September 21

Soil Washing and Metadata 9/19/2018

Rationale: wash soil samples and produce a direct isolated sample and an enriched isolated sample.

Process:

Soil Washing

  1. Cleaned lab bench with CiDecon and 70% ethanol
  2. Using aseptic techniques, added LB broth to the 15 mL mark of the tube containing ~ 2 mL of soil
  3. shook and vortexed the tube for 10 minutes
  4. Added small amount of water to get a final weight of 19.21g
  5. Centrifuged the tube with classmates
  6. Used a syringe and filter to filter supernatant into fresh tubes
    1. Used ethanol to keep the filter tip clean
    2. Ended with ~9 mL for enriched and ~1 mL for direct samples
  7. Added 0.5 mL of artho to the enriched sample
  8. Stored it in the fridge
  9. Left enriched isolated sample in the shaking incubator for 48 hours

Soil Metadata

  1. mass of dry soil and sample = 7.89g
  2. measured pH of soil
    1. added a pinch of dirt and DI to tube
    2. shook for 45 seconds
    3. held pH paper in tube for 45 seconds
    4. pH = 6.0
  3. sand/silt/clay soil dispersion
    1. Added ~8mL DI to ~4mL soil
    2. Added 3 drops of soil dispersion liquid
    3. shook for 30 seconds
    4. let sit for 48 hours

Next Steps:

Perform spot test with enriched and direct isolations. Measure sand/silt/clay dispersion and calculate % H2O.

Category: Rachel Melone | LEAVE A COMMENT
September 21

9/17/18 Picking a Plaque and Plaque Assays – Soil B

Previous Results:

  • The results of the metadata of soil composition were collected. The layers of the soil were measured by recording the markings on the tube. The total amount of soil was 16 mL with 59% silt, 37% clay, and 4% sand.
  • Plaque assay from last lab was positive and had numerous clearings, too many to count so dilutions will be necessary to know concentrations.

Objective:

  • Pick a plaque from the plaque assay made during last lab
  • Conduct a series of serial dilutions and plate the diluted lysate

Procedure:

  1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
  2. A plaque was picked by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming 10^0 dilution
  3. 10 microL of 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
  4. Step 3 was repeated to create a 10^-2 dilution
  5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
  6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
  7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
  8. All experimental tubes and the control tube were plated and left to harden for 15 min
  9. Plates were placed in incubator for 48 hours.

Results:

  • Results will not be available until next lab (9/19) to see if the phage were plated correctly and there are positive results.

Conclusion:

  • The results from the previous lab were positive and there are phage in soil sample B
  • The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

  • During the next lab (9/19) the plates with the serial dilutions will be examined for phage. Another series of dilutions will be done to ensure only one strain of phage is being tested. The same steps will be conducted.
Category: Claire Wentzlaff | LEAVE A COMMENT
September 21

Soil C Metadata

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

Category: Emily Balint | LEAVE A COMMENT
September 21

Soil Collection and Filtration Soil C

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.

Category: Emily Balint | LEAVE A COMMENT
September 21

Soil Washing and Metadata 9/21/18

Rationale: My plaque assay turned out negative, so I collected new soil. This time from a living tree rather than a dead tree, but still from Cameron Park and still sick. I also collected metadata on the soil so to find if that may be a confounding variable.

Procedure: Soil Metadata:

  1. After collecting data from tree stored in refrigerator overnight.
  2. pH tested soil and put a 4.05g sample of soil in a weight boat to let dry for

    48 hours

  3. Placed 10mL of soil in falcon tube and filled to 30mL with DI water then

    poured 3 drops of dispersion fluid and let sit under vent hood for 48 hours

  4. After 48 hours of letting percent water and soil/silt/clay analysis sit collected

    data and took pictures.

Soil Washing:

  1. Filled 15mL tube with 2mL of soil then with 10mL of LB broth.
  2. Shook for 15 minutes then spun down at 3000g for 5 minutes.
  3. Filtered supernatant with a 22nm filter then added 0.5mL of arthrobacter to

    filtered supernatant.

