September 27

09/24/2018 Purification Process continued

Objective: 

  • Acquire a concentrated sample of bacteriophages
  • Pick a plaque from the plaque assays formed after serial dilutions
  • Make a plaque assay from the picked plaque

Pre-Lab Observations:

  • Plaques were formed on all the plates ( 10^0, 10^-1, and 10^-2 were the dilutions)
  • The control plate was contaminated.

Procedure:

  1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
  2. Phage buffer was acquired from the lab instructor and microcentrifuge tips.
  3. Using the micropipette, 100μl of phage buffer was transferred to a microcentrifuge tube.
  4. In the aseptic zone, at a 90° angle, a plaque on the 10^-1 plaque assay was stabbed using a micropipette tip ( attached to the micropipette) and the tip was then put into the microcentrifuge tube with 100μl of phage buffer and stirred to properly remove bacteriophages on the tip.
  5. This microcentrifuge tube was then vortexed for 30 seconds and was labelled 10^0.
  6. One Top Agar mixture was made for the group.
  7. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
  8. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
  9. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
  10.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
  11. The vial was then set on the rack.
  12. 0.5 ml of arthrobacter was retrieved from the lab instructor
  13. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
  14. The vial was then allowed to rest on the test tube rack for 15 minutes
  15. After the 10 minutes had ended, 25 ml of the 2X TA was added to the LB broth and Cacl2.
  16. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tubes with the arthrobacter and the lysate.
  17. The contents of the test tube were then poured onto the agar plate.
  18.  Part of the top agar mixture was poured into the top agar control plate for the group.
  19. To let the top agar solidify, the plates were allowed to rest for 12 minutes.
  20. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Interpretations:

The repeated contamination of the plate is perplexing. there may be another factor causing the contamination of the plates is that the agar plate could be contaminated plates before top agar was poured onto the plates. the purification process will not be repeated a few more times to acquire more concentrated and similar phages.

Future Notes 

Check plates properly before using them and test the broth and 2x agar for potential contamination.

 

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
September 26

9.26.2018 Rewashing Soil Sample D

9.26.2018 Rewashing Soil Sample D

Rationale: At the beginning of the lab time, it was revealed that many individuals had results that did not accurately reflect what they should have. This was attributed to the fact that the Arthrobacter used for all procedures on Monday was not Arthrobacter, which made it impossible for Arthrobacter-infecting phages to interact with them. Therefore, all procedures done on Monday that involved Arthrobacter had to be redone. Since the soil sample lysate had been enriched with a problematic bacterial sample, it was necessary to rewash and enrich the new lysate from the same soil sample.

Procedure (Metadata in Italics):

  1. Established an aseptic zone.
  2. Added 2mL of soil to tube (“HMB Soil Sample D 9/26/18”)
  3. Added 10mL of LB Broth to tube
  4. Shook for 10 minutes
  5. Centrifuged sample for 15 minutes
  6. Used syringe and 22 micron filter to filter supernatant of centrifuged sample. Placed into tube labeled “HMB Soil Sample D 9.24.18”.
  7. Added 0.5mL of Arthrobacter to “HMB Enriched Soil D 9.26.18” tube to create enriched lysate.
  8. Moved enriched lysate to shaker to be used on Friday 9/28.
  9. Added 4.126g of soil to weigh boat and placed under hood to be analyzed on Friday.
  10. 4mL of soil was added to a falcon tube. 8mL of DI water was added to the falcon tube along with 3 drops of soil dispersion liquid.
  11. Shaken for 30 seconds for reexamination on Friday.
  12. Added 0.5mL of soil supernatant from the falcon tube to the pH tube. Added DI water until the pH tube was full. Used pH paper to determine the pH was 5.5 after matching with chart. 

