17 October 2018 ✷ Soil 5 amplification

Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was amplified using PCR to determine if the sample was worth spot testing for phage.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• 4 mL of enriched lysate was transferred to a 15 mL conical vial and centrifuged at 3000 g for 5 minutes. 1 mL of the separated supernatant was placed in a microcentrifuge tube and heated. 1 µL of soil sample 5’s “phage DNA” was added to 3 PCR tubes. Volumes can be seen below.

• The DNA samples were then cycled through PCR and stored until Monday.

Observations, results, data

The water used in the PCR tubes was DI water instead of doubly purified water, which shouldn’t ruin the PCR but may make some impact.

Metadata from Monday was finalized and recorded in Monday’s post.

interpretations, conclusion, next steps

The primers in each of the tubes should bind to any DNA known to an arthrobacterphage, meaning the use of a gel electrophoresis should result in the visual confirmation of phage DNA in the samples. If a sample is positive, it means that one of the DNA samples within it was positive, so a spot test or plaque assay would be conducted to determine which was positive.

Next lab, a gel electrophoresis will be run to test for phage DNA.

15 October 2018 ✷ Enrichment and Metadata, soil 5

Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was filtered and enriched with LB broth in order to aid the phage isolation process.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• An empty weighboat was massed and then filled with “wet” soil and massed. The weighboat and soil were left to dry overnight under the fume hood.
• 4 mL of soil was added to a falcon tube and mixed with 8 mL of DI water and 3 drops of soil dispersion fluid and shaken for 30 seconds. The mixture was left to settle overnight.
• 10 mL of LB broth was added to 2 mL of soil and vortexed for 10 minutes. The mixture was then centrifuged at 3,000g for 10 minutes. The liquid that rose to the top was filtered through a 22 micron syringe filter into 2 vials: one for a direct lysate (~1 mL, refrigerated) and one for an enriched lysate (~5 mL, added 0.5 mL arthro and stored in shaking incubator).
• A small amount of soil was added to a pH test tube and mixed with DI water and shaken for 10 seconds. The pH was taken after allowing the mixture to settle for 45 seconds.

Observations, results, data

pH: 6.0

Mass of weigh boat: 2.03 g

Mass of wet soil + weigh boat: 6.83 g

Mass of wet soil: 4.80 g

Mass of dry soil + weigh boat: 5.98 g

Mass of dry soil: 3.95 g

Mass of water: 0.85 g

mass of water/mass of wet soil*100: 14.2%

% sand, silt, clay:

0.5 mL clay = 12.5%

0.5 mL silt = 12.5%

3.0 mL sand  = 75%

Interpretations, conclusion, next steps

A plaque assay will be conducted in the next lab in order to test for the presence of plaques.

By the fifth attempt at isolating a phage, the human error involved has been greatly reduced. Additionally, each group member tested a different sample, so the test for phage needs to be done with as little human error (+ contamination) as possible in order to guarantee a reliable result.

10 October 2018 ✷ Try #5

Rationale: New soil was collected because the spot test from Soil 4 was negative. 

Procedure

• The lab was out of sample collection bags, so rubber gloves were used in place of bags to store collected soil.
• Soil was collected from a red oak in front of LL Sams apartment complex, then a basketball hoop, and an electric pole. Different than all other collections, a shovel was used to dig up the soil instead of a plastic scooper.
• Circumference, crown width, and height were all determined with a ruler (for the first two) and comparison of a known height for the height of the tree.
• Collected soil was then stored in a refrigerator over night.

data, observations, results

The shovel used to collect sample 5 was also used to collect soil from other places from other groups. This might mean cross-contamination which could yield a spot test positive for phage.

 the height of the electric pole was estimated to be 5.3 m. The circumference at 137 cm was found to be 144 cm and the crown spread was 30 cm.

The basketball hoop had a circumference of 5o cm, a crown spread of 103 cm, and a height of 3 m.

The soil samples taken from the electric pole and the basketball hoop were taken as a last-ditch effort to see if a correlation between tree and soil even existed.

Interpretation, conclusion, next steps

Next lab period, metadata from the soil will be collected and the soil will be washed and enriched.

The soil taken in this lab period was collected in three locations. One location was a real tree consistent with Group 6’s research question, while the other two were more of a last-ditch effort to locate a phage to study.

Gloves were also used as an apparatus to store the soil, and hopefully this does not harm the sample and has no effect on the viability of the sample.

Additionally, hopefully the use of a shared shovel does not affect the outcome of the soil’s content.

8 October 2018 ✷ Spot Test, Soil 4

Rationale: A spot test was conducted in order to test for the presence of phage.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• The enriched lysate was filtered with a 22 micron syringe filter.
• A plate was mixed with the following concentrations and volumes: (one plate for a control plate, and a “double” plate was mixed to share between 2 group members.

• 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	–	1.0 mL	–

• The plates were poured and 10 µL of phage buffer, direct lysate, and filtered enriched lysate were added to the plate and the plates were incubated overnight.

observations, results, data

The plates were poured while they were slightly too cold, so the plates didn’t completely harden. This shouldn’t have a huge effect on a spot test, but a plaque assay wouldn’t be effective in this condition.

interpretation, conclusion, next steps

If this spot test is negative, a new soil sample will be collected. If it is positive, a plaque assay will be run and a plaque will be picked to isolate a phage.

