12 September 2018 ✷ Metadata Conclusion & Spot Test

Metadata (% silt, % sand, % clay, % water, and pH) was collected in order to further examine the links between tree type and phage presence. Today it will be finalized by recording final results. A spot test will be performed to indicate the presence of phage from the soil collection.

Procedure

• The enriched lysate was spun at 3000g for 5 minutes.
• The dried soil from Monday’s lab was massed and the percent water was calculated (see SEA Bears Day 5).
• I worked with one of my lab partners to mix the agar solutions for our plates and a control plate. We cleaned the workspace with CiDecon and 70% ethanol to promote an aseptic environment and then lit an alcohol burner. The control plate was made separately, but our two plates were made together, thus the volumes of the components were doubled. LB Broth was added first, then Calcium Chloride, followed by arthro to the spot test plates (not added to the control plate) and finally top agar. The volumes and concentrations of the components can be seen below.

• component	volume (control)	concentration	volume (spot test)	concentration
  2X Top Agar	2.5 mL	1X	5 mL	1X
  LB Broth	2 mL	–	4 mL	–
  1M Calcium Chloride	23 µL	4.5 M	45 µL	4.5 M
  Arthro	0 mL	–	0.5 mL	–

• The test plate was poured immedietally to prevent it from hardening in the conical vial; it was allowed to set for 10 minutes.
• The spot test plates were poured next from a serological pipette, which was also used to mix the mixture. Each plate was allowed to set for 10 minutes.
• The enriched sample was filtered through a 22 micron syringe filter and stored in a microcentrifuge tube. 5 µL of the filtered enriched sample was added to the designated space on the plate. 5 µL of phage buffer was added to the plate as a control, and 5 µL of the direct sample was added to the plate.
• The excess samples were stored in the refrigerator and the plates were placed in an incubator to sit until Monday.
• Because the % silt/sand/clay falcon tube was difficult to read, it was reshaken and repoured to attempt to get more accurate results for this metadata.

Observations/results/data

The “recipe” for a spot test plate requires 0.5 mL arthro for EACH plate, and we forgot to double this quantity when we mixed our plates. Thus, there may not be enough arthrobacter on the plate for the phage to infect and kill to make a noticeable plaque on the plate.

Results from the metadata collection are recorded on Day 5’s post.

Interpretations/Conclusion/Next Steps

The presence of arthrobacter in the plate is important for the plaques to form, and the limited amount in my plate may pose an issue. If results are inconclusive, I will run a plaque assay with my enriched sample in order to confirm the presence of a phage.

10 September 2018 ✷ Metadata & Enrichment

Metadata (% silt, % sand, % clay, % water, and pH) was collected in order to further examine the links between tree type and phage presence. While it isn’t imperative to the research process, the metadata may provide some interesting connections between phage presence in different groups/locations. Additionally, the soil was cleaned and enriched to help isolate any phage that may have been present in the original sample.

Procedure

• First, the workspace was cleaned with CiDecon and 70% ethanol and then an alcohol burner was lit to promote an aseptic work environment.
• 2 mL of soil was added to a 15 mL conical vial and then 10 mL of LB Broth was added to the same vial (reaching 12 mL line on tube). The vial was then shaken for 5 minutes, vortexed for 5 minutes, and shaken by hand again for a final 5 minutes. The vial was massed at the end of the 15 minutes and found to be 19.329 g.
• The vial was taken to be centrifuged at 10,000g for 10 minutes. In the time the Dr. Adair had my sample, I worked on the metadata.

% Water

1. I found the mass of a dry, empty weigh boat to be 2.31 g. I added some of my soil sample to the boat and found the mass of the soil + weigh boat to be 8.62 g. The soil/weigh boat was placed uncovered under the fume hood in the lab to dry until Wednesday, when it will hopefully be dried out and massed again.