Observations:

  • Mass of tube while shaking was equal to 18.03g.
  • At the end of filtering, a very small amount of white foam dripped from the filter into the lysate tube. It is unknown if the amount or composition will have significant effects in any way.
  • As observed on Wednesday, the soil sample was very dark after adding LB Broth and sand had already begun to settle out before it was placed in the centrifuge.
  • Mass of weigh boat: 2.325g.

Data Summary from Today: pH=5.5

Next Steps/Conclusions:

  • The next step for processing this soil sample would be to perform a spot test and plaque assay. This procedure was intended to occur during this lab entry, but the delay due to the issue with Arthrobacter delayed the schedule of the lab. Therefore, the procedures will be completed on Monday.
Category: Henry Burns | LEAVE A COMMENT
September 26

Soil Washing Redo and Metadata 09.26.18

Rationale)

To wash Soil D again in order to produce an enriched lysate not created with contaminated arthro, I will also collect soil metadata.

Procedures)

  1. Setup an aseptic zone
  2. Added 2mL of Soil D to a 15mL vial labeled “NMN Soil D+ 9.26.18”
  3. Added 10mL of LB broth to the vial, shook for 10 minutes. Took the mass.
  4. Centrifuged for 15 minutes
  5. Retrieved a weigh boat and labeled “NMN %H2O”, took the empty mass, then added 4g of Soil D and placed under the vent hood.
  6. Got a falcon tube and added 4mL of Soil, filled to 12.5mL with DI water, and added 3 drops of soil dispersion liquid; covered and shook until mixed, let the tube settle for 48 hours.
  7. Added 1-2mL of falcon tube liquid to a pH vial, shook to combine, tested pH. pH=5.5
  8. Used a .22 micron syringe filter to filter the supernatant into a 50mL conical vial labeled “NMN 9.26.18 Soil D Enriched Lysate”
  9. Added .5mL of arthro to the vial
  10. Placed the vial on the shaker table for 48 hours.
  11. Stored the bag with Soil D in the walk-in refrigerator.

Observations/Data)

The mass of the vial “NMN Soil D+ 9.26.18” was 18.042g. The empty mass of the weigh tray was 2.331g. The mass of Soil D in the tray was 4g. The pH of the soil was 5.5. I observed that the LB broth we used was clear and not contaminated, additionally, the arthro I used was also non-contaminated. The Falcon tube I used had a red asterisk on it.

Conclusions/Next Steps)

I can conclude that the soil from which I collected is acidic in nature. The next steps will be to conduct a plaque assay and spot test with the enriched lysate I produced today and the direct isolation I produced Monday in the effort to isolate a phage from Soil D, which can then be analyzed later.

 

Category: Nathan Newton | LEAVE A COMMENT
September 26

Plaque Assay 9/26/18

Rationale: Due the arthrobacter being contaminated Monday based off of the positive control we will have to reconduct the plaque assay.

Procedure:

  1. Pipetted 10μL of lysate into 0.4mL of arthrobacter. Only used 0.4mL ofarthrobacter because not enough due to contamination of supply. Then

    waited for 10 minutes before beginning to make top agar.

  2. Using aseptic technique and fresh pipettes each time put 8.4mL of LB brothin a 50mL tube, then 90μL of CaCl2, then 10mL of 2X TA.
  3. Found a contaminant in TA supply so threw out the top agar we made andisolated the 2X TA from the other bottles.
  4. Grabbed a new bottle of TA and labeled Group 2 and began making a newbatch of top agar using the same procedure as #2.
  5. Pipetted 4.5mL into tube with arthrobacter and lysate and pipetted up anddown to mix.
  6. Poured contents into plate while keeping the top as close to the bottom aspossible as to not have any contaminants fall in.
  7. Waited 10 minutes to solidify then inverted and incubated for 48 hours.

Observations:
Another negative plaque assay. Was angry so accidentally threw in trash before taking picture. On the bright side at least the control was clear.

Interpretations and Next Steps: Going forward we need to be more diligent about keeping lid of the plate open for as short a time as possible. Also, now that we have found a contaminant in the TA we need to check the TA against the light before putting in the tube. We should also be more careful about the sterile pipette coming in contact with anything then putting it in the supply of anything whether it be LB broth or TA.