Time is running out to locate a phage, so if one isn’t isolated soon, Group 6 will have to adopt a plaque from another group to study.

3 October 2018 ✷ Enriching Soil 4

Rationale: Soil was enriched in order to isolate a phage.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• 2 mL of soil was added to a tube and 10 mL of LB broth was added and the vial was vortexed for 15 minutes. It was then centrifuged for 10 min at 10,000 g.
• A small amount of soil was placed in a pH vial and DI water was added; the vial was shaken for 30 seconds and a pH test strip was inserted for 45 seconds and then read immediately.
• The centrifuged sample was removed with a syringe and filtered through a 22 micron syringe filter into 2 vials: one for a “direct” lysate and one for an “enriched” sample. 0.5 mL of arthrobacter was added to the “enriched” sample and stored in a shaking incubator until Monday. The direct sample was refrigerated.

observations, results, data

pH: 5.5  

% sand, silt, clay

total: 6 mL

clay: 0.5 mL = 8.3%

silt: 5 mL = 83.4 %

sand: 0.5 mL = 8.3%

interpretations, conclusions, next steps

The silt percentage is higher in sample 4 than in other soil samples collected by group 6. Perhaps this will result in the discovery of a phage since it is a new and unique quality to the collected soil samples.

In the next lab period, a spot test will be conducted in order to test for the presence of a phage.

1 October 2018 ✷ Soil 4

Rationale: A fourth soil sample will be collected because the spot tests and plaque assay within Group 6 resulted in negative results, indicating that there is likely no phage present in the soil.

Procedure

• Soil was collected in three vials and two baggies from a red oak tree outside of the Baylor Sciences Building.\
• Back in the lab, the test for percent water and percents sand, silt, and clay were started (results will be determined in the following lab period).
• To find percent mass, an empty weighboat was massed, then soil was added, and the soil and weighboat were massed together. The boat was left to dry under the fume hood.
• To find percent sand, silt, and clay, 4 mL of soil was put into a falcon tube and 8 mL of DI water was added, along with 3 drops of dispersion liquid. The solution was shaken for 30 seconds and allowed to sit overnight.

observations, results, data

Mass of empty weighboat: 2.32 g

Mass of weighboat + wet soil: 6.77

Mass of weighboat + dry soil: 6.66

tree data:

interpretations, conclusions, next steps

% water: mass of water/mass of wet soil = (wet-dry/wet-weighboat)*100 = (6.77-6.66 g/6.77-2.32 g)*100 = 2.47% water

In the next lab period, the metadata tests will be concluded and the soil sample will be washed and enriched.

The tree selected for this soil collection was chosen because of its location. The research question is still comparing red and white oak trees, however the location is shifting throughout Baylor’s campus. Tree 4 is the healthiest of the trees that have been sampled by Group 6, so hopefully phage is present in the soil surrounding its roots.

26 September 2018 ✷ Spot Test #3, again

Rationale: A spot test will be performed to test for the presence of phage from the third soil sample collected because the data from the previous lab period was contaminated and thought to have used the incorrect type of bacteria.

Procedure

• The workspace was cleaned with CiDecon and 70% ethanol
• A tube with enough plate mix for two tests was mixed (fourth column) and a control plate mix was made (column 2), until it was discovered that there was not enough arthrobacter in the lab for two test plates. At this point, the single “control” mix became the tube of plate mix for the spot tests of two people and the plate had 5 circles for a negative control, two direct lysates, and two enriched lysates. The volumes and concentrations of their components can be seen below.

• component	volume (single)	concentration	volume (double)	concentration
  2X Top Agar	2.5 mL	1X	5 mL	1X
  LB Broth	2.1 mL	–	4.2 mL	–
  1M Calcium Chloride	23 µL	4.5 M	45 µL	4.5 M
  Arthro	400 µL	–	0	–

• The plates were poured immediately and were allowed to set for 10 minutes.
• The enriched sample was filtered last lab period and used for the spot test. 5 µL of the filtered enriched sample was added to the designated space on the plate for each group member. 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 for each group member.
• The plate was then incubated until Monday.

observations, results, data

There may be confusion with positive results in this test because two peoples’ samples were tested on the same plate due to lack of resources. However, because both lysates came from soil collected from the same tree, it is likely that if one is positive, both will be positive for phage.

interpretations. conclusion, next steps

One group member ran a plaque assay and two ran spot tests. If all 3 members receive negative results back, a new soil sample will need to be collected to find a phage. If positive results are seen, a plaque will be picked and a phage will be isolated.

Arthrobacterphage only infects arthrobacter, so the strain of bacteria accidentally used on Monday would need to be slightly different in order for the positive controls, which were remarkably strong titers. to be negative.

24 September 2018 ✷ Spot Test #3

Rationale: A spot test will be performed to test for the presence of phage from the third soil sample collected.

