10/1/18

Rational:

To collect a new soil sample and record metadata on the tree the soil was collected from. This new soil sample will then be used to perform a new plaque assay.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• New soil sample was found at McLane Stadium
  • Trunk circumference- 33.7 cm
  • Canopy length- 122.6 cm
  • Tree height- 457.2 cm
• Collected soil sample D 2 ft from the base of the tree
• Cleaned lab desk with CiDecon and ethanol

Conclusion:

The plaque assay for soil C had no plaque and the control was contaminated as well. As a result a new soil sample had to be collected. Next lab I will collect soil metadata and filter soil D so that I can perform a new plaque assay.

Fig.1.D – Shows the tree where soil sample D was taken from.

Fig.2.D – Shows browning on the tree’s leaves at the edges the reason (whether it is disease or lack of water) is unknown.

Fig.3.D – The size and shape of leaves on this tree indicate that it is a Chesnut Oak (a type of White Oak).

Fig.4.D – The white spot on the right-hand side of the plate shows that there was contamination.

Fig.5.D – Shows no sign of any plaque and on the left-hand side there is a spot that is slightly lighter than the rest of the plate which could indicate contamination as well.

9/26/18

Rational:

A new plaque assay will be performed as the last plaque assay was not done with arthrobacter and some other bacteria instead. So a new plaque assay will be performed in order to check for the presence of arthrobacter phage.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Set up and aseptic zone
• Put 10 ML FS lysate into 400 ML arthro
• Put 2 mL LB broth into TA mixture
• Added 22.5 ML 1M CaCl2 to the TA mixture
• Added another .1  mL LB broth to the TA mixture
• Added 400 ML arthro and lysate to the TA mixture
• Added 2.5 mL TA
• Poured on plate and waited 10 minutes
• Put 2.1 mL LB broth to control TA mixture
• Added 22.5 ML 1M CaCl2 to control
• Added 2.5 mL TA to the control
• Poured on the plate and waited 10 minutes
• Put plates in incubator at 26 C at 3:30
• Cleaned lab desk with CiDecon and ethanol

Conclusion:

The plates from 9/24 were not able to comfirm the presence or absence of arthrobacter phage in soil C. This is because the bacteria that was used was not the arthrobacter that has been used, so any plaques seen would be from the presence of of a different type of phage. Contamination was also seen on both the control and plaque assay plates this in itself would have required that I new plaque assay be done. Next lab I will check for plaque on plaque assay soil C 2. If there is plaque then I will start purification and if there is not then I will collect a new soil sample and filter it and collect metadata. If there is contamination I will do another plaque assay.

Fig.7.C – This image shows the plaque assay for soil C                   Fig.8.C – This image shows the control plate for soil    (done with an unknown bacteria not arthrobacter). The               C which also has an unknown bacteria growing on it.  white spots show contamination.                                                        This plate is covered in white spots indicating                                                                                                                                  contamination (may be difficult to see).

Fig.9.C – This shows the control plate for plaque                            Fig.10.C – This image shows the plate for soil C        assay soil C 2. The yellow circles in indicate where                         plaque assay 2. The yellow circle shows where a          bubbles are located to prevent them from being                             bubble is to prevent it from being mistaken as a          mistaken for plaque.                                                                              plaque.

9/24/18

Rational:

To do a plaque assay in order to see if soil C has any arthrobacter phage. Record results results of soil C metadata in order to compare with soil B metadata.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Dry soil C with weigh boat- 5.25 g
  • Dry soil C- 2.93 g
  • Percent Water- 47.9%
• Sand- 3.5 mL     Silt- 2.5 mL     Clay- 4 mL
  • Percent Sand- 35%     Percent Silt- 25%     Percent Clay- 40%
• Cleaned syringe filter to prevent contamination
• Filtered enriched lysate with the syringe filter
• Put 10 ML FS lysate into .5 arthro
• Put 2 mL LB broth into control and plaque assay TA mixtures
• Added 22.5 ML 1M CaCl2 to TA mixture for soil C plaque assay and control
• Added .5 mL arthro and FS lysate to plaque assay TA mixture
• Added 2.5 mL TA to TA mixture for soil C and control
• Poured onto soil C plaque assay and control plate and let sit dor 10 minutes
• Put plates in incubator at 26 C at 3:40-2:30 9/26