Category: Sriram Avirneni | LEAVE A COMMENT
September 26

September 26, 2018 Plaque Assay Soil C

Rationale: After finding contaminated results in the previous lab, the purpose of this lab is to run another plaque assay.

Description of Procedures:

  1. The work station was cleaned using aseptic technique and an ethanol burner was lit.
  2. A pellet was seen in the bottom of the enriched lysate, so the lysate was spun again in the centrifuge for 5 minutes at 10,000 x g.
  3. While spinning, enough top agar solution was made for four plates. 90 ul of CaCl2 and 8.4 ml of LB Broth were added to a tube.
  4. The lysate was then filtered using a 0.22 ul syringe filter.
  5. Next, 10 ul of lysate was added to 0.4 ml of arthrobacter and was allowed to sit for 10 minutes.
  6. 2x TA was then added to the top agar solution and pipetted up and down to mix. 4.5 ml of the solution was poured into the arthrobacter and then poured directly onto a plate labeled LIP 9-26-18 Plaque Assay. 4.5 ml of the solution was poured directly onto a plate labeled Control LIP EAG SS 9-26-18.
  7. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator.
  8. The work station was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results:

  • Observations
    • The enriched solution had a pellet at the bottom and was a murky color, so it was re-spun and re-filtered.
    • There were a few bubbles towards the center of the experimental plate.

Interpretations/Next Steps/Conclusions:

The experiment was complete. The next step will be to check for plaques in the next lab. If there are plaques, they will be picked. If not, a spot test will be run.

 

Contaminated Plates:

Category: Lucy Potts | LEAVE A COMMENT
September 26

Lab Day 10: Results + Redo

Rationale

Redo plaque assay due to possible contamination of Arthro from lab day 9. Other group members will redo spot test.

Detailed Procedure

  1. Took 10 microliters of filtered enriched lysate and added to 400 microliters of Athro. Sat for 15 mins
  2. Took 4.2 mL LB Broth, 45 microliters of 1M CaCl2, and 5 mL of 2X TA into a 50 mL vial.
  3. Took half of solution from step 2 and poured into control plate
  4. Took other half and added solution from step 1 and poured into plaque assay plate
  5. Both plates sat for 15 mins and was inverted into the incubator.

Conclusions+Observations/Results/Next Steps

Control plates from group members’ spot test and plaque assay were both contaminated, but showed negative results. There seems to be contamination of Arthro from class discussion. When pouring control plate, there was a bubble on the plate that did not pop. Next steps are to wait for the results. If no plaque are present for both spot test and plaque assay, then new soil will have to be found.

Category: Soo-Un Jeong | LEAVE A COMMENT
September 26

9/24/18 Round three of purification

Rationale: Perform a second third round of purification since the first third round failed, since the LB Broth was contaminated. The goal of the purification

Question: Why did the control fail for the last two experiments?

  • The last two experiments, the control were both negative.
  • One reason why this could have been negative since the same LB broth was used for both experiments. The eight groups in the class labeled there own LB broth so that the groups in the class could see which groups had the contaminated control. This experiment was done on the side, and the class quickly saw which groups had a contaminated LB broth.
  • This led to the redo of the third round of purification.

Procedure:   

