Rationale:

The purpose of today’s lab was to check on the plates that were set in the incubator the day prior to observe them before any bacteria was able to recolonize. If the plates were lysed or highly webbed, they were to be flooded immediately to gather the lysate while phage was still present.

Results 11/12/18:

• Plates had almost completely lysed with very few indicators of any bacterial lawn surviving. This is good as it indicates high phage presence and it was decided to flood each plate and combine lysates to calculate titer in the next lab.
• 

  20 µL Plaque Assay 11/12/18

  

  15 µL Plaque Assay 11/12/18

  

  10 µL Plaque Assay 11/12/18

Materials:

• 24 mL of Phage Buffer
• Vacuum Top Filter
• Serological Pipette

Procedure:

1. Began by establishing an aseptic zone.
2. Added 8 mL of phage buffer to each of the three plates.
3. Once added, plates were set on the shaker to gather phage particles for approximately an hour and a half.
4. After time had passed, plates were removed from the shaker and a vacuum top filter was assembled under the fume hood of the lab.
5. The lysates from each plate that had been gathered from the flooding process was then poured and filtered through the 0.22 micron filter into a new conical vial.
6. Then the lysate gathered from 11/07/18 flood procedure was transferred to the new conical vial, yielding a total 25 mL of flooded lysate.
7. Lysate was then labeled and stored in the fridge for next lab.

Results/Data:

• Although 24 mL of Phage buffer was added to the plates, only 20 mL of lysate was recovered from the flooding process, the remaining 5 mL of lysate came from the 11/07/18 lysate that was combined with the other 3 floods. Lysates were flooded for a little over an hour and a half, allowing plenty of phage particles to be picked up from the lysed plate. This should increase the titer of the lysate and the large volume of lysate allows for DNA extraction to be possible once a high enough titer has been acquired.

Conclusions:

Once factor that could have contributed to the loss of about 4 mL of potential lysate is reabsorption of the phage buffer by the top agar. The reason why plates were flooded so quickly was to avoid any Arthrobacter regrowing and recolonizing as phage begins to die out due to the lysed plate. There should be still be phage present on the lysed plate, just unable to reproduce due to the lack of hosts available.

Next Steps:

The next steps for this experiment are to spot titer out the new combined lysate to determine a new titer after 3 separate floods.

Rationale:

The purpose of today’s lab was to move towards obtaining a high titer of lysate for TEM and DNA extraction. Currently the lysate is both at a low volume and a low titer so flooding and spot titering is necessary to move forward with the experiment.

Results:

• The plate made with the lysate gathered from flooding last week had completely lysed, the plate with the 60 µL of diluted lysate had split. After closer examination it was determined that the plate had completely lysed, leaving the phage to die off, allowing for the Arthrobacter to recolonize and regrow onto the plate.
• It was decided to run another plaque assay with 3 different volumes of lysate instead of the 60 µL used last time to hopefully web a plate well enough to flood.
• 

  PA Made with 60 µL 11/07/18

  

  Flood TA Control 11/07/18

  

  60 µL TA Control 11/07/18

  

  FLood PA Regrowth 11/07/18

Materials

• LB Broth
• 2X TA
• Calcium Chloride
• Flooded Lysate
• Agar Plates

Procedure:

1. Established an aseptic zone
2. Ran 3 different plaque assay procedures with 3 different volumes of the flooded lysate: 10 µL, 15 µL, and 20 µL of lysate.
3. To run a plaque assay, 2.0 mL of LB broth was added to 3 separate conical vials as well as 2.5 mL of LB broth into a 4th vial for the top agar control.
4. After LB broth, 22.5 µL of calcium chloride was added to all 4 of the conical vials.
5. Then, 3 separate 0.5 mL of Arthrobacter was combined with the different volumes of lysate and left to infect for 15 minutes.
6. Once the 15 minutes were up, the infected Arthrobacter was combined with the LB Broth and 2.5 mL was added to each of the conical vials.
7. After combining, the top agar was then plated immediately into their respective plates and left to solidify for about 15 minutes.
8. Once the 15 minutes were up, the plates were then flipped and stored in the incubator.

Results and Data:

The experiment went with no errors and none of the components appeared to have any visual signs of contamination during the experiment. The top agar did not have any bubbles during plating, as a new procedure involving the serological pipette was used to plate the top agar.

