11.26.2018 DNA Extraction Initiation

Rationale: Since results observed by Lathan and sent to me showed that the lysate possessed had a titer of 1e9, the lysate was officially a high titer. Therefore, it is possible to perform the DNA extraction procedure on the lysate to progress with analysis of bacteriophage as part of Claire’s group. Also, since the TEM was available for use, it was found to be beneficial to visualize the bacteriophage using the microscope. The DNA extraction procedure will allow for the genome of the phage to be analyzed if it is selected by the class.

Procedure:

1. Aseptic zone established
2. 10mL of lysate labelled “HMB from CEW 10^0 Lysate 11/14/18” was placed into a conical tube.
3. 40µL of nuclease mix was added to the lysate
4. 4mL of phage precipitant solution was added to the lysate
5. Lysate was placed on shaker at 35 degrees Celsius for 30 minutes
6. Lysate was let sit at room temperature for 45 minutes
7. Lysate was centrifuged at 7,500g for 20 minutes.
8. Eliminated supernatant from sample and rinsed sink.
9. Stored in refrigerator overnight.
10. Cleaned bench.

Results:

• These results date back to Monday, November 19 and the procedures performed on this day. As seen, the lysate was strong enough to completely lyse the spots through the 10^-5 dilution, and had plaques form through the 10^-7 dilution. After calculation, the lysate was found to be 1×10^9 pfu/mL. Therefore, a high titer lysate had been obtained! The control also showed no signs of contamination, which confirms that the results could be considered valid.
• The electron microscope revealed very present bacteriophages. The phages had a head with a diameter of 52nm and a tail of 100nm.

Observations:

• After the phage precipitant solution was added, the lysate appeared to have smaller particles or granules appeared to be seen. They dissipated more with shaking.
• Phages appear on the TEM in the shape of a lollipop with a dark head and banded tail.
• TEM showed phages with a grainy background. Small movements had large effects, and the images moved slightly due to how the electrons interacted with the plastic film.

Conclusions/Next Steps:

• Since there were plaques present on the 10^-7 dilution, it can be concluded through calculations that there is a high titer lysate present. This is helpful in furthering the processing of the lysate, and allowed the DNA extraction process to begin. The TEM also showed the morphology and size of the bacteriophages from the sample. Since procedures today did not show definitive results, no further conclusions are available to be reported. The next steps in this procedure would be to finish the DNA extraction process, which will be done on Wednesday.

11.14.18 Dilutions and Spot Test for Titer

Rationale: Since plates overnight had lysed all of the arthrobacter on the plate, phage buffer was added to the plates to flood them to collect bacteriophage that was present on the plate. Today, it was necessary to process this by creating dilutions and running a spot test to determine titer, as it is necessary to discover whether or not a high titer lysate has been obtained.

Procedure:

1. Aseptic zone was established
2. Filter column was used to filter the phage buffer from the plate into a tube labeled “HMB from CEW 10^0 lysate 11/14”
3. Dilutions from 10^0 to 10^-9 were created by adding 90µL of phage buffer to 9 different tubes and adding 10µL of the previous dilution into the tube (10µL 10^0 into 10^-1, 10µL 10^-1 into 10^-2, etc)
4. 2mL of LB Broth was added to a tube containing 0.5mL Arthrobacter
5. 22.5µL of CaCl2 was added to the tube with Arthrobacter
6. 2.5mL 2X Top Agar was added to the tube with arthrobacter, then poured over a plate with 9 distinct sections labeled after briefly swishing
7. Plate was let sit for 10 minutes
8. 10µL of each dilution was added to plate on the respective section in the form of a drop
9. Plate was let sit for another 10 minutes, then placed in the incubator overnight.

Observations:

• 2X Top Agar used was not at the optimal temperature, which led to some slippage of the overlay. Many of the 2X Top Agars in the incubator were contaminated however, so options were limited.
• Drops seemed to absorb better than they have in previous spot tests, which could be a result of the problematic top agar overlay.
• Overlays that are likely to slip seem to be darker yellow in color and have more grainy surfaces.

