Previous Results:

• Webbed plates from last lab had numerous plaques, but were not webbed.

Objective:

• To correctly calculate the titers of the plates from last lab and decide which lysates should be increased for a webbed plate
• Plate a “10^0 #4” plaque assay and webbed plate

Procedure: Calculations

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. “10^0 #2” and “10^0 #3” plates were obtained and 5 plaques on each plate were measured using a dissecting scope. The diameters were averaged to prepare for titer calculations.
   1. “10^0 #2” avg plaque area = 2.66 mm^2
   2. “10^0#3” avg plaque area = 0.83 mm^2
3. Titers were calculated with the following equations:
   1. “10^0 #2” had 48 plaques on the plate
      1. (48 pfu/10 microliters) x (10^3 microliters/mL) x (10^0) = 4.8 x 10^3 mL = Low Titer
   2. “10^0 #3” had 15 plaques on the plate
      1. (15 pfu/10 microliters) x (10^3 microliters/mL) x (10^0) = 1.5 x 10^3 mL = Low Titer
4. The volume needed to web a plate for each lysate was calculated:
   1. “10^0 #2” = 2133.3 plaques/(4.8 x 10^3) mL = 440 microliters
   2. “10^0 #3” = 6836.7 plaques/(1.5 x 10^3) mL = 4560 microliters
5. The “10^0 #3” volume needed to web was too high, and therefore not plated. “10^0 #2” lysate was then left to create a webbed plate using a higher volume (in next procedure)

Procedure: “10^0 #4” Lysate and Webbed

1. Aseptic technique was still used
2. Overlay Agar was made in 50 mL tube using 6 mL LB broth, 7.5 mL 2x TA, and 67.5 microliters CaCl2
3. To make the “10^0 #4” plaque assay 10 microliters of lysate was added to 0.5 mL Arthrobacter and allowed to infect for 10 min
4. To make the webbed plate, 64 micro liters of “10^0 #2” lysate was added to 0.5 mL Arthro and infected for 10 min
5. 4.5 mL of Overlay Agar was added to each Arthro tube for the experimental tubes, leaving 5 mL of Overlay Agar in the 50 mL of the control plate
6. All three Agars were plated and left to harden for 15 min, then incubated for 48 hours

Results:

• The titers of both the “10^0 #2” and the “10^0 #3” plates were low titers and will need to be increased in order to move on in the procedure.

Next Steps:

• During next lab the webbed plate and “10^0 #4” plate will be examines for positive results. If the plate is webbed, then the next step in the procedure will occur. If not, then the titer of the “10^0 #4” plate will be calculated and a webbed plate will be created from that lysate.

I was unable to be in lab 10/17/18 due to illness, therefore I was unable to collected data and results on my own. I received pictures of the plates that were created in lab 10/15/18 and my lab partners flooded one of the plates so the phage could be collected during the next lab.

Previous Results:

• The “webbed 10^0 #1” plate created 10/15 was not completely webbed, meaning there wasn’t enough lysate added to the Overlay mixture to web the plate
• The 2 plaque assays were positive with plaque clearings on both
• Control had no contamination

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and Ethanol burner
2. The “webbed 10^0 #1” plate was obtained
3. 8 mL of phage buffer was added to the plate to flood it
4. Plate was sealed with parafilm and placed in the fridge until next lab

Results:

• Experiment was no completed, therefore there are no results to report

Next Steps:

• During the next lab the phage buffer that was used to flood the plate will be collected and filtered. The titers of the “10^0 #2” and “10^0 #3” plates will be calculated. Plates will be made using the amount of lysate found from the results of the calculations.

Previous Results:

• No plaque assays were created during last lab, only titer calculation and lysate collection was conducted, therefore there are no results to report.

Objective:

• To correctly plate a 10^0 lysate webbed plate for phage extraction later on
• Correctly plate 10^0 lysate plaque assays to be prepared if the 10^0 webbed plate is not webbed

Procedure: Webbed Plate

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and Ethanol burner
2. 15 mL tube containing “10^0 #1” lysate was obtained
3. Using titer/volume calculations from last lab, 64 microL of lysate was added to 0.5 mL of Arthrobacter and left to infect for 10 min
4. Overlay Agar was created using 2 mL LB broth, 2 mL 2xTA, 22.5 microL CaCl2, and 0.5 mL Arthrobacter infected with phage
5. Mixture was mixed then poured on a plate labeled “Webbed 10^0 #1” (since the lysate was from the 10^0 #1 lysate)
6. Plate was left to harden for 15 min then incubated for 48 hours

Procedure: Plaque Assays

1. Aseptic technique was used
2. 15 mL tubes containing “10^0 #2” and “10^0 #3” lysate were obtained
3. 10 microL of each lysate were pipetted into separate tubes 0f 0.5 mL Arthrobacter and left to infect for 10 min
4. Overlay Agar was created for the 2 experimental plates and a control plate using 6 mL LB broth, 6 mL 2xTA, and 67.5 microL CaCl2
5. The Overlay Agar was separated equally into 3 separate 50 mL tubes
6. 2 infected Arthrobacter tubes were poured into respected Overlay Agar mixtures
7. The 3 plates were poured and left to harden for 15 min, then incubated for 48 hours

Results:

• The experiment was not completed during this lab, therefore there are no results to report.

