Date: Monday, November 26th, 2018

Title: Titer Calculation and Plaque Assay

Rationale: The purpose of today’s lab is to calculate the titer of the lysate using a previous plaque assay and then to attempt to web a plate using the titer calculations.

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. Plates from last lab were evaluated.
3. The plaque assay from last lab had 133 plaques present, equivalent to 133 plaque forming units (pfu).
4. Diameters of the plate and the plaques were measured using a ruler and a microscope.
   1. Plate Diameter: 85 mm
   2. Average Plaque Diameter: 1.5 mm
5. The area of the plate and the plaques were calculated using A=πr^2
   1. Plate area: A=πr^2 =π(42.5)^2 =5674.5 mm^2
   2. Plaque area: A=πr^2 =π(.75)^2 =1.767 mm^2
6. The area of the plate was divided by the area of the plaques in order to calculate how many plaques would be necessary to web a plate.
   1. (5674.5 mm^2)/(1.767 mm^2) =3211 pfu
7. The titer of the lysate was determined using the formula Titer=(pfu x dilution factor x 1000 μL)/(μL lysate used x 1 mL)
   1. Titer =(133 pfu x 10^8 x 1000 μL)/(10 μL x 1 mL) =1.33 x 10^12 pfu/mL
8. The volume needed to web a plate was determined using the following math:
   1. (3211 pfu x 1 mL x 1000 μL)/(1.33 x 10^12 pfu x 1 mL) =2 x 10^-6 μL lysate needed to web a plate
9. Since the lysate was diluted by factors of 10, 2 x 10^-6 μL of 10^0 lysate equates to 20 μL of 10^-7 lysate.
10. A plaque assay and top agar control were set up using the following formula:
    1. 4 mL LB broth
    2. 45 μL 1M CaCl2
    3. 5 mL 2x Top Agar
11. 20 μL of the 10^-7 lysate was added to a culture tube containing .5 mL arthrobacter and left to infect for 15 minutes.
12. 4.5 mL of top agar solution was added to a control plate.
13. 4.5 mL of top agar solution was added to the culture tube solution and pipetted to mix before being transferred to a plate.
14. The plates were left for 15 minutes to solidify, inverted, and placed in an incubator for the next 48 hours.

Observations: The top agar control from last experiment was free of contamination. The solution in the plate made during this lab session was touched by accident and therefore might have contamination. The arthrobacter may out-compete the contaminants. This will be evaluated next lab. The titer of the lysate is very high, at 1.33 x 10^12. A highly diluted sample will need to be used to web plates.

Results: This lab yielded a plaque assay that will be webbed if successful and a top agar control to be evaluated next lab.

Next Step: The next step is to evaluate the plates from this experiment and flood a plate if it is webbed. If the plate is not webbed, a titer calculation might need to be reevaluated and a new plaque assay will be made with a different volume of lysate in order to web one.

Date: Monday, November 19th, 2018

Title: High Titer Plaque Assay

Rationale: The purpose of today’s lab is to make a plaque assay using a dilution that will form plaques without lysing a plate completely.

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. Plates from last lab were evaluated.
3. The 10^-8 dilution was the only spot that had formed plaques that were countable, so that dilution was selected.
4. Top agar for a plaque assay was setup using the following formula:
   1. 4 mL LB Broth
   2. 5 mL 2x Top Agar
   3. 45 microliters 1M CaCl2
5. A culture tube with .5 mL arthrobacter was mixed with 10 microliters of 10^-8 lysate and left to infect for 15 minutes.
6. 4.5 mL of the top agar solution was added to a top agar control plate and left to sit for 15 minutes.
7. 4.5 mL of the top agar solution was added to the culture tube, pipetted, and added to a plate.
8. The plates were inverted and left to incubate over night.

Observations: The majority of the dilutions tested on the spot test were too high of a titer to make individual plaques, so the 10^-8 lysate was used in order to make a plate that a titer calculation can be made with. There was contamination on the top agar control from last experiment.

Results: This experiment yielded a plaque assay and a top agar control that can be evaluated in the future.

Next Steps: The next step is to calculate the titer and volume needed to web a plate using the new plaque assay.

