Additional note from Wednesday (8/30):

After further examination, it became very clear that there was Arthrobacter growth on our section of the top agar control plate. Unfortunately, this indicates that some amount of contamination must have occurred during the procedure. To avoid contamination in the future, taking care to keep instruments separate and avoiding any possibility of introducing contaminants to controls should rectify further problems with this.

Rationale: We obtained an additional sample of soil titled “Soil Sample B” in order to begin compiling data to answer our overarching research question.

Research Question: For Groups 3 and 4, we concluded that our research question to guide our soil collection would be old trees as compared with newer trees that needed to be transplanted or were recently planted. As a class, our idea is to come together after dividing into four groups to determine whether or not there are any trends or correlations based on the conclusions and data of each group that can guide us to a more definite question that the whole class can agree upon.

“Is there a significant difference in phage presence between old trees and new trees (that needed to be transplanted or were planted recently)?”

Procedure:

1. Class gathered to brainstorm and evaluate hypotheses.
2. Each table of two groups liked relatively different ideas, so each group went and began to collect soil to gather evidence from their own questions in order to develop correlations that could focus a class-wide study.
3. Groups 3 and 4 (making up one table group) concluded that the question we would begin to pursue was “does the amount of time a tree has been planted or if has been transplanted influence the phages in the surrounding soil?”
4. Therefore, group 3 found each found three separate wild oak trees and took 1/4 of a collection bag of soil from each tree along with one leaf. The bag was labelled “HMB Soil Sample B 9/5/18”.
5. The online metadata survey was taken to give additional context about the selected trees beyond the initial research question.
6. Soil in bag was brought back to the lab. 2mL of the sample from the bag was placed in a 15mL conical tube. The conical tube now containing soil, leaf, and excess soil were all placed in the bag and submitted to be placed in the refrigerator until Monday.

Metadata/Observations:

• Tree type: Live oak
• Tree Size: Approximately 15m tall, well greater than 200cm in girth.
• Canopy Size: Large, approximately 33m
• Health: Tree appeared to be very healthy. Branches all had leaves and the trunk showed very little evidence of decay.
• Soil: Moist, obtained from under grass next to roots.

***Pictures submitted via survey.

Conclusions/Next Steps: The dialogue surrounding the scientific question was constructive, and ideally, each group will be able to contribute correlation between their variables. The next lab procedure will involve washing the soil gathered today to obtain direct and enriched lysates that will be used to conduct a spot test and plaque assay. Depending on the results of the tests (spot and plaque assay), we will be able to begin to support or reject the research question surrounding transplant status and age of tree.

8.29.18- Plaque Assay

Results from Monday: After 48 hours in the incubator, the results from the Spot Test were ready to be analyzed. Upon analysis, the plate had no trace of plaque. The negative control plate properly displayed a bacterial lawn with no disruption, but neither enriched lysate nor direct isolate caused any formation of plaque on the experimental plate. The negative control section of the experimental plate also showed no plaque formation or disruption of bacterial lawn, which was to be expected because any other result would have been indicative of contamination.

Rationale: Plaque Assay test needed to be done to determine whether or not results from the Spot Test on Soil Sample A could be confirmed by presence or lack of a plaque (since the description above shows that Monday’s Spot Test showed negative results for presence of a bacteriophage, a result to confirm this would be a lack of plaque on the Plaque Assay). In conjunction, the Plaque Assay was performed to gain further comprehension of the procedure and how to perform each step.

Procedure:

1. Lab space cleaned with CiDecon (sprayed on surface and wiped until dry) and 70% ethanol (sprayed on surface and wiped until 75% dry).
2. Ethanol burner lit to establish aseptic zone. Each addition to tubes was done in this aseptic zone in order to prevent particles from falling into the samples and causing contamination.
3. (In aseptic zone) Tube with 0.5mL of Arthro was obtained and labelled HMB Arthro 8/29/18.
4. (In aseptic zone) 10μL of lysate added to tube with 0.5mL of Arthro. This mixture was set to the side to give the Arthro and lysate to interact while the rest of the experiment was set up.
5. 50mL Conical tube was obtained.
6. (In aseptic zone) 8mL LB Broth was added to the Conical Tube
7. (In aseptic zone) 90μL CaCl2 added to the Conical Tube
8. Plate with agar was obtained and labelled “HMB Plaque Assay 8/29/18”.
9. (In aseptic zone) 10mL of 2X Top Agar was added to the Conical Tube with LB Broth and CaCl2
10. 2X Top Agar, LB Broth, CaCl2 was mixed in Conical Tube (components of overlay solution) by gently shaking the tube back and forth.
11. (In aseptic zone) 4.5mL of overlay solution was added into tube containing 0.5mL of Arthro and lysate. Mixture was swirled to mix.
12. (In aseptic zone) Overlay solution was poured onto plate labelled “HMB Plaque Assay 8/29/18” and let settle.
13. 1mL of overlay solution from Conical Tube added to Control plate shared by groups 3, 4, 7, and 8. This was done due to the shortage of plates in the lab because of contamination.
14. Overlay on experimental plate was allowed to solidify and was stored in the incubator to be checked on Friday.