Conclusions:

The difference of lysates included into the top agar should reduce the presence of phage particles in the top agar, increasing the probability of having a properly webbed plate. If the plates do lyse again, they will need to be immediately flooded to capture the phage particles present before the phage dies off and Arthrobacter regrows and recolonizes.

Next Steps:

The next steps for this experiment are to flood the plates as soon as possible to obtain as many phage particles from the plate as possible. The lysates gathered will then all be combined into one conical vial to gather a high volume of lysate to calculate a titer from. The goal is to continue moving towards a high titer of lysate.

Rationale:

The purpose of today’s experiment was to flood and web a plate using the calculated lysate from titer calculations.

Results from 11/05/18

• All of the plates were positive for plaques up to the 10^-2. There was also contamination present on the top agar control. It was decided that it was best to continue with the amplification process by webbing and flooding a plate due to the time constraints present.
  

  10^0 Plaque Assay 11/05/18

  

  10^-1 Plaque Assay 11/05/18

  

  10^-2 Plaque Assay 11/05/18

  

  Contaminated Top Agar 11/05/18

   

Materials: 

• Phage Buffer for flooding and Plaque Picking
• Diluted Lysate
• Agar Plates
• Calcium Chloride
• Arthrobacter
• LB Broth
• 2X TA

Procedure:

1. Established an aseptic zone.
2. Calculated titer of lysate using the equation #pfu/volume of lysate in µL * (10^3 µL/mL) *10^0. 10^0 dilution was used as it had the most clear plaques and offered the most consistent phage morphology.
3. Once titer was calculated, 10 plaques were measured for the radius, and the plate’s radius was measured to determine how many plaques were needed to web a plate using the equation #Plaques Needed = Area of Plate / Area of Plaques
4. Using the # of Plaques Needed to web a plate, the equation: Volume of Lysate (mL) = (# of plaques needed to web / titer)
5. Once the volume was calculated, it was decided that the 10^0 plaque assay from 11/05/18 had to be flooded, and a plaque assay had to be performed from the remaining 60 µL of that lysate.
6. To flood the plate, 8-mL of phage buffer was added to the top of the agar and the plate was closed and left on a shaker to shake for an hour.
7. A plaque assay was then run with the 60 µL lysate.
8. 2.0 mL of LB broth was added to one conical vial and 2.5 mL of LB broth was added to another.
9. 22.5 µL of Calcium chloride was added to both conical vials.
10. The remaining 60 µL of lysate was combined with 500 µL of Arthrobacter and left to infect for 15 minutes.
11. After approximately 15 minutes, the infected lysate was added to the conical vial with 2.0 mL of LB broth in it as the other is our top agar control.
12. Then 2.5 mL of 2X TA was added to both conical vials and plated immediately.
13. The plates were then left to solidify.
14. After solid, plates were flipped and inserted into the incubator.
15. After the 1 hour, the flooded plate was removed from the shaker and the new lysate was gathered and filtered through a 0.22 micron syringe filter to create a new filtered lysate.
16. A plaque assay procedure identical to the previous one was then run, using 30 µL of lysate instead of 60 µL.

Results and Data:

• The calculated titer of the lysate was 1.5*10^4 pfu/mL
• The Average radius of plaques gathered was 0.05 cm and the radius of the plates was 4.25 cm.
• The calculated area of the plaques were 0.007854 cm^2 with the area of the plate being 56.745 cm^2.
• The number of plaques needed to web the plate was 7.225*10^3 plaques, which meant that a volume of 481 µL of lysate would be needed.

Conclusions:

Due to the extremely low titer of lysate, an extremely high volume of lysate was needed to lyse a plate. There was not enough of the diluted lysate to web this plate, so it was decided to flood the 10^0 plate and gather a high volume of lysate from that, and run another plaque assay with the remaining 60 µL of lysate. Once the plaque assay is complete, that plate is to be flooded as well and the lysates are to be combined and the titer will be calculated from the two and the amplification process will continue.

Next Steps:

The next steps are to flood both plates and calculate the new lysate required to web a plate. Have to continue working towards a high titer of lysate.

Rationale:

The purpose of today’s lab experiment was to analyze the plaque assays performed on 10/31/18 and continue with purifying the phage particles to get a consistent morphology. We are working towards obtaining high titer lysates.