Conclusion:

The soil C metadata showed that soil C was clay according to the soil texture triangle. This is a slightly different soil type than soil B which was clay loam although the percent sand and silt change by only 5% and clay change by 10%. On the other hand the percent water in soil C (47.9%) is significantly greater than soil B (16.2%). Next lab I will check for plaque if there is a plaque then I will start purification. If there is contamination then I will redo my plaque assay and check for plaque again. If there is no plaque then I will get a new soil sample and filter it and collect metadata.

Fig.5.C – This shows the plaque assay for soil B. The                 Fig.6.C – This image shows the control plate for soil C.    yellow circles indicate the location of bubbles in the                 The yellow circles on the image indicate the location of  agar to prevent them from being mistaken as plaque.               bubbles that could later be mistaken as plaque.

9/21/18

Rational:

To get metadat such as percent soil, silt, and clay, percent water, and pH to later help explain any results and trends found.

Procedure:

• Cleaned the lab dest with CiDecon and ethanol
• Added 10 mL of soil C to a falcon tube
• Added DI water to the 30 mL mark
• Added 3 drops of soil dispersion
• Shook for 30 seconds and then placed it under the hood
• Weighed empty weigh boat- 2.32 g
• Weighed weigh boat with wet soil- 5.62 g
  •  Weight of wet soil- 3.3 g
• Placed soil under the hood
• Put soil in pH vial
• filled with DI water
• Shook tube for 10 seconds
• Let sit for 2 minutes
• Checked pH with pH paper- pH of 6
• Cleaned lab desk with CiDecon and ethanol

Conclusion

Next lab a plaque assay will be preformed to test for arthrobacter phage in soil C. The pH of soil C was the same as the pH of soil B and the rest of the metadata will be compared next lab when the results are recorded.

Fig.3.C – Shows the pH of soil C is about 6

9/19/18

Rational:

Plaque assay 2 soil B showed not plaque or contamination. A new soil samplw was collected and filtered to prepare for a new plaque assay to check for phage.

Procedure:

• Found a post Oak behind East Village for Kathryn to use
• Found a chesnut Oak in Earle court yard
  • Trunk circumference- 42.4 cm
  •  Canopy length- 238.6 cm
  • Tree height-
• Collected soil 2 ft from the trunk
• Put 2 mL soil C into tube from the sample collected
• Put 10 mL LB broth in the tube and shook for 15 minutes
• centrifuged the tube at 5,000 G for 5 minutes
• Filtered the sample and added 0.5 mL arthro
• Put in shaking incubator at 4:10 (26 C)
• Cleaned desk with CiDecon and ethanol

Conclusion:

Next lab metadata will be collected on soil C that can be looked at later to help explain results and trends. On monday a new plaque assay will be preformed which will test for the presence of arthrobacter phage in soil C.

Fig.7.B – Plaque assay 2 for soil B shows no sign of a                 Fig.8.B – Control plate shows no sign of contamination. plaque or contamination.

Fig.1.C – Shows the tree that soil sample C was taken                 Fig.2.C – Shows a leaf from the Chesnut Oak the soil      from.                                                                                                       sample C was taken from.

9/12/18

Rational:

To do a plaque assay to see if there’s any arthrobacter phage in soil B. I will also be calculating the sample’s water percentage and percent of sand, silt, and clay based on the results from lab on 9/10.

Procedure:

• Weight of the dry soil in the weigh boat- 5.33 g (weight of dry soil- 2.9)
• Weight of enriched lysate 21.6 g
• Percent water- 16.18%
• The 10 mL soil sample contained 4 mL sand (40%), 3 mL silt (30%), and 3 mL clay (30%)
• Spun the enriched lysate at 3,000 g for 5 minutes
• Filtered the enriched lysate
• Put 10 ML FS lysate into .5 mL Arthro
• Put 2 mL LB broth into the TA mixture
• Put 2 mL LB broth into vial for the control mixture
• Added 22.5 ML 1M CaCl2 to the TA mixture and 22.5 ML 1 M CaCl2 to the control TA
• Added .5 Arthro to my TA mixture
• Added 2.5 TA to the TA mixture and control TA
• Poured my mixture onto my plate and lets set for 10 minutes
• Poured control TA onto the control plate and let set for 10 minutes
• Placed in the incubator at 26 degrees Celcius at 3:45 9/12
• Cleaned lab dab desk with CiDecon and ethanol at the beginning and the end of lab

