September 25

9.21.18 Possible Phage Analysis

9.21.18 Possible Phage Analysis

Results from Wednesday, 9/19: Plaque Assay experimental plate shows one clearing that could have been caused by phage. Both the control plate predicted to show contamination and the control plate expected to not show contamination showed contamination. Spot test showed a very small clearing that appeared similar to the one found on the plaque assay.

Rationale: Results from Wednesday, 9/19 showed that there was a very small clearing on the Plaque Assay test completed. Therefore, to determine whether or not a phage was the cause of the clearing, a new plaque assay was completed to test the composition of the clearing.

Procedure:

  1. Aseptic zone established.
  2. Plates were removed from incubator and analyzed.
  3. 100μL of Phage Buffer was placed in microcentrifuge tube (“HMB PPL”)
  4. Pipette tip was used to take up plaque from plate, added to phage buffer and mixed.
  5. 2mL LB Broth added to control and experimental tubes
  6. 22.5μL CaCl2 added to control and experimental tubes
  7. 10μL of Phage Buffer and Possible phage solution added to Arthrobacter. Let sit for 10 minutes.
  8. Added 2.5mL top agar was added to control tube and plated mixture after swishing about.
  9. 0.5mL Arthro and possible phage solution added to experimental tube
  10. Added 2.5mL 2X Top Agar to experimental tube. Swished. Plated overlay. Let sit for 15 minutes before incubating until Monday 9/24.

Observations:

  • Both control plates that were used showed signs of contamination. One of the control plates was expected to show contamination, but the other should not have. This likely would have been from contaminated 2X Top Agar solution, which was not changed between the two samples.
  • A small clearing was observed on the plaque assay. It appeared to be turbid, and its size was quite small. These factors made it uncertain whether or not a plaque was present because there was not an obvious presence.

Next Steps:

  • The next step for this experiment is to observe the results on Monday and follow the next proper step in picking and diluting (if plaques are present) or obtain a new soil sample (if no plaques are present on the plaque assay).

Conclusions:

  • Systematic contamination of control plates is likely occurring from repeated use of contaminated LB Broth and Top Agar. Many of the control plates completed in group 3 in the past weeks have been contaminated in addition to other incidents of contamination across the laboratory , so it is not unrealistic to conclude that it is likely that there is a common source.
  • There likely will not be a plaque on the plate on Monday. While it is possible that a phage caused the small clearing or plaque, it is strange that there was not any other sign of phage. Furthermore, the contaminated control plate introduces a situation where bacterial competition could have caused the clearing. Therefore, it was necessary to test with another plaque assay to determine whether or not phage was present.
Category: Henry Burns | LEAVE A COMMENT
September 24

Lab Day 9: Spot Test + Plaque Assay

Rationale

Perform both spot test and plaque assay from Soil C without any trace of contamination. Record results of soil metadata from last lab day to determine the type of soil Soil C is. Two members from group 6 did spot test while one did plaque assay. Each test had an individual control plate.

Detailed Procedure

  1. Syringe filtered enriched lysate.
  2. Took 10 microliters of filtered enriched lysate and added to 0.5 mL Arthro. Sat for 15 mins.
  3. During 15 mins, took 4.0 mL LB Broth, 45 microliters of 1M CaCl2, and 5.0 mL of 2X TA into separate 50 mL vial. Mixed through pipetting.
  4. Used sterile pipette and took half of solution from step 3 into another separate 15 mL vial. Labeled as control.
  5. After 15 mins were done, took solution from step 2 and mixed through pipetting into previous 50 mL vial.
  6. Took both vials into water bath and waited for plate to be ready.
  7. Labeled plates as “Plaque Assay” and Plaque Assay Control.”
  8. Took control vial and poured into control plate and same with other plate+vial.
  9. Waited 15 mins to solidify.
  10. Inverted plates into incubator.

Conclusions/Results/Observations

  • Clay= 75%
  • Silt= 18.75%
  • Sand= 6.25%
  • Soil C is clay based sand

Due to more experience, there were no bubble on both plates and was able to prevent possible contamination (accidental spills, not being in aseptic zone.) In the next lab day, future steps would be to check for any signs of plaque. Since within the group, both spot test and plaque assay was performed, so if no plagues were found, then a new soil will be tested next.

