Rationale: Re-testing the lysate via plaque assay with a 10^0, 10^-1 and 10^-2 dilution due to no results from the previous plates / contamination. This procedure will be using the same lysate obtained on 10/12

Procedure:

• Created an aseptic zone to prevent contamination by other bacterias
• Obtained the 10^0 lysate from the fridge and obtained four plates
• Created a 10^-1 dilution by pipetting 90μL PB and 10μL 10^0 into a microcentrifuge tube
  • 10^-2 dilution by pipetting 90μL PB and 10μL 10^-1
• Obtained a 50mL conical vial and pipetted 8mL LB Broth and 90μL CaCl2
• Added 10μL of lysate (Respectively) to 0.5mL arthrobacter
• Pipetted 10mL 2XTA into the conical vial and immediately pipetted 4.5mL into the arthrobacter + lysate vials and plated
• Allowed the plates to sit for 15 minutes and then incubated

Observations:

TA control with one spot of contamination

10^0 plate with no plaques

10^-1 plate with one plaque

Next Steps/Conclusion: This plaque assay will hopefully provide positive and measurable results, which can be used to calculate the titer strength. If able to calculate the titer strength and is high enough, then the titer will be prepared for DNA extraction

Rationale: Re-performing the plaque assay procedure for the 10^0, 10^-1, 10^-2 and 10^-3 dilutions due to contamination of the plates. Will be using the 10^0 lysate obtained from last lab

Procedure:

• Created an aseptic zone to prevent the contamination of plates
• Obtained 10^0 lysate from fridge and obtained five plates
• Created the 10^-1 lysate by pipetting 90μL PB and 10μL 10^0 into a micropipette
  • For the 10^-2, added 90μL PB and 10μL 10^-1
  • For the 10^-3, added 90μL PB and 10μL 10^-2
• Added the lysates (Respectively) to 0.5mL arthrobacter
• Obtained a 50mL conical vial and pipetted 10mL Lb Broth and 112.5μL CaCl2
• Added 12.5mL 2XTA to the conical vial and immediately pipetted 4.5mL into the respective arthrobacter+lysate vials and plated
• Allowed plates to sit for 15 minutes and then incubated
• Cleaned lab bench

Observations:

• The TA plate was contaminated with spots
• The plaque assays yielded no plaques

Next Steps/Conclusion: After this plaque assay, hopefully it will yield positive results and measurable results. The next step would be to calculate the strength of the current titer, and see if it’s high enough to continue to DNA extraction

Rationale: Filtered the PB of the flooded plate to obtain the lysate, and perform a plaque assay to determine the titer (Should be high)

Procedure:

• Create an aseptic zone to reduce the chance of contamination of other bacteria
• Obtained a 22μL syringe and filter and ran the lysate through the filter
  • Filtered the lysate into a conical vial
• Obtained 4 plates for the 10^-1, 10^-2 and 10^-3 plaque assays (+ one TA control plate)
• Created the 10^-1 by pipetting 10μL 10^0 lysate into a micro-centrifuge tube along with 90μL PB
  • Created 10^-2 by adding 10μL 10^-1 to 90μL PB
  • Created 10^-3 by adding 10μL 10^-2 to 90μL PB
• Obtained a conical vial and pipetted 8mL LB Broth and 90μL CaCl2 into the vial
• Added 10μL of lysate (Respective to each dilution) to 0.5mL arthrobacter and let them sit for 15 minutes
• Added 10mL 2XTA to the conical tube and immediately added 4.5mL to the arthrobacter+lysate tube, and plated
• Allowed plates to sit for 15 minutes and incubated
• Cleaned up work area/bench

Observations:

• The filtered lysate is slight yellow in color
• The process of filtering the flooded plate was surprisingly efficient and quick

Next Steps/Conclusion: The next step would be to see the turnout of the plaque assay, and then calculate the titer of the lysate. The lysate should already be high, but performing the plaque assay will confirm. The end goal is to prepare the lysate for DNA extraction.