Results:

• Plaque Assays from Monday (11/12) resulted in completely lysed plates through the 10^-3 dilution. This is possibly indicative of a high titer presence, which is the end goal of the experiment. Hopefully, as results are seen on Thursday (11/15) or Friday (11/16), it will be possible to view plates that confirm the presence of a high titer.
• 

Next Steps and Conclusions:
If the spot test is functional despite the slightly slipped Top Agar, it will be possible to view the results that show the presence of a high titer or not. If the overlay is not functional, it will become necessary to retry the overlay with a new combination of fresher, uncontaminated Top Agar and LB Broth. This will allow for definite conclusions to be made about the titer of the lysate.

11.12.18 Plaque Assay for Lysate from Claire

Clarification: The lysate used for experiments on Monday (11/12) was generated from plates that were flooded on Thursday (11/8). The lysate originally came from Claire’s positive lysate, and the original lysate was diluted to 10^-2. Plaque assays were made with this dilution on Wednesday (11/7), and the plates were flooded on Thursday (11/8) to make the lysate that was used for today’s (11/12) experiment.

Rationale: Since the plates that contained Claire’s lysate had been flooded by Emily on November 8, a lysate that was ready to plate was obtained. Therefore, the next step in obtaining a high titer was using plaque assays to visualize the results that would indicate whether or not the new lysate had become a high titer through the processing steps.

Procedure:

1. Aseptic zone was established.
2. Claire diluted the original lysate that was obtained from Thursday (11/8) by placing 90µL of phage buffer in 3 separate tubes and adding 10µL of sample to the first tube. 10µL of this new sample was added to the next tube, etc.
3. **For the rest of this procedure, each member of Claire’s team completed these steps independently**
4. 10µL of each of the 4 dilutions (10^0, 10^-1, 10^-2, 10^-3) was added to 0.5mL arthrobacter and let sit for 15 minutes
5. 10mL of LB Broth was added to a conical tube
6. 90µL of CaCl2 was added to the conical tube
7. 2mL of the LB Broth and CaCl2 solution was added to each tube with the arthrobacter and sample.
8. 2.5mL 2X Top Agar was added to each of the arthrobacter tubes
9. Each plate was labeled with the date, dilution of the sample, my initials, and Claire’s initials to indicate where the lysate came from.
10. Plates were let sit for 10 minutes, then placed in the incubator overnight.
11. Station was cleaned

Observations:

• No top agar overlay solutions resulted in any slipping or tearing. Measurements were more precise than in the past, so this contributed to preventing the slippage.
• Samples spent slightly more time infecting arthrobacter than normal. This could contribute to a more lysed plate than normal, but will not be confirmed until Wednesday (11/14).

Results:

• Since no plates were made after the lysate was flooded, I had no results to report. Results from today’s (11/12) plates will be able to be visualized during Wednesday’s (11/14) lab session.

Conclusions and Next Steps:

• After flooding the plates on Thursday in an attempt to increase the concentration of phage, it can be hypothesized that after another cycle of phage buffer flooding that the concentration will have gone up. The next step after creating a plaque assay to support or reject this hypothesis will be to examine the plates on Wednesday and determine the titer of the lysate used. This will allow for specific results and conclusions to be obtained from the procedures completed today (11/12).

10.31.18 Gel Electrophoresis for Claire and Lucy PCR

Rationale: Since the samples from the lysates of Claire and Lucy’s positive samples had been processed by PCR, it is now possible to visualize the results through gel electrophoresis and imaging. Therefore, the next logical step would be to use the gel procedure to do this.

Procedure:

1. Added 35mL 1X TBE to two separate Erlenmeyer flasks.
2. Added 0.7g Agarose to both flasks, then heated until bubbles formed. Once bubbles formed, tube was shaken until bubbles disappeared. Repeated until solution was created.
3. Let cool to about 50°C.
4. Added 1.7µL EtBr to both Erlenmeyer flasks. Swirled to mix
5. Poured contents of flasks onto two separate gel apparatuses that contained combs to create holes in gel.
6. Once gel had set, a small amount of TBE buffer was added to the top of both apparatuses. Comb and rubber seals were removed, and gel was set in machine.
7. 1X TBE was poured over the top of the gel and wells until gel was completely submerged.
8. 2.5µL dye was added to each sample (1-3 Experimental tubes).
9. 5µL of ladder was added to both gels (Well 1)
10. 10µL of each experimental sample was placed in wells 2-4.
11. Power source was attached to apparatus and activated. Let run for 45 minutes.
12. Used imaging to view results.

