Rationale: Filtered the flooded plates (Two of them) to obtain a new lysate (Lysate 6), and prepared serial dilutions to figure out the titer of lysate 5 and 6

Procedure:

• Created an aseptic zone to prevent contamination of the experiment / procedure
• Obtained the flooded plates from refrigeration and obtained a tube top filter
• Utilized the vacuum hose in the hood to filter the lysate(s) through a 22μL filter
  • Named the new lysate Lysate 6
• Created 10^-1, 10^-2 and 10^-3 dilutions for both lysate 5 and 6
  • Created by adding 90μL of PB to a micro-centrifuge tube and adding 10μL of the previous strength lysate
• Obtained 6 vials of 0.5mL arthrobacter and added 10μL of each lysate into each tube respectively
• Obtained a 50mL conical vial and pipetted in 14mL LB Broth, 17.5mL 2xTA and 157.5μL CaCl2
• Immediately pipetted 4.5mL into each arthrobacter + lysate tube and plated
• Allowed the plates (7 total) to settle for 15 minutes and then moved to incubation

Observations:

• For lysate 6, obtained about 10mL total of lysate
• For filtering the flooded plate, used a tube top filter rather than a syringe filter

Next Steps/Conclusion: Will be coming in on Friday to check the status of the plates. If positive, will calculate the titer strength of lysate 5 and 6. If negative, will have to repeat the procedure.

Rationale: Flooded a mostly lysed plate and creating a webbed plate from previous lab’s calculations

Procedure:

• Created an aseptic zone to prevent contamination of plates by other bacteria strains
• Obtained the lysed plate from refrigeration and obtain a 50mL conical vial of PB
• Pipetted 6mL PB into the plate and allowed to swirl on a shaker for 1 hour and 15 minutes
• During the waiting period, obtained a 50mL conical vial and pipetted 6mL of LB Broth and 76.5mL CaCl2
• Added 5.25μL of lysate into 0.5mL arthrobacter and 10μL of lysate into another 0.5mL of arthrobacter
• Pipetted 7.5mL 2xTA into the 50mL vial and pipetted 4.5mL into each arthobacter + lysate tube (Respectively)
• Immediately plated and allowed to sit for 15 minutes
• After the allotted time, moved the plates into incubation
• After the hour and 15 minutes passed, removed the flooded plate from the shaker and used a syringe filter and a 22μL filter to filter the lysate
• Placed the new lysate (Lysate 5) into refrigeration

Observations:

The 10μL webbed plate after pouring

The 5.25μL plate after pouring

Next Steps/Conclusion: During the next lab, if the webbed plates turned out lysed, will be flooding the two plates and combining their lysates to create lysate 6. After getting the lysate, will be calculating their strength by running serial dilutions

Rationale: Counted the plaques on the webbed 10^-2 plate to calculate the titer and calculated the amount of lysate needed to web the plate.

Procedure:

• Retrieved plates from incubation
• Counted plaques on the 10^-2 and calculated the strength of the titer = (1.27*10^6)
• Calculated the area of the plate with the equation πr^2 (π[42.5]^2) = (5.671*10^3)
• Calculated the area of the plaques by measuring the diameters of 10 plaques (Avg. diameter = 1.04mm)
  • Avg. radii = .52mm; used equation πr^2 (π[.52]^2) = (8.491*10^-1)
• Divided area of the plate by the area of the plaques to obtain 6.6788*10^3, which was then divided by the strength of the titer to get the amount of lysate needed to web the plate (5.25μL)
• Due to time constraints, parafilmed the 10μL plate to flood on the next lab day

Observations:

10^-2 plate with countable plaques

10^-1 with a good amount of plaques; too many plaques to count but gives an indication of how high the lysate is

10μL direct lysate which completely webbed the plate

Next Steps/Conclusion: During the next lab day, the researcher will be using the calculations found today to create a webbed plate as well as flooding the lysated plate

Rationale: After TEM imaging and determining the titer wasn’t high enough, will be continuing to raise the titer strength to easily image the phages in the lysate

Procedure:

• Created an aseptic zone to prevent contamination of plates
• Obtained the lysate from the refrigerator, as well as 4 agar plates
• Created a 10^-1 dilution and 10^-2 dilution
  • The 10^-1 was created by adding 10μL of lysate to 90μL of PB; the 10^-2 was created by adding 10μL of the 10^-1 to 90μL of PB
• Added 10μL of each lysate into 0.5mL of arthrobacter (Respectively)
• Obtained a 50mL conical vial and added in 8mL LB broth, 10mL 2xTA and 90μL CaCl2
• Immediately added 4.5mL into each arthrobacter + lysate vial (Respectively) and plated
• Allowed the plates to sit for 15 minutes and moved to incubation

Observations:

• The rest of the team was working to calculate the titer of a plaque assay and calculated the amount of lysate needed to web a plate; we are using the same lysate and will be working together to continue to raise the strength of the titer

Next Steps/Conclusion: Next lab, will check the results of the plaque assays and hopefully will be able to count plaques and determine the titer strength and determine the amount of lysate needed to web a plate.

Rationale: Prepared a TEM grid for TEM imaging of the phage

Procedure:

• Obtained the lysate from refrigeration and went down to the TEM lab
• Obtained Uranyless from Aadil and obtained a parafilm strip
• Placed the parafilm strip in a petri dish and pipetted one drop of lysate, two separate drops of water and one drop of uranyless onto the strip (Each ~20μL)
• Using surgical/precision tweezers, picked up a copper grid and placed it shiny side down onto the lysate drop for 5 minutes
  • Following the 5 minutes in the lysate, transferred the grid into the first drop of water and let sit for 2 1/2 minutes; after the allotted time, transferred the grid to the other drop of water and water for 2 1/2 minutes
• After the water bath, transferred the grid into the uranyless and let sit for 1 minute (Time sensitive) and immediately wicked off excess uranyless solution
• Grid was then loaded into the TEM machine and imaged

Observations:

An image of what we believe to be a phage; the size proportions/ratio between the tail and head are off

The parafilm with the 2 drops of water, 1 drop of lysate and 1 drop of uranyless

The grids where the copper grids reside and were transferred out of and into

The completely lysed plate with bacterial growth that came back

Next Steps/Conclusion: There were no discoveries of actual phage; the phage that was found had uncommon ratios between the tail and head. The next steps would be to continue to raise the titer of the lysate to image the phages more easily

Rationale: Flooded a plate and added both flooded PB together and ran a plaque assay

Procedure:

• Aseptic zone to prevent contamination
• Obtained webbed plate from incubation and added 4mL of PB to the plate
• Allowed the plate to swirl / sit on a shaker table for a hour and a half
• After the allotted time, obtained a syringe filter and a 22μL filter tip and filtered PB into a 25mL vial
• Obtained a 50mL conical vial and three plates
• Added 10μL lysate into 0.5mL arthrobacter and 20μL lysate into another 0.5mL arthrobacter
• Pipetted 6mL LB Broth and 67.5μL CaCl2 into the 50mL vial
• Added 7.5mL 2XTA into the 50mL conical vial and immediately aded 4.5mL into the arthrobacter + lysate vial(s) (Respectively) and immediately plated
• Allowed the plates to sit for 15 minutes and then moved to incubation

Observations:

• The plate was almost throughly lysed; there was very little lawn of bacteria without plaques
• Added a team members’ flooded PB into the PB from the previous webbed plate to obtain a total of 8mL of flooded filtered lysate

Next Steps/Conclusion: Will be checking the status of the plaque assay next lab. If positive, will be calculating the new strength of the titer and will determine if another webbed plate will be needed to continue raising the strength of the titer

Rationale: Calculated how much of the titer I needed to web a plate and produce a plaque assay that is webbed.

Procedure:

• Aseptic zone to prevent contamination of plates and bacteria
• Obtained webbed plate from incubation and prepared to count plaques
• Counted plaques present on the plate (231)
• Brought the plate over to the light microscope to measure the diameter(s) the plaques [Avg. Diameter was .8μL]
• Calculated the area of the plate [5.671*10^3] and the area of the plaques [1.12878*10^4]
• Divided plate area by plaque and then divided that number by the titer of the plate to obtain the amount of lysate needed to web a plate [488μL to web]
• Obtained previous lysate from refrigerator and added 488μL to 0.5mL of arthrobacter, let sit for 10 minutes
• Obtained a 50mL conical vial and added in 4mL of LB Broth and 45μL of CaCl2
• Added 5mL of 2XTA to the 50mL conical vial and immediately pipetted 4.5mL into the arthrobacter + lysate vial and plated
• Allowed the plates to sit for 15 minutes and then moved to incubation

Observations:

Previous result of new lysate; counted plaques and calculated webbed plate

A clean control plate!