  4. Labeled “Enriched” and placed in shaking incubator for 48 hours.

Data and Observations:
The tree from which I collected soil from was an extremely young sapling. The height was 0.56m, the circumference of the trunk was 3cm, and the canopy diameter was 26cm. There were some damaged browning leaves, but other than that the tree seemed decently healthy.

Mass of soil with water is 4.05g. Mass of sample without water is 3.42g. Mass of water lost is 0.63g divided by the total mass of 4.05g is 15.56% water. The pH of the soil was about 6.5. The soil was 70% silt 30% clay. The amount of sand was too small to measure quantitatively.

Interpretations and Next Steps: Unlike the dead tree I collected from last time this soil is slightly more acidic. Also, there is almost no sand in this soil. There is twice as much moisture in this soil as compared to the previous tree. This could be a confounding variable in the presence of phage in these two samples. I would have to test more trees to see if the lack or presence of phage was due to any of these factors or not. The next step is to conduct a plaque assay to see if this soil has phage in it.

Category: Sriram Avirneni | LEAVE A COMMENT
September 21

Plaque Assay 2 Soil B

9/17/18

Rational:

To do a second plaque assay with soil B. The plate for the first plaque assay did not have arthro added by mistake so it was not possible to determine whether the sample contained arthro or not.

Procedure:

  • Cleaned lab desk with CiDecon and ethanol
  • Set up an aseptic technique
  • Added 10 ML FS lysate to 0.5 arthro
  • Added 2 mL LB broth to control TA mixture
  • Added 2 mL LB broth to TA 2 soil B mixture
  • Added ML 1 M CaCl2 to TA mixture for the control and soil B
  • Added arthro and FS lysate to TA soil B mixture
  • Added TA (2.5 ML) to control TA and soil B mixture
  • Poured control TA onto the control plate and poured soil B TA onto another plate
  • Waited 10 minutes
  • Placed plates into incubator at 26 C at 3:30 9/17 – 2:30 9/19

Conclusion:

The plate from 9/12 had no arthro added by mistake so as a result there was not way to confirm the presence or absence of arthrobacter phage. As a result a new plaque assay was done for soil B. Next lab the plate will be checked for the presence of plaques. If there are any plaques then purification will be done on the plaques, if there is contamination then the plaque assay will be redone, and if there are no plaques then new soil will be collected so that a new plaque assay can be performed.

Questions:

  1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all three got plaque). They each did a spot test in addition to their plaque assays, but only Justin had plaque on his spot. What do you think is going on?                                                                                                                                                  It is possible that the sample that Justin had contained more phage which could lead to him having more plaque than the rest of his group. The rest of group 4 on the other hand could have had less phage so that the reduction of time for the phage to infect arthro as well as the smaller sample size could have cause no visible plaques to form.
  2. Lathan checked a purified lysate by doing a plaque assay (10 mL of lysate) of a 10^-3 lysate He counted 14 plaques. How many ML of Lathan’s 10^0 lysate should he add to web the plate (75 mm diameter) if his plaque diameter is 1 mm?                                                                                                                                                                         A(plate) = π(75 mm)^2 = 5625π mm^2     A(plaque) = π(1 mm)^2 = π mm^2                                                              5625π mm^2/π mm^2 = 5625 plaques to web plate                                                                                                        (14 pfu/1 ML * 1000 ML/1 mL) * 10^3 = 1.4 * 10^4                                                                                                     5625 pfu/1.4*10^4 pfu/mL = .40 mL to web plate

                                                     

Fig.5.B – Plaque assay 2 of soil B the yellow circle                      Fig.6.B – Control plate for plaque assay 2 soil B the      indicates the location of a bubble on the plate.                           yellow circle indicates the location of bubles on the                                                                                                                         plate.

Category: Emily Balint, Uncategorized | LEAVE A COMMENT
September 21

Soil Collection 9/17/2018

Rationale: My lab group performed a spot test and plaque assay with soil from the previous red oak tree and got negative result. We wanted to fins a new red oak tree to test the soil from.

