August 26

Soil Washing Part 2 8.24.18

Soil Washing Part 2 8.24.18

Rationale)

To continue the soil washing of Soil Sample A from 8.22.18 and achieve a direct isolation and an enriched isolation from the lysate produced, which can then be used in both a spot test and a plaque assay to determine the potential presence of phage in Soil Sample A.

Procedures)

1. Retrieve the two vials labeled Soil A from 8.22.18, which had been centrifuged by the TAs in the time between 8.22.18 and 8.24.18, and an additional .22μ filter.

2. Setup the filter under the fume hood and attach the vacuum, once setup open both vials of Soil A and using a pipette transfer the supernatant from the vials to the aperture of the filter apparatus and let filter into the 50mL conical vial attached.

3. Once filtration is complete and all the lysate is in the 50mL conical vial turn off the vacuum and open the sterile packaging of the lid to the vial. Remove the filter apparatus and quickly screw on the cap using proper technique to minimize contamination. Label the vial as NMN Soil Lysate A pt2, 8.24.18.

4. Retrieve a vial containing .5mL of arthrobacter and add it under non-aseptic conditions but with proper technique to the vial containing Soil Lysate A pt2 and quickly recap the vial. Rename the vial by adding “enriched” to its description and place the vial in the shaker.

5. Under a previously setup aseptic zone retrieve the vial of Soil Lysate A 8.22.18 and transfer its contents to a 15mL conical vial labeled Soil A Direct Isolation 8.24.18. Place this vial in the tray that is to be refrigerated by the TAs.

Observations)

The Soil A supernatant was much clearer after the second centrifuging compared to its original state, the supernatant was much more golden and translucent compared to the opaque and tan/cream colored supernatant before the second centrifuging. Soil Lysate A Enriched had about 10mL in it and Soil A Direct Isolation had approximately 5mL of lysate. There is a potential that contaminants could have gotten into Soil Lysate A Enriched when the arthrobacter was added under aseptic conditions.

Conclusions/Next Steps)

The next time I conduct soil washing greater care needs to be taken to make sure all critical steps are conducted in an aseptic manner to prevent contamination of the samples. Besides the potential that my sample was contaminated, I was able to create a Direct Isolation and Enriched Isolation from the filtration of the Soil A supernatant into lysate, which will be used in the next steps of bacteriophage isolation, plaque assays, and spot tests.

Category: Nathan Newton | LEAVE A COMMENT
August 26

8/22/18 Enrichment

Rationale: In this lab, the objective was to isolate possible bacteriophages from previously collected soil samples in labs before in a lysate.

Procedure:

  1. The first step of the experiment was to create a sterile environment using the aseptic technique. It is done so there is absolutely no contamination of the soil sample by preventing any additional microorganisms from entering the vials once opened.
  2. First, the lab bench was cleaned with cidecon and wiped dry. Following that, the bench was wiped with 70% ethanol and left to evaporate. Finally, to create an aseptic zone, a burner was lit to create a convection current of air circulating to prevent any microorganisms in the air to fall on the bench and contaminate our work space.
  3. Once the area was sterilized, LB broth was added to the soil sample up to the 35 mL mark in the 50 mL tube. This was done in the aseptic zone.
  4. After the LB broth had been added, the sample was shaken for approximately 15 minutes, it was also put on the vortex machine whenever I got tired of shaking. The sample does not need to be shaken vigorously, it is more important to keep the shaking continuous during the entire duration of the 15 minutes.
  5. After the 15 minutes were completed, I weighed my soil sample in its test tube so that I may find a partner with a similar weight for centrifugation of the soil samples.
  6. Once I had found a partner, our soil samples were inserted into the centrifuge, and spun at 3,000 revolutions per minute to separate the solid pellet from the hopefully bacteriophage full supernatant liquid at the top of the vial.
  7. The vials were then removed from the centrifuge and brought back to the classroom where the supernatant was run through a .22 micrometer filter to remove any impurities still left in the supernatant, leaving  15 milliliters of a yellow liquid behind called a lysate.
  8. The lysate was then split by two methods of isolation. The direct isolation method took 5 milliliters of the lysate and put it into a 15 milliliter vial (aseptic method again) and stored in the fridge. The enriched isolation took the leftover 10 milliliters of lysate and added 0.5 milliliters of our host arthrobacter to the vial, then stored it as well.

Observations:

  • The supernatant was a much lighter tone than the dark soil it came from.
  • The lysate was almost yellow, completely different from the black soil that we started with. This may have to do with the addition of the LB broth at the beginning of the experiment.

Results:

  • The experiment yielded two vials of lysate in the end, one with our bacterial host added into it, the other without it. The soil sample we began with had been purified and filtered to hopefully isolate a bacteriophage.