Date: Wednesday, November 14th, 2018

Title: Serial Dilutions and Spot Test with Positive Lysate

Rationale: The purpose of today’s lab is to make serial dilutions and then spot test them in order to evaluate which dilution will yield a plate that has a high titer and isn’t lysed fully.

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. Plates from last lab were evaluated and yielded negative results.
3. Flooded lysate from positive plates with high titer was borrowed from a lab partner (Gabriel Andino) and 5 mL was transferred to a new conical vial.
4. Nine microcentrifuge tubes were labelled PB for Phage Buffer and then 10^-1 through 10^-8.
5. 1 mL of phage buffer was added to the tube labelled PB.
6. 90 microliters of phage buffer was added to each other tube.
7. 10 microliters of the flooded lysate were added to the 10^-1 tube and vortexed.
8. 10 microliters from the 10^-1 tube were added to the 10^-2 tube and vortexed.
9. Step 8 was repeated until 10 microliters from the 10^-7 tube were transferred to the 10^-8 tube and vortexed.
10. Two plates were taken. One was labelled as TA control and the other was divided into nine sections, with each section being labelled after each of the microcentrifuge tubes.
11. Top agar was made using the following recipe:
    1. 4 mL LB broth
    2. 45 microliters 1M CaCl2
    3. 5 mL 2x Top Agar
12. 4.5 mL of the solution was added directly to the TA control plate.
13. 4.5 mL of the solution was added to a culture tube containing .5 mL arthrobacter and pipetted to mix the solution.
14. The culture tube was emptied into the spot test plate and left to harden for 15 minutes.
15. 10 microliters from each microcentrifuge tube were added to the corresponding sections on the spot test plate and left for another 15 minutes to allow the samples to absorb into the agar.
16. The plates were left in the incubator.

Observations: Some of the top agar and LB broth jars still seem to have contamination. From Gabriel’s experiments, the first few serial dilutions still lyse a plate fully. It could be beneficial to come in earlier after the plates have only sat in an incubator for a day so that a plate can be flooded before the plate is fully lysed.

Results: This experiment yielded a spot test with 8 different serial dilution samples as well as a top agar control.

Next Step: The next step is to choose a dilution based off the results of the spot test that has a high titer but will not lyse a plate fully. Then, a plaque assay will be made and flooded if it is webbed.

Date: Wednesday, November 7th, 2018

Title: Titer Calculations and Plaque Assay

Rationale: The purpose of today’s lab is to calculate the titer of plates with plaque and attempt to web a 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 and plaque assays were evaluated.
2. The plates yielded negative results so the experiment was combined with a partner’s who had positive results.
3. Plaques on the 10° and 10^-1 plates were counted.
   1. The 10° plate had 465 plaques present.
   2. The 10^-1 plate had 21 plaques present.
4. The following math was performed in order to determine the titer of each sample:
   1. For the 10° plate: (465 pfu ÷ 30 microliters) * (1000 microliters ÷ 1 mL) * (1, the dilution factor) = 15,500 pfu/mL.
   2. For the 10^-1 plate: (21 pfu ÷ 30 microliters) * (1000 microliters ÷ 1 mL) * (10, the dilution factor) = 7,000 pfu/mL.
5. A microscope and ruler were used in order to determine the diameter of the plates and the average diameter of plaques on each plate.
   1. The plate was determined to have a diameter of 8.5 cm.
   2. The plaques on the 10° plate were determined to have an average diameter of .1 cm.
   3. The plaques on the 10^-1 plate were determined to have an average diameter of .1 cm.
6. Using A=πr^2, the area of the plaques and the area of the plates were determined:
   1. Plates: A=πr^2=π(4.25 cm)^2=56.745 cm^2.
   2. Plaques:  A=πr^2=π(.05 cm)^2=.00785 cm^2.
7. The number of plaques needed to web a plate was determined to be 7.225×10^3 by dividing the area of the plate by the area of the plaques.
8. The amount of lysate needed to web a plate was determined for each dilution using the following math:
   1. 10° plate: (7.225×10^3 pfu) * (1 mL ÷ 15,500 pfu) = 482 microliters 10° lysate.
   2. 10^-1 plate: (7.225×10^3 pfu) * (1 mL ÷ 7,000 pfu) = 1.032 mL 10^-1 lysate.
9. A plaque assay was made for the 10^-1 dilution using the following formula:
   1. 4 mL LB broth
   2. 5 mL 2x Top Agar
   3. 45 microliters 1M CaCl2
10. 60 microliters of 10^-1 lysate was added to .5 mL arthrobacter and left for 15 minutes to infect in a culture tube.
11. 4.5 mL top agar solution was added to a control plate and 4.5 mL was added to the culture tube, pipetted, and added to a plate.
12. The plates were left for 15 minutes to harden before being incubated.