% sand, % silt, % clay

1. 4 mL of dirt was added to a falcon vial and the vial was then filled to the 12 mL line with deionized (DI) water. 3 drops of soil dispersion liquid was added, the vial was covered, and the mixture was shaken by hand for 30 seconds. The supernatant was removed and stored in a separate 50 mL conical vial. Both were stored at room temperature under the fume hood.

pH

1. A small amount of soil was added to a pH tube and then the vial was filled with DI water, closed, and shaken for 10 seconds. A pH indicator strip was inserted for 30 seconds and read immediately after. The strip was a light green consistent with a pH of 6.5 (slightly acidic).

•  Once the sample was separated via centrifuge, 3 mL was removed with a syringe and filtered through a 22 micron filter attached to the end of the syringe. This was 3 times; each time the filter was removed to collect more lysate, the ends of it were cleaned with 70% ethanol. The intent was to have 10 mL lysate in a 50 mL conical vial and 1 mL in a 15 mL conical vial, but it ended up being 8 mL in the larger vial and 0.75 mL in the smaller. The smaller vial became the direct lysate and it was stored in the refrigerator.
•  0.5 mL of arthrobacter was added to the larger vial and stored in a shaking incubator at 28 degrees Celsius until Wednesday.

Observations, Results, Data

Pictured above is a leaf collected from the tree we collected soil from and an analysis of the traits of the leaf. This is more metadata that can be utilized in future steps of answering our scientific question.

interpretations/conclusion/next steps

The % sand, silt, clay separation didn’t work as well as hoped and the test will be redone next time to get better, more usable results.

Enriching the soil sample will hopefully lead to the discovery of a phage because the increased amounts of nutrients from LB broth will allow the arthrobacter to flourish, and because the phage feeds on bacteria, increased amounts of phage should be present.

The next lab period will consist of a spot test and conclusion of the metadata collection in order to isolate a phage and analyze the conditions it was found in (i.e. wetness from water percentage).

5 September 2018 ✷ Starting Over

The first time around, soil was collected with a broader question; however, this time, we collected soil with a narrower scope and research question, which is:

“In the Texana House area of campus, is there a correlation between presence of phage and tree species?”

My table, Groups 5 and 6, collected soil from trees in the Texana area. Each member of my group collected from the same Red Oak tree in front of Rogers Engineering and Computer Science building.

Procedure:

• My group, group 5, ventured to the north part of campus to collect soil from the large red oak in front of Rogers. Group 6 collected from a nearby white oak.
• We measured the circumference of the tree at 137 cm above the ground, then measured the distance from the center of the tree that the smallest part of the crown reached and then the widest part of the crown. We added those two values and divided by 2 to get the canopy diameter.
• We estimated the height of the tree by comparing it to the height of the building it stands next to.
• We collected several leaves and stored them in baggies and noted damage to the tree.
• Using a measuring scoop, we dug up a few inches of soil next to tree roots and collected soil, 4 mL into the collection vial, and a 2 scoops into the bag with the leaves. The samples were brought back to the lab and stored in a refrigerator.

Observations, data, results

Included below are the measurements of the tree, as well as physical images of the tree we collected soil from. This tree appears to be much older than other trees we passed on campus. Additionally, the ground surrounding the roots of the tree was grassy and didn’t have mulch around it, as the last tree we collected from did. Rather, it was more clay-like and I am worried that will pose an issue when we clean the soil to enrich it and isolate a phage. If enough broth cannot be filtered through the dirt, it will be very difficult to test for the presence of a phage because of the limited sample size.

These images of the tree we collected soil from show the damage on the trunk. The leaves are somwhat dead, the trunk has damage, and some branches are broken. This isn’t necessarily a variable we are testing, but it is something to take note of as we proceed through our research.

interpretation, conclusion, next steps

The next step is to wash the soil as we did in day 1; the purpose of that is to isolate any phage into a lysate that can be manipulated to test for the presence of phage. However, this washing process will be slightly altered from the initial attempt as to hopefully be more successful and efficient.