Procedure

• The workspace was cleaned with CiDecon and 70% ethanol
• A tube with enough plate mix for two tests was mixed (fourth column) and a control plate mix was made (column 2). The volumes and concentrations of their 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	–	1.0 mL	–

• The plates were poured immediately and were allowed to set for 10 minutes.
• The enriched sample was filtered through a 22 micron syringe filter and stored in a microcentrifuge tube for later use. 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 needed again.

observations, results, data

Previously, a test for percent sand, silt, and clay was performed for further metadata. 

The solid within the vial reached the 2 mL line. The lowest layer, sand, reached 0.25 mL, silt reached 0.5 mL (0.5-0.25=0.25 mL silt), and clay was the largest (2.0-0.5=1.5 mL).

% Sand = (0.25 mL)/2.0 mL * 100 = 12.5%

% Silt = (0.25 mL)/2.0 mL * 100 = 12.5%

% Clay = (1.5 mL)/2.0 mL * 100 = 75%

UPDATED: the control plate had contamination and the spot test was negative.

interpretations, conclusions, next steps

The positive control plate was also negative, indicating that there was an error with the test. It is likely that there was an issue with the “arthrobacter” used in the test because the results that should’ve been positive were negative. Thus, the same test will be repeated in the next lab period because results from Day 9 are not usable.

17 September 2018 ✷ Third Try’s a Charm?

Within Lab Group 6, two spot tests and one plaque assay were run, and all yielded negative results (see below). Thus, it was decided it was highly improbable that the sample was positive, so new soil was collected (again) in the same fashion as the previous time in order to try and locate a phage.

Lathan’s Questions

1. The sample Justin used likely had a higher phage concentration in it (a stronger titer).
2. 

Procedure

• As in the previous attempt at locating a phage, a soil sample was collected from a red oak outside of the Baylor Science Building (the difference from the last collection is the location of the tree).
• Soil was collected from 2 ft away from the base of the tree, but no specific data was collected other than photos of the tree. Circumference, crown diameter, and tree damage data were collected.  These data and images of the tree can be seen below.
• Upon returning to the lab, a small sample of the newly collected soil was set aside in a previously weighed weighboat to perform a test for percent water. It was left to dry under the fume hood until Wednesday’s lab period.
• The remaining soil was refrigerated until Wednesday.

Observations/results/data

• Circumference of tree: 102 cm; crown spread: 375 cm; some broken branches

• component	mass (g)
  empty weigh boat	2.41
  weigh boat + wet soil	6.66
  wet soil (6.66-weigh boat)	4.25
  dry soil + weigh boat	6.39
  dry soil (___-2.41)	3.98
  Water (wet soil – dry soil)	0.27
  % water (water/wet soil)	6.4% water

• Unlike the last tree, the most recent tree had more grass covering the base of the tree/roots. The correlation between this and phage presence is unknown, but hopefully it leads to the discovery of a phage.

Interpretations/conclusions/next steps

• The soil collected today will be enriched next time and then spot tested in order to hopefully isolate a phage. The percent mass test will be concluded and more metadata, including percent sand, silt, and clay will also be conducted.
• The soil collected today is significantly less sticky (less clay) than the last soil sample. Again, the correlation is not known at this time, but this may possibly be a better environment for phages to infect arthrobacter and yield a positive spot test for presence of phage.

19 September 2018 ✷ Third Enrichment + Metadata

Soil will be washed and enriched in order to help isolate a phage (if there is one) and metadata will help compare positive/negative results with other groups’ data in order to look for trends.

Procedure

• The workspace was cleaned with CiDecon and 70% ethanol and an alcohol burner was lit to promote an aseptic environment.
• 2 mL of soil was collected in a vial and roughly 10 mL of LB Broth was added and the vial was vortexed for 15 minutes to mix. The sample was then centrifuged for 10 minutes at 10,000g.
• In the meantime, the mass from the drying % Water test was massed and percent water was calculated to be 6.4% (see Day 7).
• Another vial was filled with 4 mL of soil and roughly 8 mL of DI water. 3 drops of soil dispersion fluid was added, then the sample was shaken for 30 seconds, decanted into a falcon tube, and stored until Monday. This is the test for percent sand, silt, and clay.
• pH was determined by placing a small amount of dirt in a pH tube and adding DI water, shaking to mix, and inserting a pH strip, which was compared to the test kit’s indicator chart.
• After the enrichment sample was done spinning, it was filtered with a 20 micron syringe filter and 9 mL was set aside to be enriched and 1 mL was set aside to be the direct sample. The direct lysate was refrigerated.
• 0.5 mL arthrobacter was added to the “enriched” sample and left in a shaking incubator until Monday.

Observatios/Results/Data

pH= 6.0

This sample is more acidic than the previous sample.

Interpretations, Conclusion, Next Steps

Next time, a spot test will be run in order to determine if phage is present or not.

Overall, the repetition of the same procedure three times because of two negative results has led to more confidence in the tests and hopefully the third attempt yields a positive result.

Both Group 5 and Group 6 had all negative results from the initial test location. This attempt compared the same types of trees in a different location, which limits the variables at play and only changes the research question from “near North Village” to “near the Science Building.”