Category: Soo-Un Jeong | LEAVE A COMMENT
September 24

September 24th 2018 Plaque Assay and Soil Metadata- Soil C

Rationale: The purpose of this lab is to perform a plaque assay with Soil C to isolate possible plaques, and to collect soil metadata.

Description of Procedures:

  1. The work station was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
  2. 10 ul of lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
  3. The top agar solution was then made with 8 ml of LB broth and 90 ul of CaCl2, to make enough top agar for four plates. The 2x TA was not yet added, because it would solidify before the arthrobacter had sat long enough.
  4. Next, the % water was calculated. The dry soil was found to be 5.331 g. Therefore the mass lost was 0.519 g. With the original mass of 3.53, the % water was calculated to be 14.7%.
  5. The composition of the soil was determined next. Out of the 7 ml of soil, 5.5 ml were sand, 1.0 ml was silt, and 0.5 ml was clay. The percent compositions are listed under data.
  6. Next, 10 ml of 2x TA was added to the TA solution.
  7. 4.5 ml of the top agar solution was added to each tube of arthrobacter, and this mixture was quickly poured onto a petri dish. The rest of the top agar solution was poured onto a control plate. The experimental plate was labeled LIP 9-24-18 Soil C, and the control was labeled Control LIP SS EAG 9-24-18.
  8. The plates were allowed to sit for 15 minutes and then inverted and stored in the incubator until the next lab.
  9. The work station was cleaned using aseptic technique and all materials were stored or properly disposed of.

Observations/Data/Results:

  • Observation:
    • When the plates were poured, there were less bubbles than before. Only one was seen on the experimental plate.
    • The control plate had more bubbles.
  • Data:
    • % Water- 14.7%
    • % Sand- 78.6%
    • % Silt- 14.28%
    • % Clay- 7.14%

Interpretations/Next Steps/Conclusions:

The plaque assay and soil metadata were completed for Soil C. The next step will be to check for plaques in the next lab. If plaques are found, they will need to be picked. If no plaques are found, a spot test will be performed to test Soil C once more.

Category: Lucy Potts | LEAVE A COMMENT
September 24

Plaque Assay 9/24/18

Rationale: Conducting a plaque assay to see if the soil sample I collected has a phage in it. Then we can move on to the next steps of purification.

Procedure:

  1. Pipetted 10μL of lysate into 0.5mL of arthrobacter. Then waited for 10

    minutes before beginning to make top agar.

  2. Using aseptic technique and fresh pipettes each time put 10mL of LB broth

    in a 50mL tube, then 112.5μL of CaCl2, then 12mL of 2X TA.

  3. Pipetted 4.5mL into tube with arthrobacter and lysate and pipetted up and

    down to mix.

  4. Poured contents into plate while keeping the top as close to the bottom as

    possible as to not have any contaminants fall in.

  5. Waited 10 minutes to solidify then inverted and incubated for 48 hours.

Observations:
The positive control ran by Lathan turned out negative meaning that the arthrobacter supply was contaminated. However, our negative control plate still turned out contaminated.

Interpretations and Next Steps:
There must be some other source of contamination for the control sample as it looks the same as the last plate which was contaminated. We need to find the cause of this because it will throw off our results every single time. (After looking back from 9/26 lab the cause of contamination was the contaminant in the TA.) Now we have to make another plaque assay next class since the arthrobacter we used wasn’t actually arthrobacter since it was contaminated by another bacteria.

Category: Sriram Avirneni | LEAVE A COMMENT
September 23

Metadata for Soil C (09/21/18)

Rationale:

In open lab, the procedures to collect metadata such as percent water, percent sand, percent silt, percent clay, and pH will be performed.

Procedure:

  1. Started off by cleaning the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Next, 10 mL of soil C was placed into a falcon tube labeled “KEA 9/21/18 Soil C.”
  3. The “KEA 9/21/18 Soil C” falcon tube was filled with de-ionized (DI) water to the 30 mL mark.
  4. Three drops of texture dispersion liquid were added to the “KEA 9/21/18 Soil C” falcon tube.
  5. The “KEA 9/21/18 Soil C” falcon tube was covered by a glove and shaken for 30 seconds.
  6. In a weigh boat, approximately 3 grams of soil C was added and weighed.
    • This weigh boat was labeled “KEA 9/21/18 Soil C.”
  1. Both the “KEA 9/21/18 Soil C” falcon tube and “KEA 9/21/18 Soil C” weigh boat were placed under the flume hood.
  2. To test the pH, a small amount of soil C was placed into a pH vial and it was filled the rest of the way with DI water.
  3. The pH vial was shaken for 10 seconds and then settled out for 2 minutes.
  4. Then, pH paper was used to determine the pH of soil C.
  5. The pH vial was rinsed out with DI water and returned.
  6. The counter area was cleaned with CiDecan and EtOH (70%).