Because all three members of my group collected from the same tree, we have more solid “proof” in our discoveries because the results should be, in a sense, triple checked. This, however, means that our sample pool is slightly smaller than other groups may be because we only tested one tree while others could’ve tested 3 trees.

Finally, the damage or “health” will be minimally considered in the research surrounding our phages. While it is interesting, the focus of our question is on species of tree and not health of the tree. It is certainly something to think about, but it isn’t receiving our attention for the time being.

29 August 2018 ✷ Plaque Assay

Because no plaques were present in my spot test (negative spot test), only 1 plaque assay will be run as to practice running this type of test (using filtered enriched lysate).

Procedure:

• CiDecon and 70% Ethanol were used to wash the workspace and prepare the aseptic environment. An alcohol burner was lit to clean the air.
• The plate was made–see table below for volume and concentration values. The LB broth was added first (4x the amount was made because the lab group consisted of 3 people with the addition of a control plate). Next, we added the calcium chloride. 2 mL of the mixture was removed for the test plate and put in a separate vial; 2.5 mL of top agar was added last so it wouldn’t harden while we were adding the other components. The tube was inverted to mix the components and then pipetted onto the dish.
  

  component	volume	concentration
  2X Top Agar	2.5 mL	1.05X
  LB Broth	2.0 mL	–
  1M Calcium Chloride	22.5 µL	4.5 µM
  1000 µM*V=4500µL*4.5µM = 22.5 µL
  2X*2.5 mL=M*4.5mL = 1 M

• 10 µL of filtered enriched lysate was added to a tube of arthrobacter and allowed to sit for 15 minutes.
• The remaining 7.5 mL of TA was added to the shared vial and mixed; then 5 mL was transferred to a vial for Rachel and 5 mL was transferred to a vial for Lauren. After 15 minutes, the arthro mix was added to the TA vial, inverted to mix, and poured into the petri dish. After the plate solidified, it was inverted and placed in an incubator until 2 pm on Wednesday, September 5.

Observations, Results, Data:

• Because the last plate I made was contaminated, I took extra care to ensure ALL work was done aseptically.
• However, since the last test was negative, it is highly unlikely that the plaque assay performed today will produce any positive results.
• Of the 24 people in the class, 9 had positive spot tests.
• UPDATED: after sitting in an incubator for nearly a week, the assay yielded a negative result, meaning no phage was found. However, there didn’t seem to be any contamination, which is an improvement from last week’s experiment.

Interpretations, conclusion, next steps:

• Because the bacteria (and potentially a phage) are mixed before entering the plate, the chance of infection is higher on a plaque assay than on a spot test, thus this test may be more telling of the presence of a phage in a sample. However, a control plate is necessary to ensure the agar isn’t contaminated because the phage may cover the entire plate rather than just a small circle as in the spot test.
• Though the chances for my particular plate are low, there is still a possibility my plaque assay may yield a positive result (phage present).
• The negative result from the spot test will be used in answering the class’s overarching scientific question regarding the presence of phages in relation to the types of oak trees on Baylor’s campus.
• In the future, more soil will need to be collected from another tree in hopes of isolating a new phage to continue research on.

27 August 2018 ✷ Spot Test

A spot test is being performed to check for the presence of phage.

Scientific Question:

Which types of Oak tree does arthrobacter phage inhabit more frequently?

Procedure:

• Workspace was cleaned with CiDecon and 70% Ethanol and an alcohol burner was lit to promote an aseptic work environment.
• My petri dish was labeled with my initials and the date, in addition to 3 penny-sized circles (one labeled “negative control,” one labeled “enriched,” and the last labeled “direct.”
• An additional petri dish was set up for my lab group; its purpose is to test the agar my group made.
• In the process of making the plate, my group made 2 tubes: one for the control plate, and one with enough for our three plates (both used the same LB broth). SEE TABLE. The Top Agar was added last because it hardens as it cools and it was important to add the other components first as to be time efficient.