Observations:

• The ladder seemed quite faint compared with the samples in the wells.
• The samples were not allowed as much time to move down the gel as they were in previous experiments, as the timing worked better for us to stop ours slightly early to correspond with other gels being run.

Results:

• The samples displayed negative results on the imaging tool. There was no banding that was displayed above where the dye ran to on the gel, which would have indicated a positive result.

Henry, Nathan Gel 10-31-189ldnx

Conclusion and Next Steps:

• Since the PCR procedure coupled with Gel electrophoresis yielded yet another negative result on a known positive sample, it can be concluded that there must be something wrong with the procedure being used. This would explain why no positive results had been obtained, and a solution is required before PCR testing can be considered reliable once more. The next step will be to do work in the mini-teams assigned by Lathan. We will be helping Claire achieve a high titer lysate with her plates.

10.29.18 PCR for Claire and Lucy

Rationale: Since no phage had been obtained from any samples previously run, it was found most pertinent to help with other samples that had more promise of developing a high titer lysate. Therefore, a PCR test was run for samples from Claire and Lucy to help verify the PCR procedure was working properly.

Procedure:

10.24.28 Gel Electrophoresis Sample E

Rationale: Since sample had been put through the process of PCR, the results of whether or not phage DNA is present can be processed by using gel electrophoresis and matching bands to visualize results. Therefore, it was necessary to use the gel electrophoresis process to determine the presence of phage.

Procedure

1. Added 35mL 1X TBE to two separate Erlenmeyer flasks.
2. Added 0.7g Agarose to both flasks, then heated until bubbles formed. Once bubbles formed, tube was shaken until bubbles disappeared. Repeated until solution was created.
3. Let cool to about 50°C.
4. Added 1.7µL EtBr to both Erlenmeyer flasks. Swirled to mix
5. Poured contents of flasks onto two separate gel apparatuses that contained combs to create holes in gel.
6. Once gel had set, a small amount of TBE buffer was added to the top of both apparatuses. Comb and rubber seals were removed, and gel was set in machine.
7. 1X TBE was poured over the top of the gel and wells until gel was completely submerged.
8. 2.5µL dye was added to each sample (1-3 Negative Controls, 1-3 Positive Controls, 1-3 Experimental, and 1-3 Henry Experimental).
9. 5µL of ladder was added to both gels (well 4 on gel 1 and well 1 on gel 2)
10. 10µL of each negative control sample was added to the left side of the ladder (wells 1-3) on gel 1
11.  10µL of each positive control sample was added to the right side of the ladder (wells 5-8) on gel 1
12. 10µL of each experimental sample was placed in wells 2-4.
13. 10µL of each Henry experimental sample was placed in wells 6-8.
14. Power source was attached to apparatus and activated. Let run for 45 minutes.
15. Used imaging to view results.

Observations:

• The gel surrounding second negative control well was pierced, causing not all of the sample to be placed into the well. There was enough remaining in the well so the effects should not be overly dramatic.
• Gel had a blue tint when it was solid, helpful to know when it is discouraged to touch the gel that contains EtBr.

Results:

• The gels run showed no presence of bands that would indicate phage on both the negative control and experimental wells, but they showed a very present band on one of the positive control wells, a faint band on one of the positive control wells, and no banding on the third positive control.

Conclusions:

• Since no banding was seen in the experimental wells for the fifth soil sample, it is very likely that there is no phage present. There may be a problem with the PCR procedure, as there have been no positive results since the beginning of its use, but it is overwhelmingly likely if that is not the case that there are no phages in soil sample e. The next steps will be obtaining a new soil sample unless a known positive sample is put through the PCR procedure and results display negative. In that case, the sample would need to be redone with an improved PCR procedure. 

10.22.18 PCR Test of Soil Sample E

Rationale: Since more inconclusive test results were obtained and no filters were available to test another soil sample, it was found to be important to use PCR to definitively determine whether or not phage particles were present in Soil Sample E.