Results 10/31/18:

• Plates were all plaque positive, but unfortunately the 10^-2 dilution had several air bubbles that had popped on the top agar, making the results inconclusive. Going to continue with the final round of dilutions to get a consistent plaque morphology and pick a plaque from the 10^-1 diluted plate.
• 

  10^-1 Dilution 10/31/18

  

  10^0 Dilution 10/31/18

  

  10^-2 Dilution 10/31/18

  

  TA Control 10/31/18

Materials:

• Phage Buffer
• Plaque to Pick
• LB Broth
• 2X TA
• Calcium Chloride

Procedure:

1. Established an aseptic zone.
2. To pick a plaque, used a micropipette tip to poke 1 plaque from the 10^-1 plaque assay (avoiding bacterial lawn beneath) and inserted tip into 100-μL of phage buffer to release phage particles.  This was the new 10^0 dilution.
3. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
4. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
5. Once diluted, 3 separate plaque assays were run with each dilution.
6. 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL was added to a fourth top agar control.
7. Next, 22.5-μL of calcium chloride was added to each of the conical vials.
8. 30-μL of each of the diluted lysates were combined with 3 separate 500-µL quantities of Arthrobacter and left to infect for approximately 15 minutes..
9. After about 15 minutes, the diluted lysates were combined with their respective LB Broth mixtures and 2.5-mL of 2X TA was added to each of the conical vials.
10. Swirled and plated top agars immediately.
11. Once solidified, they were added to the incubator until the next lab.
12. Calculated titer by counting plaque formation on the 10^-1 dilution from 10/31/18 and used the equation Titer = #pfu/volume of lysate in µL * (10^3 µL/mL) *10^1

Results and Data:

• The plaque assays were performed with no error during the experiment except for slight air bubble formation along the side of the plate.
• The calculated titer of the current lysate used to make the 10^1 dilution on 10/31/18 was 3.167*10^4 pfu/mL.

Conclusions:

• Overall, the calculated titer was on the lower end of the spectrum, which is consistent with the rest of the class. The phage concentration that was begun with was not very high initially as it required more than 10 µL of lysate to even show any plaque formation on any diluted plaque assays. Also, the titer is low due to the fact that there have been no webbing and flooding involved that could potentially give rise to high titer lysates as the lysate has not moved beyond the purification process.

Next Steps:

The next steps in this experiment are to analyze the results from this plaque assay, calculate a new titer, and move towards webbing and flooding a plate.

Rationale:

The goal of today’s lab was to continue the purification process and work to getting a high titer lysate as soon as possible. The lab as a whole has not yielded very many positive phage results and does not have high enough titer lysates for TEM and sequencing.

Results from 10/29/18:

• Plaque assays for the enriched lysate, 10^0, and 10^-1 dilutions were positive for plaques.
• The 10^-2 dilution plaque assay, as expected, was contaminated due to the different top agar used in the experiment.
• Enriched lysate was littered with plaques, 10^0 had roughly 15 small plaques, and the 10^-1 had 1 plaque.
• 

Materials:

• Phage Buffer
• Plaque to Pick
• LB Broth
• 2X TA
• Calcium Chloride

Procedure: 

1. Established an aseptic zone.
2. To pick a plaque, used a micropipette tip to poke 1 plaque from the 10^-1 plaque assay (avoiding bacterial lawn beneath) and inserted tip into 100-μL of phage buffer to release phage particles.  This was the new 10^0 dilution.
3. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
4. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
5. Once diluted, 3 separate plaque assays were run with each dilution.
6. 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL was added to a fourth top agar control.
7. Next, 22.5-μL of calcium chloride was added to each of the conical vials.
8. 30-μL of each of the diluted lysates were combined with 3 separate 500-µL quantities of Arthrobacter and left to infect for approximately 15 minutes..
9. After about 15 minutes, the diluted lysates were combined with their respective LB Broth mixtures and 2.5-mL of 2X TA was added to each of the conical vials.
10. Swirled and plated top agars immediately.
11. Once solidified, they were added to the incubator until the next lab.

Results/Data:

• The procedure went smoothly, and top agar did not solidify before plating. Lysate was decreased slightly from the previous experiment, but still should be able to yield enough phage particles to show on a plaque assay.

Conclusions:

• Phage concentration in the original lysate was not very high to begin with, and this explains the repeated negative plaque assay results with diluted lysates. All of the phage had been diluted out and there simply was not enough to form a plaque. Although the plaque amount was relatively small on these dilutions, there were still plaques to be picked to continue the purification process to help work towards amplification and flooding plates.

