Rationale

Obtain results from soil metadata from Lab day 11. Start enrichment for soil sample D and prevent contamination as much as possible.

Detailed Procedure: Soil Enrichment + Metadata

LB Broth was added to the 15 mL vial from Lab day 11 up to the 12 mL mark. The vial shook fr 15 mins through the vortex. Weight was taken and was recorded as 19.48 g. Vial was paired with another and was put in a centrifuge for around 5-10 mins. During the wait, a pinch of soil was taken from the ziploc bag and was put into a pH vial. D.I water filled the rest of the vial was shook for 10 secs. pH paper was put inside for 45 secs and recorded the pH. After the centrifuge, the supernatant was syringe filtered into a separate 50 mL vial. 0.5 mL Arthro was added after.

Results

• Sand= 20%
• Silt= 15%
• Clay= 65%
• pH= 5.5
• plate after 48 hrs= 6.21 g

Observations/Conclusions/Next steps

Direct sample from the filtered supernatant was not made because the next lab day will only have the plaque assay to be performed while other group members will do spot testing. Prevention of contamination seemed to go well. Different LB Broth was used and used pipette to transfer LB Broth into the 15 mL vial instead of pouring the broth in the vial. In order to prepare for possible future negative results, new soil from near the bear habitat will be taken and brought to the lab.

Rationale

Check to see if any plaque remains. If not, why/how does our results mean to the group question. Find new soil. Control plate from Lab day 10 was contaminated, but showed negative results from plaque assay plate. Other group members did spot test and had negative results and contaminated control plate

Detailed Procedure

Group 6 dug up new soil from a red oak in front of the Baylor Science Building’s parking lot. Soil was dug up around 2 feet away from the tree, near the roots. Soil was added into a plastic bag and a 15 mL vial, up to the 2 mL mark. To start on the soil metadata, a plate was weighed to be 2.47 g. Soil was added onto the plate and was a final weight of 6.66 g. Soil was added into a 50 mL vial up to the 10 mL mark and had D.I water added up to the 30 mL mark. 3 drops of soil disperser solution was added and shook for 30 secs.

Results

• weight of plate= 2.47 g
• weight of plate + soil= 6.66 g

Conclusion

New soil’s metadata must be done by next lab day to determine which type of soil we collected. Soil enrichment will also be done by next lab day as well as pH of the soil.

(not sure if I put it here?) How I spend my time after class= study with group of friends at moody doing work/study and hang out till moody closes.

Rationale

Redo plaque assay due to possible contamination of Arthro from lab day 9. Other group members will redo spot test.

Detailed Procedure

1. Took 10 microliters of filtered enriched lysate and added to 400 microliters of Athro. Sat for 15 mins
2. Took 4.2 mL LB Broth, 45 microliters of 1M CaCl2, and 5 mL of 2X TA into a 50 mL vial.
3. Took half of solution from step 2 and poured into control plate
4. Took other half and added solution from step 1 and poured into plaque assay plate
5. Both plates sat for 15 mins and was inverted into the incubator.

Conclusions+Observations/Results/Next Steps

Control plates from group members’ spot test and plaque assay were both contaminated, but showed negative results. There seems to be contamination of Arthro from class discussion. When pouring control plate, there was a bubble on the plate that did not pop. Next steps are to wait for the results. If no plaque are present for both spot test and plaque assay, then new soil will have to be found.

Rationale

Perform both spot test and plaque assay from Soil C without any trace of contamination. Record results of soil metadata from last lab day to determine the type of soil Soil C is. Two members from group 6 did spot test while one did plaque assay. Each test had an individual control plate.

Detailed Procedure

1. Syringe filtered enriched lysate.
2. Took 10 microliters of filtered enriched lysate and added to 0.5 mL Arthro. Sat for 15 mins.
3. During 15 mins, took 4.0 mL LB Broth, 45 microliters of 1M CaCl2, and 5.0 mL of 2X TA into separate 50 mL vial. Mixed through pipetting.
4. Used sterile pipette and took half of solution from step 3 into another separate 15 mL vial. Labeled as control.
5. After 15 mins were done, took solution from step 2 and mixed through pipetting into previous 50 mL vial.
6. Took both vials into water bath and waited for plate to be ready.
7. Labeled plates as “Plaque Assay” and Plaque Assay Control.”
8. Took control vial and poured into control plate and same with other plate+vial.
9. Waited 15 mins to solidify.
10. Inverted plates into incubator.