Rationale:

Calculate the titer of the most recent plaque assay, as well as calculate how much lysate is needed to web a plate. The end goal is to create a high titer

Procedure:

• Created an aseptic zone to prevent contamination from bacteria
• Obtained my 10/8 plaque assay and control plate
• Drew quadrants onto the plate to count the amount of plaques (570 plaques total)
• Brought the plate over to the light microscope to measure the average radii of the plaques and radius of the plate
  • Measured 10 plaques of various sizes, and then took the average of their diameters; divided the average diameter by two to get the average radii of the plaques (Avg. radii = 0.5675 mm)
  • Measured the diameter of the plate and divided by two to get the radius (Radius of plate = 42.5 mm)
• Calculated the titer of the plate by dividing number of plaques by the total lysate (570/10μL)
  • Then multiplied that number of 1000 to convert from μL to mL (Titer = 5.7 X 10^4)
• Calculated the area of the plate ((π[42.6]^2)=5.671X10^3) and area of the plaques ((π[.5675]^2)=3.2205 X 10^-1
• Divided the plate area by plaque area (5.671 X 10^3 / 1.709 X 10^4=1.7609 X 10^4
• Divided 1.7609 X 10^4 by 5.7 X 10^4 and multiplied by 1000 (converting from μL to mL) and resulted with 308 mL to web a plate
• Consulted with Lathan, who advised to flood the current plate
• Obtained a 50mL conical vial of PB, and pipetted 6 mL PB into the plaque assay plate
• Parafilmed the plate and then left to refrigerate

Observations:

The positive 10/8 control plate

The 10/8 plaque assay w/ numerous plaques

The drawn quadrant on the plate for counting the plaques (Total 570)

Parafilmed plate after 6mL PB was added in

Calculations to find the titer and how much lysate to web the plate

Conclusion/ Next Steps:

Will be going to open lab on 10/12 to check on the flooded plate, and will be running the PB through a 22μL filter to isolate the bacteriophage. Then will be performing another plaque assay to confirm a high titer

Rationale:

Went back to the most recent plaque assay (Made on 10/1) and picked a plaque to create another plaque assay since the one conducted on 10/3 was contaminated.

Procedure:

• Created an aseptic zone to prevent the possible contamination of the plate / lab from bacteria
• Obtained the 10/1 plaque assay from the freezer
• Prepared a microcentrifuge vial with 100μL PB
• Picked a plaque from the plaque assay and pipetted into the vial (This is the 10^0)
• Obtained a 50 mL conical vial and added in 4mL LB Broth and 45 mL CaCl2 (Enough for two plates)
• Took the 10^0 and added 10μL into 0.5mL Arthrobacter and allowed to sit for 15 minutes
• Added 5mL 2X TA into the conical vial, and immediately transferred 4.5mL to the arthrobacter+lysate vial and plated
• The plates were allowed to sit for 15 minutes and then incubated

Observations:

The contaminated TA control plate

The contaminated 10/3 10^0 plate with no plaques visible

The 10/1 plaque assay with the plaque I picked circled

Next Steps/Conclusion:

After this plaque assay, will be calculating the titer of the plate and calculating how much of the titer would be needed to web a plate. If there is contamination, then the plaque assay step will be repeated

Abstract:

Calculate the titer of the plate to be able to web a plate; count the plaques on the plate, as well as measure the plaques and plate, and then calculate the titer

Procedure:

