26 November 2018 ✷ Pick a Plaque again

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque was picked from the picked plaque run 11/19 because the titer was too low.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• Plaque 1 on the plate from the prior lab session was picked and swirled into 30 µL of  phage buffer. This mixture was added to 0.5 mL of arthrobacter and allowed to sit for 15 minutes while the plates were made.
• A plate for 1 plaque assay and a control plate were made with the following concentrations and volumes:

• component	volume	final concentration
  LB Broth	4 mL	–
  2X Top Agar	5 mL	1X
  1M Calcium Chloride	45 µL	4.5 µM

• The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated until Wednesday at 37 degrees celsius.

Observation, Results, Data

3 plaques were seen on the plate that was picked in today’s lab and each was picked and plated. This will help isolate a single phage type from the sample and purify it so it may be studied.

Interpretations, Conclusion, Next Steps

In the following lab periods, the presence of a webbed plate will mean that the lysate is a high titer and can be studied more closely using TEM. The current titer is very low, which may indicate a weaker phage or phage presence.

19 November 2018 ✷ Pick a Plaque

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque was picked from the prior plaque assay and run in a plaque assay again.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• The lone plaque on the plate from the prior lab session was picked and swirled into 30 µL of  phage buffer. 50 µL of this mixture was added to 0.5 mL of arthrobacter and allowed to sit for 15 minutes while the plates were made.
• 
• A plate for 1 plaque assay and a control plate were made with the following concentrations and volumes:

• component	volume	final concentration
  LB Broth	4 mL	–
  2X Top Agar	5 mL	1X
  1M Calcium Chloride	50 µL	4.5 µM

• The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated until Tuesday at 37 degrees celsius.
• On Tuesday, the plate was removed from the incubator, wrapped with parafilm and frozen until the following Monday.

Observations, results, data

The plate had several plaques following the plaque assay, but they were quite small and far from being a webbed plate. This sample will need to be amplified quite a bit in order to get a webbed plate.

Interpretations, conclusion, next steps

The sample seems weak because it produced a very small amount of plaques (that were very small themselves). Additionally, after picking a plaque the first time, the plaques were lost, so the phage sample in this soil isn’t the strongest phage. The sample will need to be amplified by picking plaques until the plate is webbed and there is a high titer.

14 November 2018 ✷ Plaque Assay

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque assay was run with the newly enriched portion of her sample in order to confirm the presence of a phage.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• 30 µL of filtered enriched lysate was added to 0.5 mL of arthrobacter and allowed to infect for 15 minutes before being combined with the plate mix for a plaque assay.
• A plate for a 2 plaque assays and a control plate were made with the following concentrations and volumes:

• component	volume	final concentration
  LB Broth	6 mL	–
  2X Top Agar	7.5 mL	1X
  1M Calcium Chloride	68 µL	4.5 µM

• The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated until Monday at 27 degrees celsius.

Observations, results, data

The soil sample used for this plaque assay was positive when Melissa tested it, so the plaque assay should be positive when it is examined on Monday in lab.

Interpretations, conclusion, next steps

The plaque assay will likely be positive because the soil sample produced a positive plaque assay in the past. This being said, a plaque will be picked from the plaque assay and then re-run to try and isolate a phage and amplify its DNA.

12 November 2018 ✷ re-enriching Soil 4

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. However, the plaques were lost in the last plaque assay, so the soil was re-enriched to isolate the phage again.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• 5 mL of Soil 4 from Melissa’s collection on 10/5/18 was added to a conical vial and filled with 5 mL of LB broth and vortexed for 15 minutes.
• The sample was then spun at 3000 g for 10 minutes and the supernatant was removed via syringe.
• The supernatant was then filtered with a 22 micron syringe filter and 0.5 mL of arthrobacter was added to the vial. The sample was then incubated in a shaking incubator until Wednesday.

Observations, results, data

There were a few spots on the plate that may have been plaques, but the size and number is far less than the plate in which the plaques were picked from, so the experiment was repeated.

Interpretations, Conclusions, Next Steps

The soil sample used provided a positive plaque assay when Melissa carried out the experiment, but not when members of Group 6 tried, it was inconclusive. So, this is either the result of human error or other factors, such as the phage only existing in some parts of the soil and not others.