Observations and Metadata:

  • The empty weigh boat weighed 3.40 grams. The weigh boat with the wet soil sample weighed 6.39 grams.
  • The pH of the soil sample was 6.00. The picture below shows the pH paper used.

Next Steps:

On Monday, the rest of the soil metadata will be determined. Also, a plaque assay will be run to test whether or not there is a bacteriophage in the soil sample that specifically targets Arthrobacter.

Category: Kathryn Adkins | LEAVE A COMMENT
September 21

Spot Test Using Plaque Assay and Soil Washing 9/19/18

Rationale

A spot test will be conducted using the possible plaques obtained from the plaque assay on 9/17/18. More soil and soil metadata will be collected as well.

Procedure

  1. Use the aseptic technique to clean the area.
  2. 10o µL of phage buffer was pipetted into 3 micro test tubes. The tip of a pipet was then placed into each possible phage from the previous plaque assay and the placed back into each micro test tube. Set aside the tubes.
  3. Pipet 2 mL of LB broth into a vial along with 0.5 mL of Arthrobacter, 22.5 µL of CaCl2, and 2 mL of 2X TA. The vial was poured onto a plate and the plate sat for 10 minutes.
  4. After 10 minutes had passed, 5 µL of each micro test tube was micro pipetted onto each quadrant of the petri dish, along with 5 µL of phage buffer into a control quadrant. The plate then sat for another 10 minutes.
  5. New soil was collected by lab partners during this time.
  6. Soil washing then began by placing 2 mL of soil and 9 mL of LB broth into a vial. The vial was shaken for 10 minutes to mix.
  7. The vial was then massed and centrifuged for 10 minutes.
  8. While the soil was centrifuging, % water calculations began by massing a weigh boat, adding soil, and then massing the weigh boat again. The weigh boat was then placed in the fume hood for 48 hours.
  9. The percent sand/silt/clay of the soil was also measured by adding 10 mL of soil to a falcon tube. The tube was filled to the 30 mL mark with DI water and 3 drops of soil dispersion liquid was added. The tube was covered and shaken for 30 seconds. After, it was placed in the fume hood for 48 hours.
  10. To measure the pH, 1 mL of soil was placed into a vial and then filled with DI water. The vial was shaken for 30 seconds and then sat for 10 minutes. After 10 minutes passed, pH paper was put in the vial and the pH was measured.
  11. After the tube containing soil was centrifuged, the supernatant was filtered using a syringe and syringe filter to produce 6.5 mL of enriched isolation.
  12. 0.5 mL of Arthrobacter was added and the tube was incubated.

Observations

The mass of the tube before being centrifuged was 17.18 g. The mass of the weigh boat was 2.32 g and the mass of the soil and weigh boat was 6.20 g. The pH of the soil was 6. This soil contained more debris than the previous soil collected.

Conclusions

The results of the spot test will be observed on Monday. From there, we will see if the previous soil sample can be used or if the new sample will be need to be tested.

Category: Emily Gaw | LEAVE A COMMENT
September 21

Lathan’s Questions week of 9/21/2018

Rationale: answer Lathan’s questions

  1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaques assays but only Justin had plaque on his spot. What do you think is going on?

Justin probably has the highest concentration of phage in his sample

2. Lathan checked a purified lysate by doing a plaque assay (10 µL of lysate) of a 10^(-3) lysate. He counted 14 plaques. How               many µL of Lathans 10^0 lysate should he add to web a plate (75 mm in diameter) if his average plaque diameter is 1mm?

( 14 pfu / 10 µL   ) * ( 1000 µL / 1 mL ) * 10^3 = 1.4×10^6 pfu/mL

Area of plate / Area of plaque = ( pi * 37.5^2 ) / (pi * 0.5^2) = 5625 pfu

5625 pfu / 1.4×10^6 pfu/mL = 0.0040 mL = 4 µL lysate

Category: Uncategorized | LEAVE A COMMENT
September 21

Plaque Assay and Sand/Silt/Clay Metadata 9/17/18

Rationale

A plaque assay will be conducted and data from the sand/silt/clay metadata experiment will be analyzed.