Components of Plate

control plate 

3 spot test plates

• The mixtures were poured into their respective plates and allowed to cool and harden.
• The enriched solution was filtered through a 22 micron filter into a microcentrifuge tube in order to remove arthrobacter.
• After plates hardened, a micropipette was used to place 4.4 µL of each sample into its respective circle on the plate (enriched, direct, negative control phage buffer).
• The remaining direct isolation sample was refrigerated and the plates were placed in an incubator for 46 hours.
• The lab space was then cleaned once again with CiDecon and 70% Ethanol Solution.

Observations/results/data:

• After the enriched solution was filtered, it became paler yellow.
• In the process of pouring my plate, my partner burned her finger on the burner flame and a small portion (less than 1 mL) of the agar solution spilled onto the table. Though this didn’t directly cause contamination to my plate, its possible there may be some issues with the data produced from my plate.
• UPDATED: Upon returning to lab on Wednesday, August 29, I removed the petri dish from the incubator and found no plaques, only contamination. However, our agar control plate showed no signs of contamination, thus the error had to have occurred in the process of pouring and storing our plates because each member of my lab group had contamination on her plate (see images)
• A likely source of contamination is that trying to maintain aseptic technique while having 3 people assist led to several moments where the open container we were working with left the aseptic zone and more precaution will need to be taken in future experiments.
• 

Interpretation/Conclusion/Next Steps

• The phage in each sample (if present) would infect the arthrobacter in the plate and kill it, leaving plaques that should be visible.
• If phages are present, the spot test is a way to indicate their presence; however, it does not indicate concentration.
• Instead, the use of a plaque assay next time will give further information in regards to the presence of phages from the soil sample.
• UPDATED: Because no phage was detected in the spot test, only one plaque assay will be run (with the enriched lysate) just to practice the technique. At some point in the future, another soil sample will be collected to attempt to isolate a phage.

22 August, 2018 ✷ Cleaning the Soil

After collection of soil from an oak tree in Zone 8 (near Gordon Teal Residential College), the soil was cleaned in order to isolate possible bacteriophage samples.

Procedure

• 20 mL of soil was removed from the collection vial and stored in a refrigerator for future tests. 15 mL of the original sample remained in the vial.
• The table was washed with Cidecon and 70% Ethanol solutions to promote an aseptic environment. An alcohol burner lamp was lit to clean the air surrounding the workspace.
• 20 mL of LB broth was added to the soil sample; the soil/LB broth mixture was then shaken by hand for 15 minutes to mix. At the end of 15 minutes, the vialwas massed and found to be 52.398 g.

• The vial was centrifuged at 3000 x g for several minutes. The mixture separated into 3 layers: heavier soil, a milky-yellow liquid, and some less dense wood chips at the top.

  click for centrifuged sample image
• Using a vacuum filter, the milky-yellow liquid from the center layer was filtered through a 20 micron filter. The resulting filtrate was paler yellow in color and more transparent.
• 5 mL of the lysate (filtrate) was poured into a 15 mL vial and refrigerated, and the remaining 10 mL were combined with 5 mL Arthrobacter (ATCC 21022). This sample was then stored in a shaking incubator for 5 days.

Observations/results

• As the lysate was cleaned and filtered, its color became lighter and more transparent.

Interpretations, Conclusion, Next Steps

• The purpose of this portion of the experiment was to purify the soil sample and isolate any bacteriophages in the soil sample. Thus, if any were present, they would be isolated in the enriched and direct isolation samples resulting from vacuum filtration.
• The next step is plaque assay and spot test–the potentially infected arthrobacter will be introduced to a nutrient-rich bacterial lawn and the presence of bacteriophages will be seen if plaques are present (places where the phage killed bacteria at the end of its lytic cycle).