• Created an aseptic zone to prevent contamination of the experiment from bacteria
• Obtained my previous plaque assay plate (10^0 conducted on 10/1)
• Split the plate into four quadrants to be able to count the plaques
• Counted the plaques in the plate (85 total)
• Brought the plate over to the light microscope to measure the radii of the plaques and plate
  • Measured 10 plaques and then took the average of their diameters; divided the average diameter by two for the average radius (Average radius = .445mm)
  • Measured the diameter of the plate and then divided by two for the radius (Radius of plate = 42.5mm)
• Calculated the titer of the plate by dividing the total plaques by the total lysate (85 plaques/10μL lysate)
  • Multiplied by 1000 to get from μL to mL (Titer = 8.5 X 10^3)
• Calculated the area of the plate ((π[42.6]^2)=5.671X10^3) and area of the plaques ((π[.445]^2)=6.217X10^3)
• Divided plate area by plaque area (5.671 X 10^3/6.217 X 10^3=9.121 X 10^3)
• Divided 9.121 X 10^3 by 8.5 X 10^3 and multiplied by 1000 (μL to mL) and resulted with 1,073 mL to web a plate
• Cut the recipe down since there was not enough 10^0 lysate; added 90μL to 500μL arthrobacter and let sir for 15 minutes
• Obtained a 50mL conical vial and added in 1.9mL LB Broth and 22.5μL CaCl2
• Added 2.5mL 2XTA into the vial and immediate mixed with arthrobacter+lysate and then plated
• Also created a TA control plate (2.5mL 2xTA + 2mL TA + 22.5μL CaCl2)
• Let plates sit for 15 minutes and then incubated

Observations:

The 10^0 with the 85 counted plaques

The 10^-1 with only 3 plaques

The team’s control plate

Calculations for the titer and plate

The 10^0 plate after counting and calculations

Next Steps/Conclusion:

After this titer is created, will be calculating the titer of this titer to see if it is a strong titer. If not, will titer the titer again to create a stronger titer. If the titer is strong enough, will get a webbed plate and will flood the plate

Rationale:

Passage the bacteriophage another time to further purify the bacteriophage, and to produce a countable plate

Procedure:

• Created an aseptic zone to prevent contamination of bacteria in the plates/experiment
• Picked a plaque from the 9/26 plaque assay (10^0) and pipetted into 100μL PB
• Created a 10^-1 and 10^-2 dilution by adding 90μL PB into two separate micro-centrifuge tubes and adding 10μL of the previous dilution (10^-1 created by adding 10μL 10^0 into 90μL PB, and same with 10^-2)
• Added 10μL of each lysate into 0.5mL of arthobacter
  • Allowed to sit for 15 minutes to infect the arthrobacter
• Obtained a 50mL vial and added 20mL LB Broth and 225μL CaCl2 into the vial (Multiplied recipe by 10 for 10 plates)
• Added 25mL of 2X TA into the vial, and immediately pipetted 4.5mL TA into the arthrobacter+lysate vial, and plated
• Allowed plates to sit for 15 minutes and then incubated

Observations:

• The 10^0 plate had numerous plaques, but many were split
• 10^-1 and 10^-2 had little to no plaques
• The control plate of the group was contaminated

Next Steps/Conclusion:

After this passage, the next step would be to calculate the titer of the lysate. This would be done by counting the plaques and obtaining the measurements of the plate/plaques. The bacteriophage worked with should be considerably purified, since this is the sixth(Or so) purification of the bacteriophage

Rationale:

Repeating the last plaque assay due to contaminated arthrobacter. Will be performing another purifcation plaque assay from the previous 10^0 lysate from 9/24 (Kept in refrigerator)

Procedure:

• Created an aseptic zone to prevent the possibility of bacterial contamination
• Retrieved 10^0 lysate from refrigeration and created a 10^-1 and 10^-2 dilution
  • Created by adding 90μL PB into microcentrifuge vial and adding 10μL of the previous dilution
• Added the dilutions of lysate into separate 400μL vials of arthrobacter into infect
• Obtained a 50mL vial and added in 16.8 mL LB Broth, 180μL 1M CaCl2, and 20 mL 2XTA (Enough for 9 plates)
• Immediately pipetted 4.5 mL TA mixture into the arthrobacter vial(s), shook to mix, and plated
• Let sit for 15 minutes and then incubated

