11/12/18 Titer Test Serial Dilutions and Plaque Assays

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 previously created lysate through serial dilutions.

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 Serial Dilution:

• Phage Buffer
• Microcentrifuge Tubes
• Vortex Machine
• Pipette

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
   

Then the serial dilutions were performed.

1. Five levels of dilution were created: 10^-1, 10^-2, 10^-3, 10^-4, 10^-5
2. Five microcentrifuge tubes were filled with 90 µL of phage buffer
3. 10 µL of the previously created lysate (10^0) were transferred to the 10^-1
4. The tube was vortexed to mix
5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-5 dilution

Then plaque assays on the new lysates were performed.

1. Six agar plates were labeled
2. 10 µL of each dilution were transferred into a culture tube containing .5 ml of Arthrobacter (1 dilution per tube)
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 six 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:

As can be seen in the image above, the serial dilutions produced the desired results. Our 10^-1 and 10^-2 plates were almost lysed and our 10^-3 plate was webbed. We calculated the following titer:

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.

Future:

During our next lab period we will flood our 10^-1 and 10^-2 plates to try to get a high titer lysate, in addition, we will likley also create more webbed plates that we can flood so that we will have a large quantity of high titer lysate.

11/15/18 Flooding Webbed 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 flooding the webbed plates previously created during the last lab. We will do serial dilutions and plating to test the titer of this lysate next lab. While this procedure was being done, another group was using the newly created lysate to try to create two backup webbed plates.

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 Flooding a Plate:

• Phage buffer
• Refrigerator
• Syringe Filter
• 15 ml conical vial
• 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
   

Them the four webbed plates were flooded.

1. 8 ml of phage buffer was pipetted onto each agar plate
2. The plates were placed in the refrigerator to sit for ~22 hours

Results:

The results of this lab will not be visible until Friday’s lab, but I can say that flooding the plate seemed to occur without incident.

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.

Future:

During our next lab, we will perform serial dilutions to determine the titer of the resulting lysate from this lab and Wednesday’s lab procedure. We will then determine what steps need to be taken.

11/14/18

Objective:

To create a webbed plate with all the lysate available from the 4th purification run that can be flooded.

Procedure:

1. the top agar mixture was prepared for 4 plates.
2. 8 ml of LB broth was added to a conical vial
3. 90 μl of CaCl2 was added to the conical vial.
4. Three 0.5 ml of arthrobacter samples were enriched with the extracted phages, the 10^-1 and 10^-2 dilution for 15 minutes.
5. 10 ml of 2X TA was added to the conical vial.
6. 4.5 ml of the TA mixtures was added to each enriched sample.
7. the samples and the mixture were then plated on three agar plates.
8. 4.5 ml of TA mixture was plated on another agar plate for a control plate.
9. after 15 minutes, all the plates were placed inverted in the incubator.

Analysis and Conclusion

this procedure will probably not make a fully webbed plate but it should result in a plate with a lot of plaque. the plate will be flooded and this will give us a lot of lysate.

11/12/2018

Objective

To pick and purify the phage samples and isolate a particular type of phage.

Procedure

1. 37 plaques were picked from 4th purification run and deposited in a microcentrifuge with 100μl of phage buffer.
2. After phages are mixed in the microcentrifuge tube, 10 μl of the extracted phage solution is transferred to a microcentrifuge tube with 90μl of phage buffer to acquire a 10^-1 serial dilution.
3. 10 μl of phage extract from 10^-1 dilution is transferred to a microcentrifuge tube with 90 μl of phage buffer
4. the top agar mixture was prepared for 4 plates.
5. 8 ml of LB broth was added to a conical vial
6. 90 μl of CaCl2 was added to the conical vial.
7. Three 0.5 ml of arthrobacter samples were enriched with the extracted phages, the 10^-1 and 10^-2 dilution for 15 minutes.
8. 10 ml of 2X TA was added to the conical vial.
9. 4.5 ml of the TA mixtures was added to each enriched sample.
10. the samples and the mixture were then plated on three agar plates.
11. 4.5 ml of TA mixture was plated on another agar plate for a control plate.
12. after 15 minutes, all the plates were placed inverted in the incubator.

Analysis

Due to low titer of the sample, more purification is required to isolate a particular type of phage. more lysate will be used in the future to have a plate with more plaques.

• NOVEMBER 5TH, 2018
  • OBJECTIVE:
    • Run a spot test with no contamination 
  • PROCEDURE:
    • Tables were cleaned lamps were lit
    • 2mL of lysate was filtered out using a syringe and filter
    • Then a large test tube was filled with:
      • 4mL LB broth 
      • 45 𝝁L CaCl2
      • 5mL 2x TA
    • Then 4.5mL of the 2X TA solution was added to 2 plates, one serving as the control, and the other as the test plate
    • Once the 2X TA solution was added, .5mL of arthro was then poured on to the plate
    • The plate was then swirled 
    • Once the plate solidified, 10 𝝁L of lysate was added to its designated sections
    • 10 minutes was allowed for the lysate to absorb into the plate 
    • the plate was then inverted and placed into the incubator
  • RESULTS: 
    • As seen in figure 20, the control was clear of any contaminants, and on test plate were clear of contaminants
  • CONCLUSION:
    • • The results seen show that the parts labeled “LEF” and “EB” were native for phage presence while, the spot labeled “SA” was positive for phage presence
  • NEXT STEPS: 
    • • Try to get a webbed plate with new phage
• NOVEMBER 7TH, 2018
  • OBJECTIVE: 
    • Try to figure out how many 𝝁L  of lysate will get a webbed plate 
  • PROCEDURE:
    • Tables were cleaned lamps were lit 
    • Three test tubes containing .5mL of arthro were obtained 
    • In the three tubes the following amounts of lysate were added: 20 𝝁L, 40 𝝁L, 80 𝝁L
    • In a large test tube the following were mixed 
      • 8mL LB broth
      • 90 𝝁L CaCl2
      • 10mL of 2X TA
    • Then 4.5mL of the 2X TA solution was added into each test tube of lysate and arthro and was mixed together, then poured on to a plate 
    • 4.5mL of the 2X TA solution was added to a plate to serve as the control 
    • 15 minutes was allowed for the plates to solidify 
    • The plates were then inverted and placed in an incubator 
  • RESULTS:
    • The results can be seen in figure 21
  • CONCLUSION: 
    • The results indicate that there are no phage present on any of the plates.
  • NEXT STEPS:
    • PCR will be run on the soil to determine if there is a phage