Date: Monday, October 15th, 2018

Title: Plaque Assay for Soil Sample C

Rationale: The purpose of today’s lab is to make a plaque assay for a new soil sample in order to search for plaques.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up
2. Enriched lysate was retrieved from the incubator.
3. 10 microliters of enriched lysate was added to a culture tube with .5 mL arthrobacter and left to sit for 15 minutes.
4. Agar was made for three plates with the following formula:
   1. 6 mL LB broth
   2. 7.5 mL 2x TA
   3. 67.5 microliters 1M CaCl2
5. 4.5 mL of the TA solution was added to each culture tube and the TA control plate.
6. The plates were left for 20 minutes to harden before being inverted and incubated for the next 48 hours.

Observations: One of the group members, Lucy Fisher, had a pink coloration to her enriched lysate. There has been a red bacteria travelling around the lab and has grown on some students’ plates.

Results: This experiment yielded a plaque assay that can be evaluated at a later date for plaques.

Next Steps: The next step is to evaluate the plaque assay. If it yields positive results, the next step is to pick a plaque. If negative, a new sample may have to be adopted.

Date: Friday, October 12th, 2018

Title: Soil Sample C Washing and Enrichment

Rationale: The purpose of today’s lab was to filter and enrich the soil sample in order to prepare a lysate that can be tested.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Soil Sample C was used to fill a 15 mL conical to the 2 mL mark.
3. LB broth was used to fill the conical to the 13 mL mark.
4. The 15 mL conical was vortexed for 15 minutes.
5. The conical with soil and LB broth was massed and found to be 19.470 g.
6. A second conical with the same weight was made using water in order to spin the conicals for 10 minutes at 10,000 gs.
7. 1 mL of the supernatant was filtered out using a syringe filter with a 22 micrometer filter and placed in another 15 mL conical to be used as the direct isolation.
8. ~10 mL of the supernatant was filtered out using a syringe filter with a 22 micrometer filter and placed into a 50 mL conical.
9. The lysate in the 50 mL conical was mixed with .5 mL arthrobacter and left to incubate over the next 72 hours.

Observations: At some point the 50 mL conical was possibly exposed to contamination when the lid was left open during a conversation. This occurred in an aseptic zone but will be noted and watched for.

Results: This experiment yielded an enriched and direct lysate which can be used for a spot test or plaque assay.

Next Step: The next step is to use a plaque assay or a spot test in order to test for plaques.

Date: Wednesday, October 10th, 2018

Title: Soil C Metadata

Rationale: The purpose of today’s lab was to record soil metadata for Soil Sample C collected 9-17-18.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. A pinch of Soil Sample C was added to a pH vial with DI water and shaken for ten seconds.
3. The pH vial was allowed to sit for two minutes before a strip of pH paper was added to the vial.
4. After 45 seconds, the pH paper matched a pH of 6.
5. Next, Falcon tubes were filled with soil to the 10 mL mark and DI water to the 30 mL mark.
6. 3 drops of dispersion fluid were added and the tub was shaken for 30 seconds.
7. The supernatant from the Falcon tube was added to a second Falcon tube and both tubes were left under a fume hood to allow dispersion of sand, silt, and clay.
8. To calculate percent water of the sample, a weight boat was used.
   1. Empty Weigh Boat: 2.432 g
   2. Weigh Boat with Soil: 7.452 g
   3. Wet Soil: 5.020 g
9. The weight boat with wet soil was left under a fume hood to dry out.

Observations: The plaque assay from last lab yielded negative results, meaning the plaques were either incorrectly picked or they were actually air bubbles.

Results: This lab yielded soil metadata that can be used to find trends in bacteriophages.

Next Step: The next step is to wash and enrich the soil in order to get a direct and enriched sample, then to make a spot test with the enriched sample, the direct isolation, and phage buffer to test for plaques.