Observation:

• Plaque Assay plate had many bubbles and the surface appeared to be slightly bumpy. This could be a point to examine if problems occur.
• Excess overlay solution was observed in Conical Tube. The mathematics seemed to excuse this observation (3 individuals used 4.5mL out of 18mL in tube resulting in 4.5mL leftover. 1 mL was used for the control plate, therefore allowing 3.5mL to be excess in the Conical Tube), but the discrepancy should be reported.
• Overlay solution was a golden yellow, as seen before in the Spot Test.

Results from the Plaque Assay cannot be obtained until plate has had time for bacteria to grow and be influenced by any present phages. Therefore, results from plaque assay will be found on Friday during open lab session.

Conclusions drawn: Based on the results of the Spot Test, it is more than likely that the original sample obtained does not have bacteriophages. The lack of plaque shows that there is no death of cells, which is the trademark of a bacteriophage as they reproduce. However, the Plaque Assay may be able to overturn this conclusion if it does contain plaques.

Next Steps: Control and experimental plate will be observed on Friday to determine whether or not the results of the Spot Test are supported or not. If there are no plaques present, it is confirmed that the sample did not contain bacteriophage and another sample will be needed. If there are plaques present, they will be picked and processed further.

Update: Plate was observed and confirmed the negative result for presence of a plaque or bacteriophage. The bacterial lawn was not formed particularly well (as seen in picture below), but there was still no presence of a plaque. The next steps to take from this result will be obtaining a new Soil Sample that ties in with the overarching research question to aid in developing data to answer the question.

Rationale: A spot test was performed in order to display the results of Soil Sample A. The results will how whether or not a bacteriophage or bacteriophages are present through existence of plaques on a bacterial lawn.

Scientific Research Question: Does the species of oak tree cause any discrepancy in number or type of phages found in soil samples? **Yet to be confirmed**

Procedure Performed in Lab:

1. Work site was cleaned using CiDecon (applied and wiped dry) and 70% ethanol (applied and wiped until half dry). An aseptic zone was established by setting up an ethanol burner near the center of the bench that the experiment and aseptic steps were completed close to.
2. Enriched lysate labelled as “HMB Arthro Added 8/22” was obtained along with a syringe and 22 microliter filter for the end of the syringe.
3. (Aseptic Step) 2 mL of enriched lysate was drawn into the syringe. 22 microliter filter was placed on end of syringe, then newly formed “Filter Sterilized Enriched Lysate” (abbreviated FSEL for labelling purposes) was created by moving the 2 mL of enriched lysate inside the syringe through the filter and into a microcentrifuge tube labelled “HMB FSEL 8/27/18” and “HMB” on lid. This step was done to remove existing bacteria from the enriched lysate while allowing possible bacteriophages to still pass through the filter and into the new tube.
4. Plate with agar was obtained. On the bottom, it was divided into 3 sections (enriched, direct, and negative control) and labelled “HMB Spot Test 8/27”.
5. One 50mL Conical Tube was obtained and labelled “HMB Top Agar 8/27/18”. A second Conical Tube was obtained and labelled as the negative control. Conical Tubes held components of Top Agar before the Top Agars (one with Arthro and one without) were poured out on experimental and control plates, respectively.
6. Concentration of CaCl2 was obtained for Experimental Plate containing Arthro:
   1. (1M CaCl2)(Volume)=(4.5mM)(10mL)
   2. (1000mM CaCl2)(Volume)=(4.5mM)(10000 uL)
   3. Volume= 45 microliters of CaCl2
7. Concentration of CaCl2 was obtained for Control Plate without Arthro:
   1. (1M CaCl2)(Volume)=(4.5mM)(9.5mL)
   2. (1000mM CaCl2)(Volume)=(4.5mM)(9500uL)
   3. Volume= 42.75 microliters of CaCl2
8. (Aseptic Step) Experimental Plate Containing Arthro (labelled”HMB Top Agar 8/27/18″): 4.5mL of LB Broth was added to 50mL Conical Tube. 0.5mL of Arthro was added by Lathan Lucas to 50mL Conical Tube. 45uL of CaCl2 was added to 50mL Conical Tube.
9. (Aseptic Step) Control Plate without Arthro: 4.5mL of LB Broth was added to 50mL Conical Tube. 45uL of CaCl2 was added to 50mL Conical Tube. No bacteria was added to this tube.
10. (Aseptic Step) To finish the creation of Top Agar, 5mL of Top agar solution that had been warmed in an incubator was added to both the Experimental tube and Control tube. This step was done last because Top Agar solution hardens as it cools, so as soon as it had been added to both tubes and they had been gently swirled, the 50mL Conical Tube of LB Broth, Arthro, CaCl2, and Top Agar solution was poured over the plate labelled “HMB Spot Test 8/27” and the 50 mL Conical tube containing LB Broth, CaCl2, and Top Agar was added to the negative control plate.
11. Waited 15 minutes to allow for complete solidification of Top Agar.
12. (Aseptic Step) Micropipetted 10uL of Direct Isolate, Filter Sterilized Enriched Lysate, and phage buffer into their respective sections as labelled on the bottom of plate.
13. Waited 15 minutes before placing plates into the incubator. Plates will be read on Wednesday to determine the presence of a phage or not.
14. Lab station was cleaned by disposing waste products into correct locations and storing tubes/equipment in designated places. CiDecon and 70% ethanol were used to clean the bench in similar manners as they were at the beginning of the procedure.