Figure 1: Results of the spot test from the previous class. No plaques found

Process:

  1. found a red oak in the field outside of the SLC and dug soil samples from the roots
  2. Started % H2O metadata analysis:
    1. mass of wet soil in weigh boat = 8.38 g
    2. let the wet soil sit for 48 hours

Next steps:

wash soil to get enriched and direct isolations. Continue to analyze soil metadata by taking soil pH, weighing the dry soil, and setting up a sand/silt/clay soil dispersion.

Category: Rachel Melone, Uncategorized | LEAVE A COMMENT
September 21

9-17-18 — Plaque Assay Setup and Soil Sample C Collection

Date: Monday, September 17th, 2018

Title: Plaque Assay Setup and Soil Sample C Collection

Rationale: The purpose of today’s lab is to set up a plaque assay with the lysate from Soil Sample B as well as gather more soil if the plaque assay yields negative results.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Plaque Assay Procedure:

  1. An aseptic zone was set up.
  2.  Spot tests were evaluated and found to have yielded negative results
  3. 10 microliters of the Soil Sample B enriched lysate was added to a culture tube with 5 mL ATC 21022.
  4. Culture tube was set aside for 15 minutes so phage could infect arthro.
  5. Agar was made with the following formula:
    1. 8 mL LB broth
    2. 10 mL 2x TA
    3. 90 microliters 1M CaCl2
  6. 4.5 mL of the top agar solution was added to the culture tube.
  7. TA solution with arthro and phage was added to agar plate, left to harden for 10 minutes, inverted and incubated.

Soil Sample C:

  • Tree Circumference: 380 cm
  • Small Canopy Diameter: 1606.5 cm
  • Large Canopy Diameter: 1976 cm
  • Average Canopy Diameter: 1791.25 cm
  • Tree Height: 13.68 m
  • Tree Coordinates: 31°32’59” N 97°6’57” W

Observations: Controls were all contaminated, but individual spot tests were negative for plaques and contamination. It’s unclear how the controls were contaminated. The top agar wasn’t contaminated or the individual plates would have also had contamination.

Results: This experiment yielded a plaque assay to be evaluated for possible plaques. This also yielded a new soil sample to be experimented on if the plaque assay from Soil Sample B yields negative results.

Next Steps: The next step is to evaluate the results of the plaque assay. If negative, the next step is to work with Soil Sample C and try for phage with it. If positive, the next step is serial dilutions and plaque assays.

Conclusion: From the results of testing on this sample and the soil meta data, it could become evident in what type of environment phages tend to reside, and whether there is a difference between soil around live oaks or red oaks.

In addition to the experiments, there were also two guiding questions asked of the class to consider:

  1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?
  • The likely reason is simply that the sample Justin used had the highest titer, resulting in better defined, clear spots. It’s also possible that he was more careful during his procedure, leading to less outside influences.

2. Lathan checked a purified lysate by a plaque assay using 10 microliters of 10^-3               lysate. He got 14 plaques. How many microliters of Lathan’s lysate should he add             web a plate (8 cm in diameter) if his average plaque diameter is 1mm?

  • 14 plaques divided by 10 microliters = 1.4 x 10^3 pfu per mL 10^-3 lysate. The plate radius = 40 mm, and the plaque radius = .5 mm. The area of the plate over the area of the plaque = 6.4 x 10^3 pfu to web. 6.3 x 10^3 pfu divided by 1.4 x 10^6 pfu per mL = 4.6 x 10^-3 mL 10° lysate to web = 4.6 microliters 10° lysate to web.
Category: Brandon Reider | LEAVE A COMMENT
September 21

Filtration of Enriched Lysate and Collection of Metadata (9/21/18)

Rationale:

To filter the enriched lysate using a syringe, collect the data to understand the soil’s % water and soil composition, and complete a plaque assay using the filtered enriched lysate.