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

  1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
  2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
  3. We then got an ethanol burner, and our aseptic zone was created.
  • Picked Plaque from Plaque Assay, which was performed on 9/21/18.
  • Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
  • Got two more microcentrifuge caps, labeled one cap 10^-1 and the other solution 10^-2.
  • Added 90 microliters of PB to both caps.
  • Added 10 microliters of the 10^0 solution into the 10^-1 solution.
    • The 10^0 solution has the picked plaque. Transferred this to the 10^0 solution so that this could be added to the other two caps so that three Plaque Assays could be performed.
  • Added 10 microliters of the 10^-1 solution to the 10^-2 solution.
  • All microcentrifuge caps had plaques, added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2).
  • Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
    • The formula below was used to make the solution for 9 plates (three for each of the three solutions and one for the control).
      • 20mL LB Booth (x9)
      • 22.5 microliters of Calcium Chloride (x9)
      • 25mL 2X TA (x9)
  • Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
  • Sat each plate for about 15mins to the solution solidify.
  • The remaining solution that was left in the 50mL vial was used for our control.
    • Added TA and poured that solution onto the last plate.
      • Side note: the control solidified <15 minutes.

Observations:

The experiment performed 9/21/18, the control of the experiment was contaminated. This led to the uncertainty of the plaque assays, whether or not it had plaques or not. Because of the results, the experiment was done again since the LB broth was contaminated from the last experiment. Not everyone’s control was contaminated, but only a few groups.

Next steps/Conclusions:

On Wednesday, determine wheater the plaques are plaques and make a webbed plate. Determine if the plate has a high titer or a low titer simply by doing some calculations. If the results are negative, pick a plaque from the second round of purification, or simply pick new soil.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
September 26

Soil Washing 09.24.18

Rationale)

To collect another soil sample and wash it, creating an enriched isolation that can be used to potentially isolate a phage. I will also collect metadata in regards to the soil sample I collected.

Results from Friday)

The results of the plaque assay were negative and the top agar control showed bacterial growth indicating contamination.

Procedures)

  1. Setup an aseptic zone
  2. Collected Soil D in a bag labeled “NMN 9.24.18 Soil D”
  3. Added 2mL of Soil D to a 15mL vial labeled “NMN Soil D+ 9.24.18”
  4. Added 10mL of LB broth to the vial, shook for 10 minutes. Took the mass.
  5. Centrifuged for 10 minutes
  6. Used a .22 micron syringe filter to filter the supernatant into a 50mL conical vial labeled “NMN 9.24.18 Soil D Enriched Lysate”, filtered .5mL into a micro test tube labeled “NMN 09.24.18 FDL”
  7. Added .5mL of arthro to the “Enriched” vial
  8. Placed the “Enriched” vial onto the shaker table for 48 hours.
  9. Refrigerated the “FDL” tube for 48 hours.
  10. Stored the bag with Soil D in the walk-in refrigerator.

Observations/Data)

The mass of the tube “NMN Soil D+ 9.24.18” was 18.336g. My plaque assay was negative with no plaques being formed and our top agar control showed bacterial colonies growing throughout. Once I stopped shaking my soil sample, sand began to settle out immediately.

Conclusions/Next Steps)

The next step will be to conduct a plaque assay and a spot test with the lysates produced from Soil D in the effort to isolate a phage from Soil D, in addition to collecting metadata on Soil D.

 

Category: Nathan Newton | LEAVE A COMMENT
September 26

Picking a “Plaque” and Plaque Assay 09.21.18

Rationale)

To check my plaque assay and spot test from Wednesday, if the result is positive the plaque(s) will be picked and a lysate will be produced from the plaque and used to conduct a spot test, if the result is negative the plates will be disposed of another soil sample will be collected.

Results from Wednesday)

The plaque assay and spot test from Wednesday came back with a possible positive, as the plaque assay possibly contained a plaque which could have also been an air bubble.

Procedures)

