Rationale: Spot titer performed to help calculate titer. The titer is unknown for the newly flooded lysate and high titer is needed to extract DNA from.

Procedure: Lysate was obtained from the refrigerator and was used for 11/26 experiment. TA solution was made, and serial dilutions were performed to perform a spot titer. 100µL of the flooded lysate was used for TEM image. The Formula used to make TA solution:

• 2mL LB broth
• 2.5mL 2X TA
• 22.5µL CaCl2
• 0.5 Artrhobacter

This TA solution was made in a 15mL vial and quickly poured onto the labeled plates. Plates sat for 15 minutes and then the serial diluted lysate were spotted onto the plates (10^-5 – 10^-9 and one control). The plate was placed in the incubator for 24 hours at 24 degrees Celcius and titer will be counted 11/27.

Observations: 45mL lysate obtained from 11/19 experiment. The lysate would then be used for DNA extraction for each member in group 4, but the titer of the new lysate is unknown. High titer must be obtained in order to extract DNA.

Conclusions: High titer calculations from 11/26 experiment will result in DNA extraction 11/28. If low titer, web plates and obtain newly flooded lysate, which would then need to be spotted to calculate titer.

Rationale: The plates ran last week turned out to not be webbed. They had two distinct plaque morphologies, so I will pick a plaque from each and run plaque assays to make sure this is the same phage making both types of plaque.

Procedure:

1. Filled 2 microcentrifuge tubes with 100µL of 1X Phage Buffer labeled one alpha and the other beta.
2. Picked a large plaque and placed in the tube labeled alpha. Pick a small small plaque and placed in the tube labeled beta.
3. Diluted each sample out to the 10^-4 dilution factor.
4. Enriched alpha dilutions in 0.5mL arthrobacter for 20 minutes.
5. Made top agar with 10mL of LB broth, 12.5mL of 1X TA, and 112.5µL of CaCl2.
6. Plated each mixture of alpha dilution and arthrobacter after pipetting in 4.5mL of top agar.
7. Repeated steps 4-6 with the beta dilutions.
8. Made a top agar control by plating 4.5mL of top agar.
9. Let sit then inverted and placed in the incubator.

Observations:

The marked plaque on the top right is the plaque picked for the alpha samples. The plaque picked on the bottom left is the plaque picked for the beta samples.

The results show that both alpha and beta lysates produced both large and  small plaques. The top agar was contaminated, so when the  first two dilutions of alpha lysed the plate the contamination was able to grow back in place of arthrobacter.

Conclusions  and Next Steps: This means that  there is just one type of phage in my plate making the radically different plaques. Since I have a plate closed to being webbed and a lysed plate I will flood both so I can have enough lysate to perform DNA extraction. I’m flooding the lysed plate because the plate with a lower dilution factor also lysed, but the plate with a higher dilution factor nearly webbed so there is most likely some amplification that occurred.

Results from Previous Experiment: Discovered our lysate to have a titer of 10^11.

Objectives: Complete TEM with the high titer lysate.

Procedure:

• Allocated Petri Dish and Parafilm
• Placed a strip of parafilm securely inside petri dish top
• Placed a drop of lysate, 2 separate drops of water, and a drop of Uranyl less on the parafilm
• Using precision tweezers, placed a copper grid, shiny side down, on the lysate for 5 minutes
• Moved copper grid to the first drop of water for 2.5 minutes
• Moved copper grid to the second drop of water for 2.5 minutes
• Placed copper grid on uranyl less (UL) for 1 minute, and quickly blotted an excess UL once remove
• Used tweezers to move copper grid into TEM Grid Holder

Future Plans: Complete TEM after thanksgiving break.