Next Steps:

• The next steps for this experiment are to continue with 1-2 more rounds of purification until phage yields consistent plaque morphologies. The ultimate goal is to work towards gathering a high titer lysate as quickly as possible as time is running out in the semester and a high titer lysate is needed for TEM.

Rationale: 

In today’s lab, the goal was to analyze the plaque assay results and continue with the purification process. This was the third round of plaque picking with no positive results yielded. If results from 10/24/18 were negative, aspects of the experiment would have to be modified.

Results from 10/24/18:

• Plaque assay results for all dilutions were negative, with no contamination on the top agar control.
• Phage particles could possibly be diluted out since original lysates were not very concentrated to begin with. Decided to increase the amount of lysate used in plaque assay procedure to give more phage particles in the dilution. Did not adjust any other part of the top agar solution and did not adjust any concentrations.
• 

  GJA 10^0 Dilution 10/24/18

  

  GJA 10^-1 Dilution 10/24/18

  

  GJA 10^-2 Dilution 10/24/18

  

  Top Agar Control 10/24/18

Materials:

• LB Broth
• 2X TA
• 50-µL of Diluted Lysates
• Agar Plates
• Calcium Chloride

Procedure:

1. Established an aseptic zone.
2. Gathered original lysate from 10/03/18 enrichment and filtered through 0.22 micron syringe filter
3.  3 separate plaque assays were run with each dilution as well as one top agar control and one plaque assay with the new filtered lysate.
4. To begin, 2.0-mL of LB broth was added into 4 separate conical vials and 2.5-mL added to a fifth top agar control vial.
5. Next, 22.5-μL of calcium chloride was added to the each of the conical vials.
6. 50-μL of each of the diluted lysates were combined with 4 separate 500-μL quantities of Arthrobacter and left to infect for approximately 15 minutes.
7. After about 15 minutes, the infected diluted lysates were combined with their respective labeled LB Broth mixtures and 2.5-mL of 2X TA was added to the all 5 of the conical vials.
8. Swirled and plated top agars immediately and left to solidify.
9. Once solidified, they were flipped and added to the incubator until the next lab.

Results/Data:

• Plaque assay procedure went well with no complications. The increase of lysate by 40-µL is almost negligible for the final concentration of the 1M calcium chloride and 2X TA, so those values were not adjusted. The original enriched lysate was a dark red color, seemingly contaminated from a airborne bacterium that had been found contaminating other lysates as well. Also, for the 10^-2 dilution a different top agar had to be used since there was no more left to use.

Conclusions:

• The increasing of the lysate should be able to increase the phage population to combat the serial dilution. Original phage concentration could be weak due to the small and few plaques present from the plaque assays run with the original enriched lysate. There is a possibility of the 10^-2 dilution being contaminated or not being similar to the rest of the plaque assays run as it was made with a totally different top agar.

Next Steps:

The next steps for this experiment are to check the plaque assay results in the next lab. If the dilutions are positive for plaques, the purification process will be continued by picking a plaque from one of the dilutions to further purify. If no plaques are present, then plaque assays will be rerun with an even higher lysate volume.

Rationale:

The purpose of today’s lab was to analyze the plaque assay results from the previous lab. If plaques are present, then the purification process will continue. If no plaques are present, then new serial dilutions will be formed to retest phage presence.

Results from 10/22/18:

• Plaque results were negative.
• Although results were negative, top agar control was uncontaminated, and is a clear indicator that there truly were no phage particles present.
• New serial dilutions will be run with new lysates.
• 

  10^0 PA 10/22/18

  

  10^-1 PA 10/22/18

  

  Top Agar Control 10/22/18

  

  10^-2 PA 10/22/18

   

   

Materials: 

• Phage Buffer
• 10 Circles Plaques to Pick
• LB Broth
• 2X TA
• Calcium Chloride
• Agar Plates

Procedure:

1. Established an aseptic zone.
2. Circled 10 new plaques to pick.
3. To pick a plaque, used a micropipette tip to poke 10 plaques separately the top agar and lifted out (avoid bacterial lawn beneath) and inserted tip into 100-μL of phage buffer.  This was the 10^0 dilution
4. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
5. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
6. Once diluted, 3 separate plaque assays were run with each dilution as well as one top agar control.
7. 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL added to control.
8. Next, 22.5-μL of calcium chloride was added to the conical vials.
9. 10-μL of each of the dilutions was combined with 3 separate 0.5 mL quantities of Arthrobacter and left to infect.
10. After about 15 minutes, the diluted lysates were combined with their LB Broth mixtures and 2.5-mL of 2X TA was added to the broths.
11. Swirled and plated top agars immediately.
12. Once solidified, they were added to the incubator until the next lab.