Calculations for webbing a plate

Next Steps/Conclusion: This plaque assay should provide a webbed plate that can be flooded to obtain another lysate, which will be tested for it’s strength. If the plate is contaminated, will re-run the experiment with the same amount of lysate.

Rationale: Filtered the flooded plate that has sat for 48 hours tot obtain a new lysate; conducted a plaque assay to calculate/test the titer strength.

Procedure:

• Created an aseptic zone to prevent/reduce bacterial contamination
• Obtained the previous flooded plate from the refrigerator
• Obtained a syringe filter (22μL) and filtered the PB into a 15mL conical vial
• Obtained a 50mL conical vial and added in 4mL LB Broth and 45μL CaCl2
• Added 20μL lysate to 0.5mL arthrobacter to infect
• Pipetted 5mL 2xTA into the conical vial and immediately added 4.5mL into the arthrobacter + lysate vial and plated
• Allowed plates to sit for 15 minutes and then incubated

Observations:

The new lysate obtained from filtering the PB of the flooded plate

The results of a plaque assay to observe the strength of the previous titer

10^-1 result of the previous titer

Next Steps/Conclusion: After performing this plaque assay, will be calculating the new titer strength and aiming to strengthen the titer strength. If the plate is negative / contaminated, will perform another plaque assay to check plaques and calculate the strength.

Rationale: Flooded the 10^0 plate, as well as calculated to web a plate by using more lysate.

Procedure:

• Created an aseptic zone to prevent contamination from other bacterium
• Obtained by plates from 10/22 and observed the results
• Flooded the 10^0 plate with 6mL PB and let sit for 48 hours
• Decided to run three plaque assays
  • 10^0 plate would be with 20μL lysate
  • 10^-1 would be 20μL 10^0 lysate + 80μL PB
  • 10^-2 would be 20μL 10^-1 dilution + 80μL PB
• Obtained a 50mL conical vial and added in 8mL LB Broth and 90μL CaCl2
• Added 10μL of 10^-1 and 10^-2 (Respectively) to 0.5mL arthrobacter
• Added 20μL 10^0 into 0.5mL arthrobacter
  • Let the arthrobacter+lysate sit for 10 minutes
• Added 10mL 2XTA to the conical vial and immediately pipetted 4.5mL into each arthrobacter+lysate vial and plated
• Allowed the plates to solidify and moved to incubation

Observations:

Contaminated TA control

10^-2 plate with markings on it to count the amount of plaques

10^-1 plate with a few plaques

Next Steps/Conclusion: Will be attending open lab on 10/26 to filter flooded plate and observe results of the plaque assays. If the plaque assays are positive, will calculate the titer of the lysate. If negative, will redo a plaque assay next lab.

Rationale: Re-calculate the titer strength of the lysate due to contamination of control plate and plaque assays

Procedure:

• First created an aseptic zone to prevent contamination of plates
• Obtained previous plates from incubation and observed contamination
• Obtained previous lysate from refrigeration as well as 4 petri dishes
• Created a 10^-1 dilution by adding 10μL 10^0 lysate to 90μL PB
• Created a 10^-2 dilution by adding μL 10^0 dilution to 90μL PB
• Obtained a 50mL conical vial and added in 10mL LB Broth and 112.5μL CaCl2 (Recipe was multiplied by 5 because a teammate used the same Lb Broth and TA)
• Added 10μL of lysate (Respectively) to 0.5mL Arthrobacter and let sit for 10 minutes
• Added 12.5mL 2XTA to the conical vial and immediately added 4.5mL to each arthrobacter + bacteriophage tube and plated
• Let the plates sit for 15 and incubated

Observations:

• 

  Contaminated Control Plate

  

  10^0 plate with no results; contamination prevalent

  

  Contaminated 10^-1 plate with no results

  

  Contaminated 10^-2 plate with no results

   

  Next Steps/Conclusion: The next steps would be to see if this current plaque assays turn out positive or not. If they do, calculating the titer of the plaque assay or flooding the plate would be the next course of action to create a high titer. If the plates turn out negative, then repeat the plaque assays.