Observations:

• Filter Sterilized Enriched Lysate was yellow-gold in color after being moved through the 22uL filter.
• Filter was said to cause resistance in pushing lysate out of syringe. However, during experiment, only slight resistance was felt during expulsion. If there are adverse results, this could be a reason why.
• Top Agar was a dark golden color while kept in the Conical tube before being plated.
• Drops of Direct Isolate, Filter Sterilized Enriched Lysate, and phage buffer momentarily stayed on surface of Top Agar before being absorbed. This caused Direct Isolate drop to slide to a location relatively close to the border between it and the next section, which should be observed and considered when taking results.

Results:
Test is not completed, so results have yet to be obtained. On Wednesday (after 48 hours have gone by), it will be possible to discern whether or not a phage is present on the plate by the presence or lack of plaques.

Next Steps: In the lab on Wednesday, a plaque assay will be performed in addition to analyzing the plates that were prepared by the spot test procedure. This will serve to confirm the results that will be obtained at the beginning of the lab session on Wednesday.

Rationale: Washing Soil Sample A will rid sample of waste products like dirt and bacteria and allow for possible bacteriophages found in sample to be isolated in a lysate.

Procedure:

1. Cleaned lab station using CiDecon (sprayed and fully dried) and 70% EtOH (sprayed, half-dried, and let to evaporate).
2. Obtained and lit an ethanol burner.
3. LB Broth (placed close to the burner to maintain aseptic technique) was poured into tube to 35 mL mark on Conical Tube.
4. Using a combination of hand-shaking and use of vortex machine, LB Broth and Soil Sample A (from oak tree by Baylor Science Building) were mixed for 15 minutes.
5. During mixing process, tube containing Soil Sample A and LB Broth was massed and found to be 53.454g. The purpose of this was to find another sample of similar mass (+/- 0.1g) to achieve symmetry in the centrifuge.
6. After mixing, tube containing Soil Sample A and LB Broth was centrifuged at 3000xg rotations/minute for 5 minutes.
7. Supernatant was added to top filter to remove bacteria and other particles from soil. Filtrate was obtained after it had passed through the white filter.
8. 26mL of filtrate that passed through filter was split (about 13mL left in top filter conical tube, about 13mL half placed in new 15 mL tube).
9. 0.5mL Arthrobacter was added to 50mL conical tube from top filter containing half of lysate to form an enriched isolation.  The other 15mL tube with half of lysate was left to act as the direct isolation.
10. Enriched and direct isolations were left to be shaken until Monday, 8/27.

Observations:

• After shaking process, overall volume appeared to have decreased from 35mL to 33mL – could have been due to soil absorbing LB Broth during shaking.
• Mixture was dark brown and had many bubbles immediately after shaking.
• After use of centrifuge, supernatant appeared to be a yellow-gold color that still had some soil particles and grass inside that was avoided when placing in the filter.
• After placing 0.5mL of Arthrobacter in the enriched solution, it appeared that there was a very slight amount of Arthrobacter left in the tube that did not get added to the solution. Adverse results could eventually be explained by this, however, it appeared that generally it was common for others to encounter the same situation.

Results:

• Procedure was accomplished without problems during creation of isolations. No issue with filter was had. Tangible results with more data will be possible to obtain on Monday (8/27) when a spot test could reveal any possible bacteriophages from sample.

Next Steps:

• During the next lab time, isolations will be examined to see if bacteriophages were present in the sample obtained from the soil. This will be accomplished by the spot test procedure.