Observations: Below are the results of the last experiment. The contamination is unlike contamination seen before. It has spots throughout the plate as well as tendril-like patterns throughout the plate. Most of the LB broth and top agar in the autoclave are contaminated and it’s unclear why. It’s possible the autoclave wasn’t turned on or simply didn’t kill everything. The middle plate is the 10^-1 plate borrowed from a lab partner.

Results: This experiment yielded a plaque assay that will hopefully be close to webbed so that it can be flooded.

Next Step: The next step is to flood the plate in order to get the phage from the plate into phage buffer.

Date: Monday, November 5th, 2018

Title: Plaque Picking and Serial Dilutions with Adopted Plate

Rationale: The purpose of today’s lab is pick a plaque from a lab partner’s plaque assay and set up serial dilutions in order to get 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. Plaque assays from the previous week were evaluated and found to yield inconclusive results.
3. 100 microliters of phage buffer were added to a microcentrifuge tube.
4. A pipette tip was touched into a plaque from an adopted plate and swirled in the microcentrifuge tube to add phage to solution. This was marked as 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. 8 mL LB broth
    2. 10 mL 2x Top Agar
    3. 90 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 pipetted 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 plates from last week did not fully harden and a layer of liquid solution was on top of the agar. Below are the results from the last experiment compared to the results from the partner the lysate was adopted from. It’s possible that the plates were positive for plaques and the plates were fully lysed over the weekend.

 

Results: This lab yielded three plaque assays and a top agar control that will hopefully allow plaques to form without fully lysing a plate.

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

Date: Wednesday, October 31st, 2018

Title: Soil Sample D Metadata Continued and Plaque Assay with Positive Lysate

Rationale: The purpose of today’s lab is to make a plaque assay with a lab partner’s positive lysate.

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. The phage-positive enriched lysate from a lab partner was retrieved and 1 mL was filtered to a microcentrifuge tube.
3. 40 microliters of the phage-positive lysate was added to .5 mL of arthrobacter and left to infect for 15 minutes.
4. Agar was made for two plates with the following formula:
   1. 4 mL LB broth
   2. 5 mL 2x TA
   3. 45 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 5 days.
7. The weigh boat from last lab was taken out from under the fume hood and weighed:
   1. Weigh boat with dry soil: 5.420 g
   2. Weigh boat alone: 2.435 g
   3. Dry soil: 2.985 g
8. The weight of the lost water was divided over the weight of the wet soil and multiplied by 100 to deetermine that the soil was 9.33% water.

Observations: The lysate used was from another student who had plaque assays yielding positive results. 40 microliters of lysate was used since previous plaque assays have shown that 30-50 microliters lysate yield enough plaques to cover the plate without webbing it.

Results: This experiment yielded a new 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 calculate the concentration needed to web a plate and work towards getting a high titer plate.

Date: Monday, October 29th, 2018

Title: Soil Sample D Collection and Metadata

Rationale: The purpose of today’s lab was to collect more soil and record its metadata.

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. A live oak was found at 31°32’58” N 97°6’57” W.
2. Soil was collected near its roots.
3. The tree was calculated to be the following measurements:
   1. Circumference at 137 cm: 222 cm
   2. Canopy diameter: 14.63 m
   3. Tree height: 11.279 m
4. A pinch of soil was added to a pH vial filled with DI water.
5. The vial was shaken for 10 seconds then left for 2 minutes to settle.
6. A strip of pH paper was left in the vial for 45 seconds then compared to a chart to determine that the soil pH was about 5.8.
7. Percent water calculations were started by weighing soil and the weigh boat. The calculations are as follows:
   1. Empty weigh boat: 2.435 g
   2. Weigh boat with wet soil: 5.727 g
   3. Wet soil: 3.292 g

Observations: This soil was found by a tree close to the previous samples, yet it was more acidic soil.

Results: This experiment yielded a new soil sample as well as soil metadata to compare for trends.