Conclusions/Results/Observations

• Clay= 75%
• Silt= 18.75%
• Sand= 6.25%
• Soil C is clay based sand

Due to more experience, there were no bubble on both plates and was able to prevent possible contamination (accidental spills, not being in aseptic zone.) In the next lab day, future steps would be to check for any signs of plaque. Since within the group, both spot test and plaque assay was performed, so if no plagues were found, then a new soil will be tested next.

Rationale

Complete enrichment process of new collected soil and results from soil metadata from new soil. Label group number on LB Broth and 2X TA jars to prevent future contamination.

Detailed Procedure: Soil Metadata

1. Filled soil up to 10 mL mark in a 50 mL vial and filled rest with D.I water up to 30 mL mark.
2. Shook for 10 secs and added soil dispenser solution.
3. Left to rest on rack for 48 hours.
4. Pinched soil into pH vial and filled rest with D.I water and shook for 10 secs
5. Left to rest for 2 mins
6. Pinched off 1 inch of pH paper and dipped into pH vial for 45 secs.
7. Recorded pH of soil

Detailed Procedure: Enrichment

1. Filled LB Broth into vial from Lab Day 7 up to 12 mL mark in aseptic zone
2. Shook using vortex for 15 mins.
3. Weighed vials and centrifuged for 5 mins
4. Syringe filtered supernatant (8 mL into 50 mL vial and 1 mL into 15 mL vial)
5. Poured 0.5 mL Arthro into 50 mL vial

Results

• weight of plate + soil after 48 hours= 6.48 g
• ph of soil= 6
• weight of vial before centrifuge= 19.75 g

Conclusions

After 48 hours, I will be able to determine what type of soil was collected. After incubation of enriched lysate, group 6 will be able to do spot test and plaque assay of the new soil and check to see if there are any presence of plaque.

Rationale

Check to see if any plaque remains. If not, why/how does our results mean to the group question. Find new soil. Control plate from Lab day 6 was contaminated, but showed negative results from plaque assay plate. Other group members did spt test and had negative results as well.

Detailed Procedure

1. Dug up new soil from a red oak in between the BSB and business building.
2. Filled 15 mL vial up with soil up to 2 mL mark
3. Filled bag with soil
4. Began soil metadata by weighing plate and soil

Results

• weight of plate= 2.34 g
• weight of plate + soil= 6.68 g

Group question

1. Lathan checked a purified lysate by doing a plaque assay (10 microliters lysate) of a 10^-3 lysate. He wanted 14 plaques. How many microliters of Lathan’s lysate?

Conclusion

New soil’s metadata must be done by next lab day to determine which type of soil we collected. Soil enrichment will also be done by next lab day.

Rationale

Make plaque assay and record results from Lab day 5

Detailed Procedure

1. Since I was the only one in my group without a direct lysate, I had to make a plaque assay while the rest did a spot test. (therefore I had to make my own control)
2. Add water to help balance my partner’s weight= final enriched weight= 22.38 g
3. Filtered enough enriched lysate into a 15 mL vial.
4. Took 10 microliters of enriched lysate and added to 0.5 m Arthro vial
5. Mixed it well and let sit for 10 mins
6. During that time, took 2.0 mL LB Broth, 23 microliters of 1M CaCl2, and 2.5 mL 2X TA and mix well through pipetting in a separate 50 mL vial
7. Took enriched lysate + Arthro mixture and added to the 50 mL vial
8. Mixed one more time through pipetting
9. Poured mixture onto a plate label “Plaque Assay B”
10. Repeat step 6 for control onto a new plate labeled “Plaque Assay Control”
11. Let both plates sit for 15 mins