11/19/2018

Objective:

• Extract lysate from the flooded plate
• Do a serial dilution for each lysate
• Make spot tests

Procedure:

1. On 11/16/18, three plaque assay plates were flooded with 8 ml of phage buffer each. The plates were then placed in the fridge
2. The phages buffer with phages from the plate was extracted and filtered from each plate.
3. 100 μl of the lysate from each plate was transferred to three microcentrifuge tubes.
4. 10 μl of lysate from each microcentrifuge tube was transferred to another microcentrifuge tube with 90 μl buffer.
5. 10 μl of lysate from each diluted microcentrifuge tube was transferred to another microcentrifuge tube with 90 μl of phage buffer.
6. 8 ml of LB broth is transferred to a conical vial.
7. 90 μl of CaCl2 was added to the vial as well.
8. 10 ml of 2X TA was added to the conical vial.
9. 4.5 ml of the Top Agar mixture was added to each of the 3 ,0.5 ml arthrobacter test tubes.
10. The mixture is then plated on 3 agar plates. 4.5 of the Top Agar mixture was added to another plate for a control.
11. Divide the three plates with arthrobacter into 4 sections.
12. After Agar has solidified, spot each plate with the dilutions of the respective lysates and phage buffer.
13. The plates were allowed to absorb the spots for 10 minutes and were then placed in the incubator.

Analysis And Conclusion:

the plates were flooded so that we can have more lysate. the spot tests will now be used to calculate the titer of the lysate on hand.

11.19.18 TEM Preparation and Redo of Titer Calculation

Rationale: Since the titer calculation plate from the last lab did not display results due to a slipped top agar, it was found to be necessary to redo this calculation to ensure a high titer lysate was present. However, it was found to be possible to continue with the procedure of TEM as each other individual that used Claire’s base lysate had found a high titer lysate from the last round of amplification. Therefore, since we all did similar procedures with similar results, it was concluded that there was a possibility that I too had a high titer lysate.

Procedure:

1. Aseptic zone established
2. 100µL of lysate was added to a microcentrifuge tube
3. Parafilm was placed on petri dish
4. 15µL of lysate was added to the parafilm at the center
5. 15µL of water was added to the parafilm to the right of the previous drop two times
6. Uranyl Acetate was added to the parafilm at the rightmost point.
7. Copper mesh was placed on the lysate for 5 minutes
8. Mesh was transferred to first water drop for 2.5 minutes
9. Step 8 was repeated with second water drop
10. Mesh was transferred to uranyl acetate for 1 minute, then dried with filter paper. Placed in B8.
11. 2mL of LB Broth was added to a tube with 0.5mL of arthrobacter and an empty conical tube
12. 22.5µL of CaCl2 was added to both tubes
13. 2.5mL of 2X Top Agar was added to both tubes, then the solutions were swished and plated on respective tubes.
14. Original lysate was used to create dilutions from 10^0 through 10^-9. These lysates were added to labeled sections on the experimental plate in 5µL drops
15. Plates were incubated overnight.
16. Station was cleaned

Results:

• Plate prepared on Wednesday (11/14) that was intended to show the titer of the lysate had issues with the consistency of the top agar. This was predicted to be due to the temperature of the agar when it was used. The plates created today seemed to confirm that the top agar had been allowed to cool for too long, which likely caused the problems with Wednesday’s plate. The same problem was not encountered today.
• Lysate titer will be reported as the plate created in this lab session is examined this week.

Observations:

• Top Agar and LB Broth was very clear and was made fresh. This will help reduce the chances of contamination.
• Drops did not settle into plate for spot test. Therefore, it was found to be necessary to place the plate in the incubator right side up with instructions to avoid flipping or moving the plate to give the drops time to settle.
• With the TEM preparation, there seemed to be confusion regarding shiny-side up and shiny-side down with the copper mesh. Any adverse results could have been caused by this confusion.

Conclusions:

• Since the top agar worked today after being taken fresh from the incubator rather than from the table, it can be concluded that the temperature caused the problems that were seen in the previous top agar. The TEM will allow for bacteriophage visualization, and the plate run today will confirm whether or not a high titer lysate has been used for the TEM.