 

Procedure:

The data for the soil composition and % water was first collected. To determine the % water, a scale was used to determine the initial and final masses which were used for calculations. For the soil composition, the different layers were observed and the volume of each layer was taken down for later use in calculations. After collecting all the data, the next step was to filter the enriched lysate. It was filtered using a syringe and 0.22 µm filter into a tube. In another tube, 1 mL of filtered enriched lysate and 0.5 Arthrobacter were combined and left alone for 10 minutes to infect. Next, 22.5 µL of CaCl2 and 2mL of LB broth were combined to form what was to become the top agar. After 10 minutes, 2.5 mL of 2X Top Agar was added to the Top Agar solution. The Arthrobacter and lysate were added to the top agar solution and poured onto the agar plate. The plate was left alone for 10 minutes for solidification. Then, the plates were placed in the incubator.

 

Results and Analysis:

% Water

(3.336/3.79)x100=88.0%

Soil Composition

Total: 4 mL

2 mL of sand

.7 mL of silt

1.3 mL of clay

(2/4)x100= 50% of sand

(.7/4)x100= 17.5% of silt

(1.3/4)x100= 32.5% of clay

Plaque Assay Plate

 

Conclusions and Future Plans:

First, data was collected from procedures completed previously (soil composition and % water) and used to perform calculations to determine the percentages. Then, the lysate was filtered using a syringe. Lysate and Arthrobacter were combined and left alone for 10 minutes. During that time, CaCl2 and LB broth were combined in a conical vial. After the 10 minutes, the Arthobacter and lysate were added to the CaCl2 and LB broth along with 2X Top Agar. The top agar was poured on a plate and left to solidify for 10 minutes then placed in the incubator.

Future Plans:

On Monday (9/23/18), the plates will be checked for plaques. If there is contamination of the control with no plaques on the plate, another plaque assay will be made. If there is no contamination along a negative plate, another soil sample will be collected in another area. If there is no contamination with a positive plate, the plaque will be picked and will be diluted using a phage buffer.

 

Category: Sabin Patel | LEAVE A COMMENT
September 21

Plaque Assay and Metadata for Soil Sample (9.17.18) 9/21/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is a presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate. The sample’s metadata is also very important for more insight into our research question since it would provide more information towards the characteristics of the soil and help us build patterns.

Soil Metadata:

Soil Composition:

  1. Remove the supernatant in the falcon tube
  2. Record the results

% Water:

  1. Weigh the petri dish
  2. Record the mass

Calculate the Soil Composition and % Water

Plaque Assay for Soil (9.17.18):

Materials:

  • Micropipette
  • Serological pipette
  • Centrifuge tube(1.5ml)
  • 50 ml conical tube
  • LB Broth
  • CaCl2(aq)
  • Sample (9.17.18) Lysate
  • 2x Top Agar
  • Agar plate
  • Arthrobacter

Procedure:

  1. Set up an Aseptic zone.
  2. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 10 min for infection.
  3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a 50 ml conical tube
  4. Add the infected lysate to the 50 ml conical tube
  5. Add 2.5 ml of 2x Top Agar to the 50 ml conical tube, pipette up and down then decant the solution to the agar plate
  6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

Soil Metadata results for Sample (9.17.18):

  1. Soil Composition: 0.5 ml Clay, 1 ml Silt, 1.5 ml Sand
  2. % Water: weigh boat=2.40g, wet soil+weigh boat=7.40g, dry soil+weigh boat= 6.77 g
  3. Soil pH = 6.0

Interpretations & Conclusions:

Soil Metadata results for Sample (9.17.18):

Soil Composition: 16.7% Clay, 33% Silt, 50% Sand

% Water: 12.6%

Soil pH = 6.0

  • From the metadata of the soil sample, the soil falls into the category of Loam, which is the typical soil type in the State of Texas.
  • The soil is suitable for gardening because of its ability to retain water, 12.6% for the sample collected.

Next Step:

If the Plaque Assay for Sample (9.17.18) shows positive results further purification process would be done, otherwise, new samples, multiwell enrichment or Plaque Assay re-run are all possible choices.

Category: Yang-En Yu | LEAVE A COMMENT