In the following lab periods, the lysate will be run through a plaque assay to identify the presence of a phage and later amplify it.

7 November 2018 ✷ Pick a Plaque Re-do

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque assay was run (pick a plaque) to amplify present phage. The first plaque assay showed heavy contamination so it will be redone in order to have a plate ready for flooding.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• The lysates from Monday’s lab were still available and there was enough arthrobacter for every group member to make a plate. One large vial was mixed with the following volumes and concentrations:

• component	volume	final concentration
  LB Broth	8 mL	–
  2X Top Agar	10 mL	1X
  1M Calcium Chloride	90 µL	4.5 µM

• 30 µL of the lysate was allowd to infect the arthrobacter for 15 minutes before being combined with 5 mL of the above mixture and poured into a plate, allowed to harden, and incubated until Monday.

Observations, results, data

The contamination seen in the plates likely came from the top agar or LB broth because several of the other groups that made plates experienced the same contamination.

There were very few plaques in the plaque assay run on Monday, so the volume of lysate was increased in the plaque assay run on Wednesday.

Interpretations, conclusion, next steps

If this plaque assay is run without contamination in the control plate, it will be amplified by flooding the plate with phage buffer in order to increase the concentration of phage in the sample.

5 November 2018 ✷ Adoption

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque assay was run (pick a plaque) to amplify present phage.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• 70 µL of phage buffer was placed into a microcentrifuge tube and a micropipette tip was used to “pick” a plaque from a plaque assay run by Melissa from her soil sample’s lysate and the tip was swirled around the tube to add phage to the buffer.
• A plate for a plaque assay and a control plate was made. However, because the lab was short on arthrobacter for this test, only Rachel’s picked plaque was run. The concentrations and volumes can be seen below:

• component	volume	final concentration
  LB Broth	6 mL	–
  2X Top Agar	7.5 mL	1X
  1M Calcium Chloride	68 µL	4.5 µM

• NOTE: the mixture shown above was meant for three plates, but because of the limited resources, only 2 plates could be made.
• The mixture was left in the hot water bath so the TA wouldn’t harden while Rachel’s lysate infected the arthrobacter for 15 minutes. Then, the arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated until Wednesday at 27 degrees celsius.

Observations, results, data

The control plate showed contamination but plaques were present on the test plate.

Interpretations, conclusion, next steps

The plaque assay will need to be repeated because of the contamination. After a clean plate and positive plaque assay are seen, the plate will be flooded in order to amplify the phage presence/titer.

Other groups’ control plates had the same contamination, so this likely wasn’t a result of human error.

31 October 2018 ✷ Soil 6 spot test

Rationale: Soil sample 6, a sample taken from near the bear habitat will be spot tested in order to test for presence of phage.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• 2 spot test plates and one control plate were made using the following table:

• 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 allowed to harden. On the first test plate, the samples from the first well (arthro added before initial incubation) were spotted from each member. On the second test plate, the control samples (no arthro initially) were spotted. The plates were then incubated at 27 degrees celsius until Monday.

Observations, Results, Data

The green contamination was spotted on both plates instead of being a control sample on the second plate. This shouldn’t have an effect on the outcome of the plate.

Interpretations, Conclusion, Next Steps

The high level of negative results seen by group 6 and by the class as a whole has led to the adoption of positive samples by members of groups that have had a low success rate. In the coming lab sessions, members of group 6 will begin working with a new (and positive) sample in order to study it more closely.

29 October 2018 ✷ Soil 6 “redo”

Rationale: Soil sample 6, a sample taken from near the bear habitat, was enriched and incubated, but one well turned green with contamination. The samples from the other wells were not visibly contaminated, but all were filtered in order to be spot tested for phage.

Procedure

• The lab table was cleaned with Cidecon and 70% ethanol.
• 
• A pipette was used to remove approximately 1 mL of the first and 1 mL of the third row fluids; each into its own microcentrifuge tube where it was centrifuged at 13300 g for 2 minutes.
• the top layer of fluid was then removed with a syringe and filtered through a syringe filter (22 micron). The samples were then stored overnight in a refrigerator.