Observations:

• The control TA plate sat for less than 15 minutes
• Surprising that the arthrobacter was the one to be contaminated, rather than the LB Broth or TA
• Lathan explained that the bacteria in our sample most likely are not arthrobacteria anymore since there were no plaques, and rather were another strain of bacteria

10^0 plate with contamination. Interesting pattern created

10^-1 contaminated plaque assay

10^-2 exhibiting contamination

The control TA with contamination

Conclusion/Next Steps:

It was unfortunate that there as contamination since after this plaque assay, the lysate would’ve been able to be used to create a titer and web a plate. It has shifted lab time one session back. The next steps would be to analyze the results from this plaque assay and hopefully be able to create/calculate a titer and web a plate

Rationale:

Performed another plaque assay on the lysate to further purify the bacteriophage

Procedure:

• Created an aseptic zone to minimize chances of bacterial contamination
• Picked a plaque from the 9/21 plaque assay (10^0 dilution) and pipetted into 100μL of PB
• Created a 10^-1 dilution by adding 10μL of lysate (10^0) to 90μL of PB
• Created a 10^-2 dilution by adding 10μL of lysate (10^-1) to 9 μL of PB
• Obtained a 50 mL conical vial and added in 18mL LB Broth and 202.5μL CaCl2 in (Calculated for 9 plates)
• Added 10μL of lysate (All three) to separate 0.5μL arthrobacter vials (Allow to infect)
• Added in 22.5 mL 2XTA to the conical vial, and immediately pipetted 4.5 mL of the TA into the bacteriophage+arthrobacter vial and plated (For all three)
• Let the plates sit for 15 minutes and then incubated

Observations:

Not as many plaques on the 10^-1, but still a considerable amount

Significant amount of plaque on the 10^0

10^-2 plaque assay with only a few plaques

The group TA control was contaminated yet again, possibly indicating something is contaminated in either the LB Broth or TA

Conclusion/Next Steps:

The lab group was able to finish this procedure without problems or using up too much time since we are repeating procedures. Next lab period, will be looking for countable plaques on the 10^0 plate to create a webbed plate

Rational:

Performed another round of plaque assays to further isolate the bacteriophage in the sample

Procedure:

• Created an aseptic zone to prevent bacteria contamination
• Picked a plaque from the 9/17 plaque assay (10^0), and added it to 100μL of PB
  • This became the new 10^0 dilution for which the plaque assay would be performed with
• Added 10μL of 10^0 to 0.5 arthrobacter
• Obtained a 50mL conical vial and added 14mL LB Broth, 22.5 μL CaCl2, and 17.5 2X TA
  • Made enough TA for seven plates; two plates per member of the group, and one control plate
• Immediately added 4.5 mL TA to the 10^0 + arthrobacter tube and allowed to mix, and then plated
• Let the plates sit for 15 minutes and then incubated

Observations:

• The 10^0 had many plaques, similar to the 9/12 plate
• The 10^-1 only had one, singular plaque
• The 10^-2 only had one, singular plaque
  

  Contaminated TA control for 9/17

  

  The 9/17 plaque assay (10^-2) with one plaque (Circled)

  

  The 9/17 plaque assay (10^0) with noticeable plaques

  

  The 9/19 plaque assay plate

  

  The 9/17 plaque assay (10^-1) with one plaque (Circled)

  

  9/19 control plate for TA

   

• Plates turned out as expected, with 10^0 with the most plaques, and 10^-1 and 10^-2 having less plaques
• Contamination of the 9/17 Control TA is concerning, and may entail a contaminated LB Broth

Conclusion/Next Steps:

The lab group was able to complete the procedure quickly since it was a repeat of the procedure of Monday’s lab. The next steps would be to see the results of the current plaque assay, and repeat another plaque assay to continue to isolate the bacteriophage