Date: Monday, October 8th, 2018

Title: Third Attempt of Purification: Second Passage Soil Sample B

Rationale: The purpose of today’s lab is to go back to an older plaque assay with more promising plaques since new plates are yielding negative results.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plaque assays from the first purification passage were taken back out and a third plaque was chosen to be picked.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into the chosen plaque and swirled in the microcentrifuge tube to add phage to solution.
5. The microcentrifuge tube was vortexed to mix phage with buffer. This was set aside for later use.
6. Agar for two plates was made using the following recipe in a 50 mL conical vial:
   1. 3.8 mL LB broth (Note: less LB broth was used due to an increase in lysate used)
   2. 5 mL 2x Top Agar
   3. 45.0 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 100 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing .5 mL microliters arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 5 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.4 mL of the top agar solution was added to a top agar control plate.
12. 4.4 mL was added to the culture tube containing the arthro and phage sample.
13. This solution was added to an agar plate and moved around to cover the plate with solution.
14. The plates were left sitting to allow the agar to harden.
15. The plates were inverted and left to incubate.

Observations: The plaque assay from last lab yielded negative results. It’s interesting that the plaques have disappeared when they should be becoming more prevalent. It’s possible that the “plaques” picked so far have been bubbles or that the plaques have been picked incorrectly. Either way, more lysate was used this time so that more phage would be available to infect the arthro lawn.

Results: This experiment yielded a new plaque assay that should have a higher titer that can either be passaged again or flooded for amplification.

Next Step: If the plaque assay yields positive results, the next step is to passage the sample for the third time. If the plaque assay yields negative results, the next step is to work with Soil Sample C.

Date: Wednesday, October 3st, 2018

Title: Second Attempt of Purification: Third Passage Soil Sample B

Rationale: The purpose of today’s lab is passage the same lysate from the previous lab using a larger sample of phage in order to raise the titer of the plate.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plaque assays from the third purification passage were taken back out and evaluated. Since there was only one plaque on the plate, the plate was stored for future experimentation.
3. Agar for 3 plates was made using the following recipe in a 50 mL conical vial:
   1. 8.0 mL LB broth
   2. 10 mL 2x Top Agar
   3. 90.0 microliters 1M CaCl2
4. The LB broth and 1M CaCl2 were added to the 50 mL conical.
5. 50 microliters of the phage and phage buffer solution from the same microcentrifuge tube used for the third passage were transferred to a culture tube containing .5 mL arthrobacter.
6. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
7. 10 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
8. 4.5 mL of the top agar solution was added to a top agar control plate.
9. 4.5 mL was added to the culture tube containing the arthro and phage sample.
10. This solution was added to an agar plate and moved around to cover the plate with solution.
11. The plates were left sitting to allow the agar to harden.
12. The plates were left to incubate without being inverted since the agar didn’t harden.

Observations: The plaque assay from the third passage only had one plaque on it. Since the sample had such a low titer and was expected to have more plaques on it, 50 microliters of lysate was used instead of the usual 10 in order to amplify results. Also, the top agar was left to sit for over 20 minutes and still did not harden. This could have been because of the temperature of the plates.

Results: This experiment yielded a new plaque assay that should have a higher titer than before.

Next Step: The next step is to evaluate the plaque assay from this third passage and begin amplification of the phage. If the plaque assay doesn’t yield positive results, the next step is to pick a different plaque from an earlier plate and begin the process of purification again.

Date: Monday, October 1st, 2018

Title: Purification: Third Passage Soil Sample B

Rationale: The purpose of today’s lab is to further passage the lysate for the third time in order to isolate a phage.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plaque assays from the second purification passage were taken back out and a new plaque was chosen to be picked.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into the chosen plaque and swirled in the microcentrifuge tube to add phage to solution.
5. The microcentrifuge tube was vortexed to mix phage with buffer. This was set aside for later use.
6. Agar for 3 plates was made using the following recipe in a 50 mL conical vial:
   1. 6.0 mL LB broth
   2. 7.5 mL 2x Top Agar
   3. 67.5 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 10 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing .5 mL arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 7.5 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.5 mL of the top agar solution was added to a top agar control plate.
12. 4.5 mL was added to the culture tube containing the arthro and phage sample.
13. This solution was added to an agar plate and moved around to cover the plate with solution.
14. The plates were left sitting to allow the agar to harden.
15. The plates were inverted and left to incubate.

Observations: The control plate from the second passage was contaminated. It’s still unclear what’s causing such consistent control plate contamination. The current theories are that either the pipette tips are being hit against a contaminated surface or that people are talking near the aseptic zone.

Results: This experiment yielded a new plaque assay that can be evaluated and either passaged again or begin to web a plate.