Data, results, observations

Percent Sand: 75%

Percent Silt: 12.5%

Percent Clay: 12.5%

percent water: 83.67%

Interpretations, Conclusion, Next Steps

The arthrobacter in the sample that turned green was likely outcompeted by a bacterium such as pseudomonas. None of the other wells were noticeably contaminated, but all samples will be spotted in the following lab period to see what takes place and if a phage is present regardless of the contaminant.

24 October 2018 ✷ Soil 6 Enrichment + Metadata

Rationale: Soil sample 6, a sample taken from near the bear habitat was enriched and tests for metadata were conducted in an attempt to isolate phage and gather data on the soil.

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 soil was vortexed for 15 minutes with 8 mL of LB broth and then centrifuged for 5 min at 3000 g.
• Percent sand, silt, and clay was tested by combining 4 mL soil with 8 mL DI water and 3 drops soil dispersion fluid and shaking for 45 seconds. That mixture was allowed to sit overnight to settle.
• Other group members performed the tests for percent water and pH. The tree was not measured because of poor weather conditions, but it will be measured and reported when the rain stops.
• 1 mL of the supernatant from the centrifuged soil was pipetted into 3 wells in a well plate and heated to 55 degrees Celsius for 6 minutes and then heated to 60 degrees Celsius for 5 minutes in order to kill off any bacteria existing in the mixture before arthro was to be added.
• 100 microliters of arthrobacter was added to two of the three wells (last one was left empty to serve as a control) and then the shaker thermostat was left to shake and incubate the bacteria overnight (27 degrees Celsius).

Observations, results, data

pH: 6.5

The soil was collected during a thunderstorm so the percent water will be much higher because of the rain. It was also very difficult to measure out when attempting the metadata tests, but this shouldn’t change percent sand, silt, clay much. It may have raised the pH because the soil itself was already so watered down from the rain.

The experimental procedure was adapted in this experiment because of a lack of materials, but new filters were recently delivered and the prior trials can be repeated as before.

Interpretations, conclusion, next steps

The arthrobacter/lysate mix will be filtered and prepared for PCR in order to amplify any present phage DNA so that it can be tested for presence of phage DNA.

22 October 2018 ✷ Soil 5 gel electrophoresis

Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was tested using gel electrophoresis in order to look for presence of phage DNA.

Procedure

• The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
• 0.8 g of agarose powder was mixed with 40 mL TBE (buffer) and microwaved. Ethidium Bromide was added and the solution was poured into a gel apparatus and a 8-prong comb was added and the gel was allowed to harden. It was then transferred to an electrophoresis tray and filled with TBE.
• The first well was used for other group members to practice. The remaining wells were as follows:
  • Well 2: “(: 1” LL Sam’s Oak DNA + Other group’s DNA + primer 1
  • Well 3: “(: 2” LL Sam’s Oak DNA + other group’s DNA + primer 2
  • Well 4: “(: 3” LL Sam’s Oak DNA + other group’s DNA + primer 3
  • Well 5: DNA Ladder
  • Well 6: “★1” DNA from 2 other groups + primer 1
  • Well 7: “★2” DNA from 2 other groups + primer 2
  • Well 8: “★3” DNA from 2 other groups + primer 3
• The gel electrophoresis was run for roughly 30 minutes at 100 V and analyzed with UV.

observations, results, data

The gel, when analyzed with the UV scanner, was negative for phage DNA. The controls all worked, so the test was effective and the soil tested did not contain phage.

The other two samples collected on the same day, from the basketball hoop and electric pole, were also negative. The use of DI water instead of DD water did not have an adverse effect on the gel.

The gel pictured above “glowed” more than other groups, but this is likely because it ran for less time than the other groups’ gels and there was a higher concentration of dye in one spot instead of being dispersed throughout the entirety of the gel.

interpretations, conclusion, next steps

It is possible that the chosen variable for group 6, red oaks, is a negative one, meaning that the scientific question “is there a correlation between the presence of phage in white vs red oaks?” is being answered with, “there are no phages near red oaks” because every sample from red oaks has been negative for phage. It is still somewhat premature to make this conclusion, but the data has not proved this statement incorrect as of yet.

In the coming days, more soil will be collected and enriched. Then, it will be run through PCR and gel electrophoresis.