Procedure

  1. Use the aseptic technique to clean the area.
  2. While working near a flame, 10 µL of filtered enriched lysate was added to a tube containing 0.5 mL of Arthrobacter. The tube was set aside for 10 minutes.
  3. 8 mLs of LB broth was placed into a vial along with 90 µL of CaCl2 and 10 mL of 2X TA.
  4. 4.5 mL of the contents in vial was pipetted into the vial containing the filtered enriched lysate and then was quickly poured onto a petri dish. It sat for 10 minutes.
  5. When the plate was sitting, the percent sand, silt, and clay in the soil was analyzed.
  6. After the plate sat for 10 minutes, it was inverted and placed in the incubator.

Observations

Air bubbles did occur during the plaque assay but they were minimal and occurred near the edges. There were also multiple layers of sand, silt, and clay as shown below and the different types were grouped together for analyzation purposes.

Total amount of soil: 10 mL

Total amount of sand: 8.25 mL

Total amount of silt: 1.25 mL

Total amount of clay: 0.5 mL

% Sand: 82.5%

% Silt: 12.5%

% Clay: 5%

Conclusion

The previous spot test conducted produced no plaques, therefore a plaque assay was conducted. The results of the assay will be observed on Wednesday, hopefully with the presence of plaques. If no plaques are produced, more soil will be collected and the process will start again

Additional Questions

  1. The tree that Justin got his soil from may have a slightly different bacteriophage than the trees that Michael and Cooper used, which led to the difference in plaque presence.

(14 pfu/10 µL) x (1000 µL/1 mL)=1400 mL

1400 mLx10^3=1400000

(37.5^2)π/(0.5^2)π=5625

5625/1400000=0.004017 mL

0.004017×1000=4.01 µL

Category: Emily Gaw | LEAVE A COMMENT
September 21

9-19-18 — Plaque Picking and Serial Dilutions

Date: Wednesday, September 19th, 2018

Title: Plaque Picking and Serial Dilutions

Rationale: The purpose of today’s lab is pick a plaque from the plaque assay and set up serial dilutions that can be plaque assayed further.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

  1. An aseptic zone was set up.
  2. Plaque assays were evaluated and plaques were marked on the plate.
  3. 100 microliters of phage buffer were added to a microcentrifuge tube.
  4. A pipette tip was touched into a plaque and swirled in the microcentrifuge tube to add phage to solution. This is the 10° serial dilution.
  5. The 10° tube was shaken to mix phage with buffer.
  6. 90 microliters of phage buffer were added to each of two other microcentrifuge tubes.
  7. 10 microliters of the 10° dilution were added to one of the tubes, marked as the 10^-1 dilution. This tube was then shaken.
  8. 10 microliters of the 10^-1 dilution were added to the last tube marked as the 10^-2 dilution. This tube was also shaken.
  9. 10 microliters of each dilution were added to different culture tubes with .5 mL ATC 21022 each.
  10. Agar was made using the following recipe:
    1. 20 mL LB broth
    2. 25 mL 2x Top Agar
    3. 225 microliters 1M CaCl2
  11. 4.5 mL of the TA solution was added to each culture tube.
  12. The culture tubes with bacteria, phage, and top agar were briefly vortexed to mix the solution.
  13. The contents of the culture tubes were added to their corresponding plates based on their dilution number.
  14. The plates were left for 15 minutes to harden before being inverted and incubated.

Observations: The control for the plaque assays was contaminated, much like the control for the spot tests before. It’s still unclear what is causing the contamination, but it’s likely that arthro is somehow getting into the TA control.

Results: The plaque assay from before yielded positive results, with upwards of 20 plaques. This was confirmed not to be contamination, and hopefully this yields actual phages that can be isolated and sequenced.

Next Steps: The next step if the serial dilutions come back positive is to web a plate and further explore the phage. The next step if the serial dilutions plaque assays come back negative is to start working on Soil Sample C. Alternatively, the next step could be to pick a second plaque from the positive plaque assay and perform serial dilutions on it.

Category: Brandon Reider | LEAVE A COMMENT
September 21

9/19/18 2nd Purification Plaque Assays – Soil B

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.
Category: Claire Wentzlaff | LEAVE A COMMENT