Fig.3.B – Shows the plate for the plaque assay for soil B             Fig.4.B – Shows the control plate for soil B after the TA after the TA was added. The yellow circle shows where              was added. The yellow circles indicate where bubbles  a bubble is on the plate.                                                                      were observed on the plate.

9/10/18

Rational:

To analyze the soil sample (type of soil, percent water, and pH). This will give metadata that could give further insight into the results that our group finds.

Procedure:

• Cleaned the lab desk with CiDecon and 70% ethanol
• Placed 2 mL of soil in a 15 mL conical vial
• Used aseptic technique to add 10 mL LB broth using a serological pipette
• Started 15 minute timer
• Shook vial for 15 minutes
• Weighed vial at 18.99g then centrifuged at 10,000 G for 5 minutes
• Added 10 mL soil to a falcon tube
• Added DI water up to 30 mL
• Added soil dispersion (3 drops)
• Shook for 30 seconds
• Let sit for 30 seconds
• Poured off supernatent into a conical tube (later poured supernatent back into the falcon tube)
• Weighed empty weigh boat- 2.43 g
• Weighed weigh boat with wet soil- 5.89 g (wet soil- 3.46 g)
• Placed weigh boat  under hood to dehydrate
• Put some soil into a pH vial and filled the rest of the tube with DI water
• Shook the tube for 10 seconds and then let it sit for 2 minutes
• Placed pH paper in the vial for 45 seconds and checked the pH (pH- 6)
• Filtered out centrifuged soil sample with a top filter
• About 7.5 mL was filtered out
• Added Arthrobacter into the tube using the aseptic technique (0.5 g Arthrobacter)
• Then placed tube into the shaking incubator at 3:50 pm 9/10 at 26 degrees Celcius (until 2:30 9/12)
• Cleaned lab desk with CiDecon and ethanol

Fig.2.B – shows the pH of soil sample B to be 6

9/7/18

Rational:

To collect soil on campus from three White Oaks with two group members (three other sample will be taken off campus by the remaining group members in Cameron Park Waco, TX)

Procedure:

• Walked around campus look for White Oaks
• Found a Bur Oak (a type of White Oak) in the Burleson Quadrangle (near Fountain Mall)
• Claire recorded data and observations about the tree before taking a soil and leaf sample
• Found another Bur Oak in Burleson Quadrangle
• I recorded data and observations about the tree and then took a soil and leaf sample
• The last tree was found in Founders Mall
• Kathryn recorded data and observations about the tree and then took a soil and leaf sample

Observations:

• The circumference was 86.5 cm, canopy width- 427.2 cm, and tree height- approx. 914.4 cm
• One area on the bark had a large population of ants on it
• Some of the leaves seemed to be turning brown
• Though some of the area around the tree was bare some was covered in thick grass

*observations only on the tree I took a sample from

Results:

Does the presence or absence of pesticides impact the amount of arthrobacter phages found in the soil

*question resulting from the discussion on 9/5

*note we did not choose trees of similar size like originally planned

Fig.1.B – The leaves of the Bur Oak I collected my soil sample from

9/5/18

Rational:

To discuss the question that our experiment will address and explore.

Procedure:

• Discussed what question we are going to test
  • some topics were specific to the trees age, whether it was transplanted or not, species and pesticides vs no pesticides as possible factors on arthrobacter.
• Our group decided to test for the presence of arthrobacter in White Oaks of similar size with pesticides (on campus) and without (off campus at Cameron Park).

Future Steps:

On Friday I will collect three soil samples from White Oaks on campus with two other group members. The remaining three samples will be collected at Cameron Park by the remaining three group members.

*The group decided to test how pesticides impact the presence of arthrobacter (specific to soil around White Oaks)