Results:

• Experiment went smoothly, pushed a little farther into the top agar to see if there was a possibility plaques were not being picked properly. Plates were plated without any issues as top agar did not solidify before plating.

Conclusions:

• The experiment was performed with good aseptic technique, and should yield results with no data skewing. A possibility for error is in the plaque picking as pushing deeper could possibly puncture the bacterial lawn below. There were no clear signs of puncturing, but it is a possibility.

Next Steps:

• The next steps are to analyze the plaque assay results for plaque presence. If plaques are present then purification will continue in hopes of obtaining a high titer lysate.

Rationale:

The purpose of todays lab was to analyze the plaque assay results from the previous lab. If plaques were present, amplification and purification processes were to be continued, but if no plaques were present, plaques would have to be picked to be diluted again.

Results from 10/15/18:

• 
• Top Agar had split due to not fully solidifying before being put in the incubator, this can be examined by the sliding of the top agar from the side of the plate.
• Top Agar control was uncontaminated.
• Plaques were not present, however plates were cracked as well.

Materials:

• Phage Buffer
• Plaques to Pick
• LB Broth
• 2X TA
• Calcium Chloride

Procedure:

1. To pick a plaque, used a micropipette tip to poke 10 plaques separately the top agar and lifted out (avoid bacterial lawn beneath) and inserted tip into 100-μL of phage buffer.  This was the 10^0 dilution
2. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
3. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
4. Once diluted, 3 separate plaque assays were run with each dilution.
5. 2.0-mL of LB broth was added into 3 separate conical vials.
6. Next, 22.5-μL of calcium chloride was added to the conical vials.
7. 10-μL of each of the dilutions was combined with 3 separate 0.5 mL quantities of Arthrobacter and left to infect.
8. After about 15 minutes, the diluted lysates were combined with their LB Broth mixtures and 2.5-mL of 2X TA was added to the broths.
9. Swirled and plated top agars immediately.
10. Once solidified, they were added to the incubator until the next lab.

Results:

Top Agar had solidified the moment the top agar had been plated, causing it to slide around the side of the plate without fully solidifying. This caused the top agar to solidify unevenly. This is going to cause the top agar to split, not allowing phage to infect.

Conclusions:

Top Agar will most likely split due to the improper solidifying that took place, which will not allow plaques to form. The top agar was not hot enough to be poured and plated effectively.

Next Steps:

The next steps will be to analyze the plaque assay results for plaque presence and morphology. Another plaque assay procedure will most likely be performed as the top agar did not solidify properly, causing the top agar to split.

Rationale:

The purpose of today’s lab was to continue with the purification and amplification process to gather consistent phage particles.

Results from 10/10/18:

• Top Agar had split, not allowing for the proper formation of plaques. Meaning the top agar had not fully solidified before being flipped and inserted into the incubator. Plaque assays had to be redone.
• 

Materials:

• LB Broth
• 2X TA
• Diluted Lysates
• Agar Plates

Procedure:

1. Established an aseptic zone.
2. Mass of dry plate was recorded from water percentage experiment.
3. Once diluted, 3 separate plaque assays were run with each dilution.
4. 2.0-mL of LB broth was added into 3 separate conical vials.
5. Next, 22.5-μL of calcium chloride was added to the each of the conical vials.
6. 10-μL of each of the dilutions was combined with 3 separate 500-mL quantities of Arthrobacter and left to infect.
7. After about 15 minutes, the diluted lysates were combined with their LB Broth mixtures and 2.5-mL of 2X TA was added to the broths.
8. Swirled and plated top agars immediately.
9. Once solidified, they were flipped and added to the incubator until the next lab.

Results/Data:

• The top agar seemed solidified before inserted into the incubator with no clear indicator splitting.
• The mass of the dry soil with plate 3.49 g, making the mass of the dry soil 1.53 g.
• The mass of the water was 1.93- 1.53 = 0.4 grams
• Water percentage = 20.7%

Conclusions:

The water percent of the soil sample was relatively low, which is unsurprising due to the relative dryness of the soil once it was collected. Soil was not extremely clay like, and was very pebbly.

Next Steps:

The next steps for this experiment are to analyze the plaque assay results for phage particles. If there are no plaques present, new plaques and dilutions will be performed to test through plaque assays again.