Detailed Procedure: Sand, Silt, Clay+ Metadata results

1. Record total mL in tube
2. Record how many mL of sand, silt, clay, and calculate the %

Observations/Results

• Sand= 50%
• Silt= 43.75%
• Clay= 0.0625%
• Soil + petri dish= 5.75 g

Next Steps

• Wait for results from plaque assay and see if there’s any presence of plaque.
• Results and comparison to rest of class will help resolve/eliminate any variables or questions we are trying to solve

enriched lysate w water

control vs exp

after 48 hours

Rationale:

• Make enriched lysate of soil B, find pH of soil and start soil metadata to find future calculations of sand, silt, and clay.

Detailed Procedure: Enriched Lysate

1. Took vial from Lab day 4 and added LB Broth up to 12 mL mark
2. Shook vial for 10 mins
3. Weigh vial= 19.04 g
4. Centrifuge for 5 mins
5. Syringe filter 7.5 mL of supernatant (did not have enough for direct)
6. Add 0.5 mL Arthro to vial

Detailed Procedure: Metadata

1. Add ~10 m of soil in a 50 mL vial.
2. Add water up to 30 mL mark
3. Shook vial for 30 secs
4. Add soil disperse solution to it and mix for a bit
5. Let sit for 48 hours
6. Took some soil and weigh it with petri dish
7. Let sit for 48 hours to dry

Detailed Procedure: pH

1. Took pinch of soil in a small pH vial and filled the rest with water
2. Shook for a bit and rip off 1 inch of pH paper
3. Dip paper for 45 secs and compare color
4. Recorded ph was green= 7.0

Observation/Results

• petri dish weight= 2.35 g
• petri dish weight + soil= 6.26 g
• after this, I ran out of all of my soil, so I have no more left to test if needed in the future.

Next steps

• calculate sand, silt, clay
• weigh petri dish when soil dries
• plaque assay with enriched because I have no direct lysate
• 

Rationale: As a class, we came up with multiple ideas for our questions, from tree species, transplant/natural trees, or environmental factors (fire.)

Detailed Procedure

1. New group and I decided to go around Texana area since a fire occured there around 4 months ago and collect from from both red and white oak trees.
2. Took ~2 mL of Soil B from a red oak tree into a 15 mL vial
3. Took a sample of a leaf and extra soil in bag
4. Labeled both bag and vial

Question

Is there a correlation between tree species of oak trees and/or fire in the area? Will there be a presence of Arthro in any of the soil samples we collected?

After two days, my plate looks cloudy compared to the control. The control plate was very clear with no sign of contamination. My plate looks like it had been contaminated (probably from my errors in Lab Day 2) and looked like some parts of TA didn’t settle properly.

Detailed Procedure

1. Took 0.5 Arthro vial from the back and added 10 microliters of enriched lysate with a pipette in the aseptic zone. Let it sit for 15 mins.
2. With rest of group, add 2.0 mL of LB Broth and 2.5 microliters of 1M CaCl2 with a pipette
3. As soon as each group members waited 15 mins, we added 2.5 mL of 2X TA.
4. Spilt above mixture equally into 3 vials.
5. Took one of the vials and added the mixture from step 1 and used pipette to mix it thoroughly.
6. My vial tilted over, which resulted in a small spill.
7. Poured the rest of the vial onto a separate plate labeled as “Plaque Assay”
8. Wait 10 mins for it to solidfy

Thoughts

• Due to a small spill in step 6, I was unable to get exactly 4.5 mL onto my plate.
• This might have caused some contamination.

Questions

• Can certain oak trees be more prone to bacteria
• Turns out the soil from the tree my group and I took was from a red oak. If we can get soil from a white oak in our area, we might be able to find plaque from tests. We would need to find another red oak and white oak to test this.
• Does the present of Arthro appear more dominant in one oak tree species than the others? If so, in this species, is there a correlation between the presence of Arthro and the present of oak wilt fungus growth?
• 

  plaque assay plate

  

  control plate from Lab Day 2. Shows no sign of contamination

  

  signs of contamination. No sign of plaque

  

  second pic of spot test results