  1. Checked plates from Wednesday
  2. Setup an aseptic zone
  3. Labeled a micro test tube “NMN 9.21.18 PPL”
  4. Filled tube with 100 microliters of phage buffer.
  5. Picked the possible plaque in the plaque assay and swirled in the phage buffer to transfer, vortexed the tube to combine.
  6. Labeled 50mL conical vial “NMN 9.21.18 Possible Plaque Assay Soil C” and other one “Top Agar Control for PA 9.21.18”
  7. Added 2mL of LB broth to both vials
  8. Added 22.5 microliters of 1M CaCl2 to both vials
  9. Added 10 microliters of “PPL” to a .5mL vial of arthro, let infect for 15 minutes.
  10. Collected two plates labeled one “9.21.18 Top Agar Control NMN, HMB” and the other “NMN 09.21.18 Possible Plaque Plaque Assay
  11. Added 2.5 of 2xTop Agar to the control tube, swirled the tube and poured into the control plate. Let sit for 15 minutes then inverted the plate and incubated until Monday.
  12. Added .5mL of infected arthro to the PA tube
  13. Added 2.5mL of 2xTop Agar to the PA tube, swirl the tube and pour into the plaque assay plate. Let the plate sit for 15 minutes then invert and incubate until Monday.

Observations/Data)

I observed that the top agars contained thin filaments and the LB broth and top agar we used appeared to be from of contamination as based upon their high clarity. I also observed possible air bubbles in the plaque assay I performed which I subsequently marked with a sharpie to make sure misidentification did not occur.

Conclusions/Next Steps)

Based upon the possible positive result from my plaque assay on Wednesday I picked the supposed plaque and conducted another plaque assay using lysate produced from the plaque. The next step will be to check to see if the plaque assay comes back positive or negative, if positive purification will continue to occur by picking a plaque and performing another plaque assay, if the result is negative another soil sample will be collected and washed in the hopes of eventually isolating a plaque.

Category: Nathan Newton | LEAVE A COMMENT
September 26

9.24.18 Obtaining and Washing New Soil Sample

9.24.18 Obtaining and Washing New Soil Sample

Results from Friday, 9/21: As suspected, the plaque assay revealed that there was no phage, indicating that the small clearings were likely caused by competition among bacterial groups or other sources. Therefore, Soil Sample C is officially negative. The control plate also was contaminated yet again, and similarly to Wednesday’s (9/19) sample, it is believed that the cause stems from a contaminated top agar or LB broth.

Rationale: Since the results from the test on Friday showed contamination on the control plate and only an extremely small mark on the plate, it was concluded that a new sample would be needed to restart the process. The washing procedure was also fit into the lab session after obtaining a new soil sample (Soil Sample D) to be ready to perform a plaque assay and spot test on Wednesday.

Procedure:

  1. Establish an aseptic zone.
  2. Collected new sample of soil from tree at Teal Residence Hall. Recorded metadata in survey.
  3. Added 2mL of soil to tube (“HMB Soil Sample D 9/24/18”)
  4. Added 10mL of LB Broth to tube
  5. Shook for 10 minutes
  6. Centrifuged sample for 10 minutes
  7. Used syringe and 22μL filter to filter supernatant of centrifuged sample. Placed into tube labeled “HMB Soil Sample D 9.24.18”.
  8. Moved 100μL of filtered lysate to microcentrifuge tube for a direct isolate.
  9. Added 0.5mL of Arthrobacter to “HMB Soil Sample D 9.24.18” tube to create enriched lysate.
  10. Moved enriched lysate to shaker to be used on Wednesday 9/26.
  11. Moved direct isolate to fridge to be used on Wednesday 9/26.

Observations:

  • Control Plate still contaminated using the last LB Broth and Top Agar combination. Will be determined if it is the cause when next plaque assay is completed, as new Top Agar and LB Broth solutions have been created.
  • Soil was very dark and already began settling out sand particles before centrifugation. Through this, it can be predicted that there will be a higher proportion of sand than normal.
  • Mass of tube with soil and LB Broth was equal to 18.376g as measured before being placed in the centrifuge.

Next Steps:

  • The enriched and direct lysates will be used for a plaque assay and spot test. These tests will show accurately whether or not phages are present in the soil obtained. Furthermore, it will be necessary to take extra care when using the new Top Agar and LB Broth to avoid contaminating the fresh samples, as this will have adverse effects on the control plates certainly and potentially the experimental plates as well.
Category: Henry Burns | LEAVE A COMMENT