Shepard Saabye
November 14th, 2018
SEA PHAGE Lab Journal

Results from the Previous Lab: The control plate was contaminated, but interestingly, it was not contaminated when Dr. Adair looked at it the day prior. I have no way to interpret that information. Another plate had some plaques form but due to contamination, Cooper and Michael’s plates were used for a plaque assay 

Objectives and Rationale: Plate Serial Dilutions in order to determine the titer of the lysate and web a plate

Procedure:

• Established Aseptic Zone
• Diluted lysate out to 10^-4 of the original lysate
• Obtained 3 vials of .5 mL Arthrobacter
• Added 10 mL LB Broth, 12.5 mL 2X TA and 112.5 uL CaCl to a single 50 mL tube
• Added 10 uL of 10^-2, 10^-3, and 10^-4 Lysate to their own respective Arthro tubes
• Added 4.5 mL of Agar mixture to each Arthro tube, and plated
• Plated remaining 4.5 mL Agar mixture on the control plate
• Waited 15 min for Top Agar to solidify
• Left plates in the incubator at 24.5 degrees Celsius

Future Plans: Calculate Titer and continue increasing the concentration of the phage in each lysate

Shepard Saabye
November 12th, 2018
SEA PHAGE Lab Journal

Results from the previous lab: One of the plates was close to webbed. The control came out a little contaminated.

Rationale and Objectives: Today the goal was to generate a new lysate, and have it prepared to calculate titer by the next lab. This is to increase the titer high enough to complete TEM.

• Procedure:
• Established Aseptic Zone
• Flooded webbed plate with 5 mL Phage Buffer
• Let sit on moving platform for 1 hour
• Diluted lysate out to 10^-3 of the original lysate
• Obtained 3 vials of .5 mL Arthrobacter
• Added 10 mL LB Broth, 12.5 mL 2X TA and 112.5 uL CaCl to a single 50 mL tube
• Added 50 uL of 10^0 Lysate to the first Arthro tube
• Added 10 uL 10^-2 Lysate to the second tube
• Added 10 uL 10^-3 Lysate to the third tube
• Added 4.5 mL of Agar mixture to each Arthro tube, and plated
• Plated remaining 4.5 mL Agar mixture on the control plate
• Waited 15 min for Top Agar to solidify
• Left plates in the incubator at 24.5 degrees Celsius

Future PLans: use the results from this lab to determine the titer of our lysate, and decide if that is high enough to continue with TEM.

11/14/18 Titer Calculations, Flooding Plates, Webbing Plates

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of calculating the titer of the lysate created last lab in addition to flooding plates from the serial test titer dilutions, and making more webbed plates to flood later.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials for a Plaque Assay:

• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• Culture tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone
   

The titer from the previous serial dilutions was calculated (also included under the updated results section of the 11/12 lab)

The 10^-1 and 10^-2 plates were flooded

• 8 ml of phage buffer was pipetted onto the agar plate.
• The plate was left on the shaker at room temperature, to slowly shake for an hour.
• At an hour, a syringe filter was used to filter the resulting lysate into a conical vial to create ~11 ml of lysate for future testing

Then plaque assays on the new lysates were performed.

1. Five agar plates were labeled
2. 10 µL of the 10^-2 dilution were transferred into each of the four culture tube containing .5 ml of Arthrobacter
3. The culture tubes were set aside for 15 minutes.

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

1. The agar was prepared according to the following recipe (makes 5 plates):
2. 4.5 ml of the agar was transferred to the plate labeled “TA control”
3. The plate was swirled and set aside
4. 4.5 ml of the agar was transferred into each culture tube
5. The resulting mixture was poured into the corresponding plates
6. The was set aside for 10 minutes to allow agar to solidify.
7. Plates were left to incubate until nest class

Results:

Update: While it wasn’t possible to see the results of the webbed plates when the lab period was finished on 11/14, it is clear that on 11/15 the plates were close to webbed, suggesting that the procedures detailed above were a sucess.

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay or spot test. This testing gave us a medium titer lysate that we will continue to amplify, we hope that by flooding all of our plates we will create enough high titer lysate to move on to TEM.

Future:

During our next lab period, we will flood our four webbed plates. We will also need to perform a titer test on the two new lysates we have created.