obtained from Baylor’s campus meaning her soil had been exposed to pesticides. 
  • SPOT TEST ANALYSIS + PLAQUE ASSAY 
  • OBJECTIVE: Conduct a plaque assay with no contamination 
  • PROCEDURE:
    • Tables were cleaned and lamp was lit
    • .10𝝁L of lysate was pipetted into a test tube containing .5mL of arthrobactor and was left to sit for 15 minutes
    • A large test tube (50mL)  was then filled with: 
      • 8mL LB broth
      • 90𝝁L CaCl
      • 10mL 2X TA
    • The control plate accidentally had 9.5 mL of the top agar solution added to the plate, therefore there was only enough for 2 group members to have the solution added to their lysate
    • Next the 4.5mL of the original solution was added to the test tube containing the lysate and arthrobactor 
    • The test-tube was then poured onto the plate and left to solidify for 15 minutes 
    • During the 15 minutes a different group member (SA) made a double batch of the TA solution and conducted a separate control
  • RESULTS: 
    • The control plate, seen in figure 9 had many spots on it from contamination, also had yellow 
    • The spot test is cloudy from contamination as seen in figure 10
    • As seen in figures 9 and 10 there is contamination on both plates. Contamination could possibly be from Micropipettes or LB.   
  • CONCLUSION: 
    • Due to the plates showing no plaques after a spot test and plaque assay, the group had decided to collect new soil samples, and keep continuing investingng the question: Do trees sprayed with pesticides have a higher concentration of phage than trees that have not been treated? (species of tree being test has yet to be determined). Also due to contamination from now on, LB bottles used will be labeled by groups, and all micro pipettes have been cleaned with ethanol to help diminish the chanced of future contamination. 
  • NEXT STEPS:
    • Collect new soil samples from Cameron Park and the Baylor campus 
  • EXTRA- Critical Thinking Problems:
    • 1)Group 4 all had plaques on their plaque assays. Justin had the most and most well defined  plaques (but all 3 had plaques). They each did a spot test in. Addition to their play assays but only Justin had plaque on his spot…what do you think is going on? I think that since Justin had the most, and most well defined plaques that his phages were probably very stable when put into the spot test so his pages were probably the most likely to survive since he had a higher concentration of pace and had “stronger” or more well preserved plaques. The other group members phages probably weren’t in high enough concentrations, or were in a “weaker” state and were unable to be put into a spot test with out falling apart.
    • 2) Lathan Checked a purified lysate by doing a play assay of a 10^-3 lysate. He counted 14 plaques. How many 𝝁L of Lathan’s 10^0 lysate should he add to web a plate (75mm in diameter) if his average plaque diameter is 1mm? .004017 mL
• SEPTEMBER 19TH 2018
  • GRAM STAINING OF CONTAMINANTS 
  • OBJECTIVE:
    • Determine the source of contamination 
  • PROCEDURE: 
    • .10 𝝁L of water was put on 2 spots onto a slide
    • Inoculating loop was dragged across plate and mixed into one water spot
      • Was done for control and play assay 
    • Water was then left to air dry
    • Then the slides were dragged over a flame multiple times to heat fix the bacteria 
    • Then crystal violet was applied to both samples for 1 minute and were then rinsed 
    • Next, potassium iodine was applied to both samples for one minute and was the washed away with water
    • Then ethanol was applied to the samples for 30 seconds, and was then washed away with water 
    • Lastly, Zafrin was applied to both sampled for 1 minute and was then washed away by water 
    • Samples were dried, then viewed under a compound microscope
  • RESULTS: 
    • As seen in figures 11 and 12 the control and plaque assay were both contaminated by a cocci gram-negative bacteria 
  • CONCLUSION:
    • It appears that arthrobactor was the possible bacteria present on both plates. It was a gram negative cocci, which correlates with arthrobactor. 
  • NEXT STEP: 
    • Collect new soil sample from Cameron Park.

FIGURES 7-12:

• SEPTEMBER 10TH, 2018
  • SOIL WASHING AND METADATA
• OBJECTIVE: To wash new soil samples collected and to perform metadata on the soil as well
• PROCEDURE:
  • Tables were cleaned 
  • Next soil samples were used to fill a test tube to the 2mL mark, filled to the 12mL mark with LB, and were then shaken and centrifuged for 15 minutes
  • The tubes were then taken to spin @10,000G for 5 min 
  • During that time 10mL of soil was added to a conical vile 
  • DI water was added to the vile until it hit the 30mL mark
  • Then 3 drops of soil dispersion fluid was added to the vile, where it was then shaken for 30 seconds 
  • The test tube of soil and broth was taken to a fume hood where the supernatant was poured into a top filter, where 10mL was filtered out 
  • Then the 10mL filtered supernatant  then has .5 mL of arthrobactor added to it and was left to incubate
  • Wet soil sample was weighed out and then left to dry 

– NEXT STEPS: Conduct spot test

• • SEPTEMBER 12TH, 2018
    • SPOT TEST
    • OBJECTIVE: Conduct a spot test without any contamination 
    • PROCEDURE: 
      • Tables were cleaned 
      • Dry soil sample was weighed out 
        • Plate without soil: 7.20g
        • Wet soil: 10.02g
        • Dry Soil: 9.45g
        • Water: 0.57g 
        • %Water: 
    • Then meta data was analyzed: 
      • • %sand: 54.02%
        • %clay: 26.44%
        • %silt: 19.54%
    • My sample was then masses and a “spin buddy” was found
    • The sample was spun at 3000G for 5 min
    • Then a syringe was used to push the spun supernatant through a .22𝝁m filter out 
    • Then a test was made with the following:
      • • 6mL LB
        • 1.5mL Arthro
        • 7.5mL 2X TA
        • 67.5𝝁L of 1M CaCl 
• 5mL of the contents of test tube were pipetted onto 3 plates and left to sit for 15 min
• After 15 min .5𝝁L of lysate of was pipetted onto plate
• Was left to incubate
• NEXT STEPS: wait to see results from spot test

• SEPTEMBER 5TH, 2018
  • QUESTION DEVELOPMENT AND PLAQUE ASSAY ANALYSIS 
  • OBJECTIVE: Develop a question for the class to investigate for the semester, and to analyze the results of the plaque assay conducted 
  • POSSIBLE QUESTIONS: 
    • Comparison of concentration of arthrobactror in:
      • Different species of oak?
      • White vs red oak? 
      • in transplanted trees vs. naturally grown trees?
        • Could compare old trees to young trees bases on girth of tree?
      • Comparison of diseased oak vs. healthy oak? 
      • Trees/oaks in an area affected by fire vs. trees/oaks in “normal” environments? 
        • Recent fire near Texana?
      • Arthrobactor has been known to be known to breakdown and metabolize pesticides so:
        • My table has decided to test oaks in Cameron park that are untreated with pesticides and compare it to oaks at Baylor that have all been exposed to some  from of pesticides 
  • RESULTS: 
    • The plaque assay ,shown in figure 6, was negative for any bacteriophages 
  • NEXT STEPS:
    • Since the spot test and plaque assay were both negative the next step is to collect soil from oaks (species of oak has yet to be determined)  from Cameron park and soil from trees treated with pesticides at Baylor and compare the concentration (or presence) of phages