Next Steps:

• During the next lab the plates will be examined. If the ” webbed 10^0 #1″ plate is completely webbed, then the phage will be extracted and the next step of the project will be conducted. If the webbed plate is not completely webbed, then the titers of the 2 10^0 plaque assays will calculated and webbed plates will be created using those calculations.

Previous Results:

• The plate that was made in lab (10/8) was positive. It was not webbed, but had more plaques than previous plates, indicating a possible high titer.

Objective:

• To correctly calculate the titer of the 10^0 plate
• Collect the phage buffer (lysate) of both plates that were created during last lab to prepare for plating in a future lab

Procedure: Titer Calculation

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and Ethanol burner
2. The 10^0 plate (made 10/8) was obtained and the number of plaques were counted. # plaques = 312
3. The titer of the lysate used was found with the following equation:
   1. (#pfu/volume used microL) x (10^3 microL/mL) x (dilution factor) = titer
   2. (312/10 microL) x (10^3 microL/mL) x (10^0) = 3.12 x 10^4 mL = low titer
4. To calculate the volume of lysate needed to web a plate, the diameter of 5 plaques were measured under the dissecting scope, then they were averaged
   • (1.5 mm + 2.0 mm + 2.0 mm + 2.1 mm + 1.9 mm) / 5 = 1.9 mm
5. The diameter of the plate was measured to be 85 mm
6. The areas of both the plaques and the plate were found
   • Plaque Area = 2.84 mm^2
   • Plate Area = 5674.5 mm^2
7. It was found that the number of plaques needed to web the plate would be 6920.12 plaques
   • 5674.5 mm^2 / 2.84 mm^2 = 1998.1 plaques
8. Then the volume of the lysate needed to web the plate was found using the following equation: (# of plaques) / (titer) = volume needed
   • 1998.1 plaques / (3.12 x 10^4 mL) = 0.064 mL = 64 microL

Procedure: Flooded Plates

1. The webbed plate and 10^0 plate (made 10/3) that were flooded 10/8 were obtained. The phage buffer of each plate was removed using 3 mL syringes
2. Phage buffer for each plate was filtered into separate 15 mL tubes for storage
3. Tubes were placed in the fridge to be plated during next lab

Results:

• The experiment was not completed, therefore there are no results to report.

Next Steps:

• During next lab two plates will be made, each plate containing one of the two 10^0 lysates that were created in lab 10/8. Then the titers of those plates will be calculated, hopefully high titers so the project can move forward.

Previous Results:

• The results from the webbed plate and the 3rd purification plate (10^0) created 10/3 were both positive. The control plate did not have contamination, so the next steps in the procedure can be completed. The webbed plate was not completely webbed, therefore the titer must be increased.

Objective:

• Create a higher titer of lysate by flooding the webbed plate and collecting phage

Procedure: Creating new 10^0 lysate

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and Ethanol burner
2. Webbed plate from (10/3) was obtained. Corner of the plate with the most visible plaques was flooded using 2 mL phage buffer. Let sit for 5 min.
3. Phage buffer was then filtered into a 15 mL tube using a 5 mL syringe and connected filter. Phage buffer with phage considered to be new 10^0 lysate
4. 10 microL of 10^0 lysate was added to 0.5 mL of Arthrobacter. Let sit while rest of Overlay Agar was mixed
5. Overlay Agar was created using 2 mL LB broth, 2.5 2x Top Agar, and 22.5 microL CaCl2. A control agar was created using same recipe
6. The 0.5 mL Arthro with lysate was added to the Overlay Agar experimental tube and plated. Control tube without Arthro was plated
7. Plates were left to harden for 15 min and incubated for 48 hours

Procedure: Flooding webbed plate and 3rd purification plate

1. 8 mL of phage buffer was pipetted into both the webbed plate and 3rd purification plate from 10/3
2. Plates were sealed with parafilm and placed in fridge for 48 hours

Results:

• Experiment was not completed, no results to report

Next Steps:

• During next lab, the 10^0 plate with new 10^0 lysate will be examined. If positive, plaques will be picked and a 2nd purification will be completed. The phage buffer that was put into the webbed plate and 3rd purification plate will be collected and plates will be made using that lysate to try and create a higher titer.