Next Step: Since this is the third passage for this sample, the next step is to prepare for amplification. However, if the titer is too low, the next step would be to passage the sample again.

Date: Wednesday, September 26th, 2018

Title: Second Attempt of Purification: Second Passage Soil Sample B

Rationale: The purpose of today’s lab is to repeat the procedure from the last lab day and passage the phage for a second time.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Situation: It was determined by experimentation that what had been used during the last lab day was not arthrobacter ATCC 21022. This was determined by trying to infect a bacterial lawn with a high titer bacteriophage. The results were negative, meaning the bacteria was not arthro. Because of this, every procedure from Monday involving arthrobacter has to be repeated. Below is an image of my second passage plaque assay with no plaques (the small dot was determined to be the location of a bubble and therefore not a plaque).

Procedure:

1. An aseptic zone was set up.
2. Plaque assays from the first purification passage were taken back out and a second plaque was chosen to be picked.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into the chosen plaque and swirled in the microcentrifuge tube to add phage to solution.
5. The microcentrifuge tube was vortexed to mix phage with buffer. This was set aside for later use.
6. Agar for four plates was made using the following recipe in a 50 mL conical vial:
   1. 8.4 mL LB broth (Note: more LB broth was used due to a decrease in available arthrobacter)
   2. 10 mL 2x Top Agar
   3. 90.0 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 10 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing 400 microliters arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 10 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.5 mL of the top agar solution was added to a top agar control plate.
12. 4.5 mL was added to the culture tube containing the arthro and phage sample.
13. This solution was added to an agar plate and moved around to cover the plate with solution.
14. The plates were left sitting to allow the agar to harden.
15. The plates were inverted and left to incubate.

Observations: The plaque assay from last lab day wasn’t usable since the bacteria in the top agar wasn’t arthrobacter. By keeping our old plaque assays, there were more plaques available to pick without having to totally repeat the whole purification process from the first passage.

Results: This experiment yielded a new plaque assay that can be evaluated and either passaged again or begin to web a plate.

Next Step: The next step is to either further passage the phage, try to web a plate and prepare for amplification, or begin the purification process again with either another plaque on my first plaque assay or with new soil.

Date: Monday, September 24th, 2018

Title: Purification: Second Passage Soil Sample B

Rationale: The purpose of today’s lab is to further passage the bacteriophage in order to purify the phage sample.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plaque assays were evaluated and plaques were marked on the plate.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into a plaque and swirled in the microcentrifuge tube to add phage to solution.
5. The microcentrifuge tube was vortexed to mix phage with buffer. This was set aside for later use.
6. Agar for four plates was made using the following recipe in a 50 mL conical vial:
   1. 8 mL LB broth
   2. 10 mL 2x Top Agar
   3. 90.0 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 10 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing .5 mL arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 10 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.5 mL of the top agar solution was added to a top agar control plate.
12. 4.5 mL was added to the culture tube containing the arthro and phage sample.
13. This solution was added to an agar plate and moved around to cover the plate with solution.
14. The plates were left sitting to allow the agar to harden.
15. The plates were inverted and left to incubate.

Observations: This plate yielded a significantly smaller number of plaques. This is probably due the purification leaving out other phage species as the plaque assays and dilutions isolate the phage more and more.

Results: This lab yielded a plaque assay that can be further evaluated in order to perform a third passage on the phage sample.

Next Steps: The next step is to evaluate the plaque assay during the next lab. If the results are positive, the next step is to passage the phage for a third, possibly final, time. If the results are negative, then the next step is to either pick a different plaque from the first passage or to start with new soil (soil sample C).