Procedure:

1. Aseptic zone was established
2. 4mL of soil sample F was added to a conical tube
3. 10mL of LB Broth was added to tube
4. Tube was shaken and vortexed for 10 minutes
5. Tube was centrifuged for 5 minutes to separate soil from supernatant
6. Attempted to filter using a 3 mL syringe and a 22µm filter. Filter did not fit, and no filters of proper size were present. Tube was labeled “HMB Soil F” and stored in refrigerator.
7. Obtained enriched lysate from soil sample E
8. 1mL enriched lysate E was added to a microcentrifuge tube
9. Microcentrifuge tube placed in 37°C container for 10 minutes to boil
10. Obtained 3 tubes with 12.5µL of Taq Polymerase in them. Tops labeled 1,2,3.
11. Added 7.5µL DDI water to each tube
12. Added 4µL Primer 1, Primer 2, and Primer 3 to corresponding tubes
13. Added 1µL of the boiled enriched lysate sample to each tube.
14. Thermocycled all tubes.
15. Cleaned and tidied bench.

Observations:

• While using the improper filter, it was easy to distinguish that the filter broke due to pressure. After the filter broke, white foam was observed on top of the sample that was forced into the conical tube. Therefore, it would be relatively safe to assume the foam was from the filter breaking. This foam was previously of unknown origin and was seen in a prior experiment.
• Mass of sample in the centrifuge was 22.767g.
• Soil added to conical tube was wet when placed in the freezer. Thus, it still retained a clay texture and while shaking, it was difficult to get all parts of the soil sample to be mixed with the LB Broth.

Results:

• The plates prepared after flooding the previous plate with unknown clearings showed no clearings other than tears in the top agar overlay. The control plate also showed signs of a different type of contamination than normal.

Conclusions:

• Since no clearings were seen on the flooded plate, it is generally thought that the plaques seen on the previous plate were due to contamination. However, since the control plate was yet again contaminated, results cannot be fully supported or disproved. Therefore, the PCR test should be useful in determining whether or not the plaques were due to phage in a setting where contamination will not have such a prevalent effect. The next step will be processing the PCR sample and running a gel to determine presence of phage.

10.17.2018 “Flooding” A Plate

Rationale: Since the plate that was produced through Monday’s tests showed many small clearings, it was found necessary to use a procedure to create a 10^0, 10^-1, and 10^-2 lysate solution to begin the process of purifying phages.

Procedure:

1. Aseptic zone was created
2. Top agar was scraped off of the experimental plate completed on Monday (10/15) and placed in a conical tube.
3. 10mL of phage buffer was placed in the conical tube.
4. Tube was shaken for one hour.
5. Centrifuged for 5 minutes at 4000rpm.
6. 1mL lysate obtained from centrifuged tube was added to a microcentrifuge tube (10^0 tube)
7. 10µL chloroform was added to the 10^0 tube.
8. 90µL phage buffer was added to two additional tubes (10^-1 and 10^-2), and the proper dilutions were made from the 10^0 tube.
9. 10µL of each tube was added to 0.5mL Arthrobacter and let sit for 15 minutes.
10. 8mL LB Broth placed in conical tube
11. 90µL CaCl2 was added to the same tube
12. 2mL LB Broth and CaCl2 solution was added to each of the three dilution tubes that contained 0.5mL arthrobacter
13. 2.5mL top agar was added to all four of the tubes. Tubes were swished, then plated on separate tubes with one being the control tube.
14. Plates let sit, then placed in incubator
15. Cleaned table.

Results:

The results from Monday show several small clearings on the plate that contained the retry of Soil Sample E. The control plate also displayed similar levels of contamination to the first time it was completed. No spot test was attempted because the top agar for only the plaque assay was contaminated, not the spot test. The small clearings appeared to be mostly uniform circles.

Observations:

• The top agar created today did not set well and appeared to have tears in it. This resulted in the plates being left right side up in the incubator.
• The control plate persisted to have contamination, so extra care was taken to ensure that the top agar used today was different and was very clear. Same protocol was used for the LB Broth.
• Lysate from after the top agar shaking process had some impurities in the lysate after centrifugation. This would have caused problems in a filter, but since none were able to be used an alternate procedure made the impurities not a problem.