Next Step: The next step is to complete metadata and wash the soil for enrichment.

Date: Wednesday, October 24th, 2018

Title: Gel Electrophoresis

Rationale: The purpose of today’s lab is to perform gel electrophoresis in order to test samples against a DNA ladder to determine whether or not the sample contains phage DNA.

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. 35 mL of TBE buffer was added to a flask.
2. .72 g agarose was added to the flask with TBE buffer.
3. The flask was placed in a microwave and cooked in intervals until the solution was clear.
4. The flask was allowed to sit until it was cool enough to hold with a bare hand comfortably.
5. 1.7 microliters of Ethidium Bromide (EtBr) was added to the flask.
6. The solution in the flask was poured into a tray and allowed to harden
7. A thin layer of TBE was added over the hardened tray so that the comb and dams could be removed without tearing the gel.
8. TBE buffer was added to the gel electrophoresis tray until it covered the gel section.
9. Four wells were used:
   1. The first well was filled with 5 microliters of DNA ladder which would serve as a match to possible DNA.
   2. The second well was filled with 10 microliters of the small tube with PM 1. 2.5 microliters of dye was added to the tube before the contents were added to the well in order to weigh down the DNA so it did not float from the well.
   3. The third well was filled with 10 microliters of the small tube with PM 2.
   4. The fourth well was filled with 10 microliters of the small tube with PM 3.
10. The gel was connected to a power source and allowed to run for 45 minutes. The gel was imaged and yielded negative results.

Observations:

The results from the gel electrophoresis did not match the expected results based on the ladder. This means that the sample in all likelihood did not contain phage DNA.

The compound EtBr is a carcinogen and must be handled carefully. Gloves were used when in contact with EtBr.

Results: This experiment yielded negative results. The PCR and gel electrophoresis did not match the DNA ladder and did not contain phage DNA.

Next Step: The next step is to either collect more soil or adopt from another group.

Date: Monday, October 22nd, 2018

Title: PCR Soil Sample C

Rationale: The purpose of today’s lab is to perform PCR to replicate and search for phage DNA in the soil 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. The enriched lysate from soil sample C was centrifuged at 3000 gs for 10 minutes.
2. 1 mL of the centrifuged lysate was transferred to a microcentrifuge tube and boiled to release possible phage DNA from capsids.
3. 6 small tubes were set up. Three were used as a negative control and three used the lysate from soil sample C as well as a partner’s lysate in order to test for phage DNA. The tubes were set up the following way:
   1. 12.5 microliters of Taq Polymerase was added to each of the 6 tubes.
   2. 4 microliters of primer mix 1, 2, and 3 were added to the negative control tubes and the experimental tubes marked with 1, 2, and 3 respectively.
   3. 6.5 microliters of ddH2O were added to each of the 6 tubes.
   4. 2 microliters of lysate (1 microliter from the soil sample C lysate and one microliter from a partner’s lysate) were added to each of the 3 experimental tubes.
   5. An extra 2 microliters of ddH2O were added to each of the 3 negative control tubes.
4. These tubes were all put into a heat cycle in order to let the primers match with possible phage DNA.

Observations: The experimental tubes marked for PM (primer mix) 2 and 3 were green while the tube marked for PM 1 was clear. This is because a different material was used in the tube. For gel electrophoresis, a dye will have to be added to the first tube in order to weigh DNA down.

Results: This experiment yielded three experimental tubes that can be tested with gel electrophoresis to search for the presence of phage DNA. This experiment also yielded three negative control tubes that can be tested against to make sure there isn’t contamination or that a procedure was done incorrectly since these tubes should result in strictly negative results.

Next Step: The next step is to use gel electrophoresis in order to check for phage DNA. If this yields negative results, the next step is to find new soil or to adopt phage/soil from another group.

Date: Wednesday, October 17th, 2018

Title: Second Attempt 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. Plaque assays were retrieved and evaluated. They were found to be inconclusive and stored in the walk-in freezer.
3. Another 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 5 days.

Observations: The plaque assay and the TA control were not contaminated and there were spots on the agar, implying the presence of phage; however, the spots were misshapen and the plate had not fully solidified. It’s possible that plaques were present and had become abnormal due to the liquidity of the plate.

Results: This experiment yielded a new 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.