Date: Wednesday, September 19th, 2018

Title: Plaque Picking and Serial Dilutions

Rationale: The purpose of today’s lab is pick a plaque from the plaque assay and set up serial dilutions that can be plaque assayed further.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plaque assays were evaluated and plaques were marked on the plate.
3. 100 microliters of phage buffer were added to a microcentrifuge tube.
4. A pipette tip was touched into a plaque and swirled in the microcentrifuge tube to add phage to solution. This is the 10° serial dilution.
5. The 10° tube was shaken to mix phage with buffer.
6. 90 microliters of phage buffer were added to each of two other microcentrifuge tubes.
7. 10 microliters of the 10° dilution were added to one of the tubes, marked as the 10^-1 dilution. This tube was then shaken.
8. 10 microliters of the 10^-1 dilution were added to the last tube marked as the 10^-2 dilution. This tube was also shaken.
9. 10 microliters of each dilution were added to different culture tubes with .5 mL ATC 21022 each.
10. Agar was made using the following recipe:
    1. 20 mL LB broth
    2. 25 mL 2x Top Agar
    3. 225 microliters 1M CaCl2
11. 4.5 mL of the TA solution was added to each culture tube.
12. The culture tubes with bacteria, phage, and top agar were briefly vortexed to mix the solution.
13. The contents of the culture tubes were added to their corresponding plates based on their dilution number.
14. The plates were left for 15 minutes to harden before being inverted and incubated.

Observations: The control for the plaque assays was contaminated, much like the control for the spot tests before. It’s still unclear what is causing the contamination, but it’s likely that arthro is somehow getting into the TA control.

Results: The plaque assay from before yielded positive results, with upwards of 20 plaques. This was confirmed not to be contamination, and hopefully this yields actual phages that can be isolated and sequenced.

Next Steps: The next step if the serial dilutions come back positive is to web a plate and further explore the phage. The next step if the serial dilutions plaque assays come back negative is to start working on Soil Sample C. Alternatively, the next step could be to pick a second plaque from the positive plaque assay and perform serial dilutions on it.

Date: Monday, September 17th, 2018

Title: Plaque Assay Setup and Soil Sample C Collection

Rationale: The purpose of today’s lab is to set up a plaque assay with the lysate from Soil Sample B as well as gather more soil if the plaque assay yields negative results.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Plaque Assay Procedure:

1. An aseptic zone was set up.
2.  Spot tests were evaluated and found to have yielded negative results
3. 10 microliters of the Soil Sample B enriched lysate was added to a culture tube with 5 mL ATC 21022.
4. Culture tube was set aside for 15 minutes so phage could infect arthro.
5. Agar was made with the following formula:
   1. 8 mL LB broth
   2. 10 mL 2x TA
   3. 90 microliters 1M CaCl2
6. 4.5 mL of the top agar solution was added to the culture tube.
7. TA solution with arthro and phage was added to agar plate, left to harden for 10 minutes, inverted and incubated.

Soil Sample C:

• Tree Circumference: 380 cm
• Small Canopy Diameter: 1606.5 cm
• Large Canopy Diameter: 1976 cm
• Average Canopy Diameter: 1791.25 cm
• Tree Height: 13.68 m
• Tree Coordinates: 31°32’59” N 97°6’57” W

Observations: Controls were all contaminated, but individual spot tests were negative for plaques and contamination. It’s unclear how the controls were contaminated. The top agar wasn’t contaminated or the individual plates would have also had contamination.

Results: This experiment yielded a plaque assay to be evaluated for possible plaques. This also yielded a new soil sample to be experimented on if the plaque assay from Soil Sample B yields negative results.

Next Steps: The next step is to evaluate the results of the plaque assay. If negative, the next step is to work with Soil Sample C and try for phage with it. If positive, the next step is serial dilutions and plaque assays.

Conclusion: From the results of testing on this sample and the soil meta data, it could become evident in what type of environment phages tend to reside, and whether there is a difference between soil around live oaks or red oaks.

In addition to the experiments, there were also two guiding questions asked of the class to consider:

1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?

• The likely reason is simply that the sample Justin used had the highest titer, resulting in better defined, clear spots. It’s also possible that he was more careful during his procedure, leading to less outside influences.

2. Lathan checked a purified lysate by a plaque assay using 10 microliters of 10^-3               lysate. He got 14 plaques. How many microliters of Lathan’s lysate should he add             web a plate (8 cm in diameter) if his average plaque diameter is 1mm?

• 14 plaques divided by 10 microliters = 1.4 x 10^3 pfu per mL 10^-3 lysate. The plate radius = 40 mm, and the plaque radius = .5 mm. The area of the plate over the area of the plaque = 6.4 x 10^3 pfu to web. 6.3 x 10^3 pfu divided by 1.4 x 10^6 pfu per mL = 4.6 x 10^-3 mL 10° lysate to web = 4.6 microliters 10° lysate to web.