Conclusion and Next Steps:

• Since the clearings on the plate appeared to be roughly uniform, the plate was flooded to obtain as much phage as possible as part of amplification. The next step will be to examine the plates the were prepared today to confirm firstly that phage is present and secondly to begin to determine the best method to create a high titer lysate.

10.15.18 Retry of Plaque Assay

Rationale: Since there was no plaque present on the plaque assay completed on Friday (10/12), but there was contamination on the control plate, it was necessary to retry the test to obtain a sample without contamination. The contamination could have been causing a false negative result, therefore it would be beneficial to ensure that the result was accurate with a repeated test.

Procedure:

1. Aseptic zone established.
2. 2mL of LB Broth was added to two tubes (control and experimental).
3. 10µL of possible phage/phage buffer solution to 0.5mL arthrobacter. Let sit for 15 minutes.
4. 22.5µL of CaCl2 was added to both tubes.
5. 2.5mL 2X top agar was added to control tube and was plated on control plate (“HMB Plaque Assay Control 10/15/18”).
6. 0.5mL arthrobacter/phage buffer/ possible phage solutoin was added to experimental tube
7.  2.5mL 2X top agar was added to experimental tube and plated on “HMB Plaque Assay 10/15/18”.

Results:

• The plaque assay from Friday (10/12) showed no sign of plaques or clearings. However, the control plate showed significant signs of contamination. The contamination was in isolated colonies, not lawn growth, but nevertheless it was still very present.

Observations:

• There was a small area of no growth on the control plate. This could have been due to uneven top agar spreading or an alternate source.
• There was a small amount of top agar overlay on the control plate created today that went up the side of the plate due to spreading. Important to note for further examination.
• Both top agar and LB Broth used appeared to be the correct color and clear, signifying that they are free of contamination.

Conclusions and Next Steps:

• Since there was contamination, there could be no official conclusions drawn about the contents of soil sample E. Further conclusions will be able to be made on Wednesday when it is apparent whether or not there are clearings if there is a control plate free of contamination to accompany the plaque assay. The next step is to reanalyze on Wednesday (10/17), and a soil sample will be obtained in the event that the negative result from today is confirmed.

10.12.18 Plaque Assay

Rationale: Since the plates that contained a spot test and plaque assay from Soil Sample E displayed phenomena that resembled plaque, it was necessary to pick the areas and complete another plaque assay to confirm the presence of phage.

Procedure:

1. Aseptic zone was established.
2. Results from tests on Wednesday (10/10) were examined.
3. Plaque was picked and placed in 100µL phage buffer, then vortexed.
4. 10µL of phage buffer was added to 0.5mL arthrobacter. Let sit for 15 mins.
5. 2mL of LB Broth was added to tube.
6. 22.5µL CaCl2 was added to tube.
7. 2.5mL 2X Top Agar added to tube.
8. Plated on “HMB Plaque Assay 10/12/18”. Let sit for 10 minutes, then placed in incubator.
9. 2.0mL LB added to Control tube.
10. 22.5µL CaCl2 to Control tube.
11. 2.5mL 2X Top Agar added to control tube and plated overlay solution on plate (“HMB NMN 10.12.18 TAC PA”). Let sit for 10 minutes, then incubated.

Results from Wednesday, 10/10:

• As seen in the included pictures, there are clearings on the plaque assay and there is no growth on the control plate. However, on the spot test, there are no clearings found on any of the three sections. There is a strange pattern between the phage buffer and the enriched lysate section.

Observations:

• When creating the plate with the possible phage, there appeared to be a white substance in the overlay solution that was plated.
• Overlay solution set quickly and appeared to be smooth before placing in the incubator.
• The clearings on the plaque assay appeared to be clear, not turbid. If there a phage is confirmed, these clearings were caused by the lytic cycle rather than the lysogenic cycle.

Conclusions:

• Since there were plaques on the plate, it is likely that they were caused by bacteriophage, as there was no contamination on the control plate. A test will be run to confirm, but if the plaques observed on the initial plaque assay reappear, it would be a safe assumption that phage was present. These observations and predictions will be supported or rejected on Monday (10/15) after the confirmatory plaque assay is done.