Objective:

• Conduct a third series of serial dilutions for the 10^0 plate to see if the contaminated results from (9/26) effected the previous results
• Create a webbed plate to prepare for plate flooding

Procedure: 3rd Purification of 10^0

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked from the 10^0 plate (created 9/24) by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
3. The dilution was poured into a tube of 0.5 mL Arthrobacter to allow phage to interact with bacteria
4. A mixture of Overlay Agar was made for the experimental and control plate using 4 mL of LB broth, 45 microL CaCl2, 5 mL 2xTA. The mixture was divided into 2 50 mL tubes
5. The mixture of Arthrobacter with the dilution was then added to the 50 mL tube of  Experimental Overlay Agar
6. The experimental tube and the control tube were plated and left to harden for 15 min
7. Plates were placed in incubator for 48 hours.

Procedure: Webbed Plate

1. The webbed plate was made under the same aseptic conditions
2. Using the results from the titer calculations (10/1), the left over 90 microL of 10^0 lysate (created 9/24 for 3rd purification) was poured into a tube of 0.5 mL Arthrobacter to allow phage to interact with bacteria
3. The Overlay Agar for the webbed plate was created using 1.9 mL LB broth, 22.5 CaCl2, 2.5 mL 2xTA. Mixed in 50 mL tube
4. The 0.5 mL of Arthrobacter and the dilution was added to the 50 mL tube
5. The plate was left to harden for 15 min, placed in incubator of 48 hours

Results:

• This step in the experiment is not complete, therefore there are no results to report

Next Steps:

• During the next lab (10/8), both the 3rd purification plate and the webbed plate will be examined for positive results. The control plate will be examined for contamination. If the webbed plate has positive results, then the plate will be flooded to collect the phage for further testing. If the webbed plate is not completely webbed, then titer calculations will be reviewed and modified.

Previous Results:

• The plaque assays from the 3rd purification (9/26) were examined. The control plate had a few spots for bacterial growth, indicating the sample was contaminated.
• All three plaque assays (10^0, 10^-1, and 10^-2) were all positive with plaques.

Objective:

• Correctly calculation the titer of the plate and prepare for a webbed plate

Procedure:

• The 3rd purification plate containing the 10^0 dilution was examined and the number of plaques totaled 15 on the plate
• To calculate the titer of the dilution the following equation was used:
  • (# pfu / volume used in microL) x (10^3 mciroL / mL) x dilution factor
  • (15 / 10 microL) x (10^3 microL / mL) x 10^0 = 1.5 x 10^3 pfu/mL
  • Low titer
• The diameter of 5 plaques were measured under the dissecting scope, then they were averaged
  • (1 mm + 0.8 mm + 1.5 mm + 0.8 mm + 1 mm) / 5 = 1.02 mm
• The diameter of the plate was measured to be 85 mm
• The areas of both the plaques and the plate were found
  • Plaque Area = 0.82 mm^2
  • Plate Area = 5674.5 mm^2
• It was found that the number of plaques needed to web the plate would be 6920.12 plaques
  • 5674.5 mm^2 / 0.82 mm^2 = 6920.12 plaques
• Then the volume of the lysate needed to web the plate was found using the following equation: (# of plaques) / (titer) = volume needed
  • 6920.12 plaques / (1.5 x 10^3 mL) = 4.61 mL

Results:

• After calculations it was determined that 4.61 mL of lysate will be needed to web the plate

Next Steps:

• During next lab, a webbed plate will be made using the remaining sample of lysate from the 10^0 dilution made in lab (9/24). If results are positive, then the plate will be flooded to collect the phage to move on to the next procedure.

Previous Results:

•  The plaque assays and control plate (9/24) were all found to be contaminated. After examining other plates, it was concluded that the stocks of LB broth and 2x Top Agar used during the last plating were contaminated. The previous procedure and plaque assays would have to be redone.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a third series of serial dilutions and plate the diluted lysates with phage

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked from the 10^0 plate by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
3. 10 microL of new 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (10/1)) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were inconclusive
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (10/1)) the plates with the third round of serial dilutions will be examined for phage. If positive, then the experiment will move on to the next steps of calculating a high titer to create a webbed plate.

Previous Results:

• Plaque assays from second purification that were created during the last lab (9/19) were all positive and the control did not have any contamination.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a third series of serial dilutions and plate the diluted lysates with phage

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked from the 10^0 plate by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
3. 10 microL of new 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (9/26) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were positive
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (9/26) the plates with the third round of serial dilutions will be examined for phage. If positive, then the experiment will move on to the next steps of calculating a high titer to create a webbed plate.

Previous Results:

• Plaque assays from different dilutions created during the last lab (9/17) were all positive and the control did not have any contamination.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a second series of serial dilutions and plate the diluted lysates with phage

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked from the 10^0 plate by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
3. 10 microL of new 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (9/24) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were positive
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (9/24) the plates with the second round of serial dilutions will be examined for phage. A third series of dilutions will be done to ensure only one strain of phage is being tested. The same steps will be conducted.