Conclusions/Next Steps

  • The next steps are to examine the lysate to see if we successfully isolated a bacteriophage within our soil sample. If there were any bacteriophages present, the arthrobacter introduced in the enriched isolation should have multiplied by using the bacterial host introduced into the vial. The next steps will be to analyze the enriched and direct isolation lysates to look for the presence of bacteriophages.
Category: Gabriel Andino | LEAVE A COMMENT
August 25

8.22.18- Washing of Original Soil Sample

Rationale: Washing Soil Sample A will rid sample of waste products like dirt and bacteria and allow for possible bacteriophages found in sample to be isolated in a lysate.

Procedure:

  1. Cleaned lab station using CiDecon (sprayed and fully dried) and 70% EtOH (sprayed, half-dried, and let to evaporate).
  2. Obtained and lit an ethanol burner.
  3. LB Broth (placed close to the burner to maintain aseptic technique) was poured into tube to 35 mL mark on Conical Tube.
  4. Using a combination of hand-shaking and use of vortex machine, LB Broth and Soil Sample A (from oak tree by Baylor Science Building) were mixed for 15 minutes.
  5. During mixing process, tube containing Soil Sample A and LB Broth was massed and found to be 53.454g. The purpose of this was to find another sample of similar mass (+/- 0.1g) to achieve symmetry in the centrifuge.
  6. After mixing, tube containing Soil Sample A and LB Broth was centrifuged at 3000xg rotations/minute for 5 minutes.
  7. Supernatant was added to top filter to remove bacteria and other particles from soil. Filtrate was obtained after it had passed through the white filter.
  8. 26mL of filtrate that passed through filter was split (about 13mL left in top filter conical tube, about 13mL half placed in new 15 mL tube).
  9. 0.5mL Arthrobacter was added to 50mL conical tube from top filter containing half of lysate to form an enriched isolation.  The other 15mL tube with half of lysate was left to act as the direct isolation.
  10. Enriched and direct isolations were left to be shaken until Monday, 8/27.

Observations:

  • After shaking process, overall volume appeared to have decreased from 35mL to 33mL – could have been due to soil absorbing LB Broth during shaking.
  • Mixture was dark brown and had many bubbles immediately after shaking.
  • After use of centrifuge, supernatant appeared to be a yellow-gold color that still had some soil particles and grass inside that was avoided when placing in the filter.
  • After placing 0.5mL of Arthrobacter in the enriched solution, it appeared that there was a very slight amount of Arthrobacter left in the tube that did not get added to the solution. Adverse results could eventually be explained by this, however, it appeared that generally it was common for others to encounter the same situation.

Results:

  • Procedure was accomplished without problems during creation of isolations. No issue with filter was had. Tangible results with more data will be possible to obtain on Monday (8/27) when a spot test could reveal any possible bacteriophages from sample.

Next Steps:

  • During the next lab time, isolations will be examined to see if bacteriophages were present in the sample obtained from the soil. This will be accomplished by the spot test procedure.
Category: Henry Burns | LEAVE A COMMENT
August 25

8/22/2018- Washing

8/20/2018

soil washing

Objective: todays goal is to extract the phages from the soil that was collected on 8/20/2018 , labeled soil sample A.

Supplies: sample collected on Monday, 50 ml test tube, centrifuge, LB Broth, pippetes

Procedure:1) set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%) ( wipe on the desk and let it evaporate) after clearing the table.

2) lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

3) take the tube filled with the collected dirt and add LB broth into the tube to 35 ml.

4) shake the test tube for 15 minutes using hand and vortex machine

5) put the test tube into the centrifuge  at 3000g for 5 minutes.

6) ran out of time so the sample was stored in the fridge to continue the process on Friday.

Results: the end product was the large particles deposited at the bottom of the tube. the solution was still very dark and dense.

the tree from which the soil sample was extracted is located below. the mass of the test tube after the LB broth was added was 51.105g and the LB  broth was poured into the solution at 3:09 pm.

Analysis: due to the dense nature of the supernatant  in the test tube, it seemed that it was going to take a long time to filter the lysate from the supernatant. the aseptic zone was properly created and maintained.

Future notes: manage time more effectively and work faster so as to finish this process at the same time. Keeping the supernatant solution idle for a few days might affect the time it takes to separate the lysate from the supernatant because some of the solid might mix again  with the part of the tube containing the phages.

 

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
August 25

08/24/2018- Enrichment

8/24/2018

enrichment

Objective: todays goal is to extract the phages from the soil that was collected on 8/20/2018, soil sample A.

Supplies: sample collected on Monday in 50 ml test tube, pipettes, tube top vacuum filter, shaking incubator, vacuum pump, 15 ml vials, 0.5 ml Arthrobacter host

Procedure:

1) set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%)( wipe on the desk and let it evaporate) after clearing the table.

2) lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

3) divide the supernatant solution into two 15 ml vials and put it into the desk centrifuge for 17 minutes.

3) set up the tube top vacuum filter, put in the vacuum tube and put in the supernatant from the two vials using a pipette .

4) turn on the vacuum pump and let the supernatant filter.

5) After filtration, take out the lysate(solution filtered) and dispose of the waste appropriately .

6) Keep 10 ml of lysate in the tube and move the rest into an 15 ml vial in the aseptic zone.

7) put the lysate in the 15 ml vial in the fridge for direct isolation.

8) add the Arthrobacter host to the 10 ml of filtered lysate for enrichment.

9) put the enriched sample into the shaking incubator at 38 degrees Celsius for 76 hours

Results: the end product was the large particles deposited at the bottom of the tube. the solution was still very dark and dense.

Analysis: the lysate was filtered faster than initially predicted due to the second time use of the centrifuge. the aseptic zone was properly maintained and so far no apparent events that could have caused contamination. the filtered lysate that was acquired was 15.5 ml. the diagram for the process is below

enriched sample is labelled: ADP 08/24/2018 enriched ; and direct isolation sample is labelled: ADP 08/24/2018

Future notes: dividing the supernatant into the two vials and putting them into the centrifuge helps a lot with the washing process. next lab we will be to do the spot test and observe the plaque assay in the petri dish, which will be made from the enriched sample to test the presence of phages in the soil. we will know if there are phages in the soil if there are blank spots on the dish because that would represent dead bacteria, which will be the bacteria that were infected by bacteriophages.

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
August 25

August 24, 2018 Direct Lysate- Soil A

Rationale: To create a direct lysate sample for the experiment from the soil, without adding arthrobacter.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Procedure:

  1. The sample of supernatant that had not yet been filtered was spun in a centrifuge for 5 minutes at approximately 4000 x g.
  2. This sample was then put into a 0.22 um filter using a pipette and filtered. Approximately 7.35 mL of lysate was obtained from filtering the supernatant.
  3. This lysate was then transferred into a 15 mL tube, not in an aseptic zone. This sample will be refrigerated until Monday and will eventually be plated.
  4. The used materials were then properly disposed of and the work station was cleaned.

Observations/Results/Data:

Observations:

  • After being spun in the centrifuge a second time, the filtering process was much quicker.
  • The supernatant was cleared than it had been before the second spin.
  • The transferring of the direct lysate to a smaller container was not done in an aseptic zone.
  • Approximately 7.35 mL was obtained of direct lysate.

Results:

The procedure from Wednesday was completed, with the direct lysate sample having now been obtained. This sample is approximately 7.35 mL.

The sample will be stored in the refrigerator.

The tube was labeled LIP 8-24-18 Direct Lysate

Interpretations/Conclusions/Next Steps:

The procedure was now complete. The second spin in the centrifuge made it possible to quickly filter the rest of the supernatant and obtain the direct sample. The next step is to shake the enriched lysate until Monday. On Monday, the next step will be a spot test to determine if any arthrobacter bacteriophages have been found.

 

Category: Lucy Potts | LEAVE A COMMENT
August 25

August 22, 2018 Soil Washing and Enrichment- Soil A

Rationale: To isolate the possible bacteriophages from the soil through the process of soil washing, and to collect lysate for enriched and direct samples.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Experimental Procedures:

  1. In this procedure, aseptic technique was used to avoid contamination as much as possible. CiDecon and 70% Ethanol were used to clean the work space. A 100% Ethanol burner was used to form an aseptic zone.
  2. The soil sample that was collected from the oak tree in Zone 4 was mixed with LB broth in a 50 mL tube. The tube contained approximately 13 mL of soil and was filled to the 35 mL mark with LB broth.
  3. After adding the LB broth, the mass of the sample and the tube was found to be 53.332 grams. The tube was then shaken for 15 minutes to mix the mixture.
  4. The class tubes were then paired with a tube that had a mass within +/- 0.1 g of the mass of their tube.
  5. The tubes were then put into the centrifuge and spun at 3000 x g for 5 minutes.
  6. After this process was complete, the supernatant was transferred into a 0.22 um filter using a pipette and filtered with a vacuum pump. Approximately 12.5 mL of lysate were obtained after filtering.
  7. 0.5 mL of arthrobacter was then added to the lysate to create the enriched isolation, which will be shaken at room temperature until Monday, August 27. The rest of the supernatant was too thick and needed to be transferred to a 15 mL tube and put through the centrifuge again. This will be done on Friday, August 24.
  8. The work station was then cleaned and all materials used were disposed of properly.

Observations/Results/Data: 

Observations:

  • moist soil
  • when shaken, bubbles formed
  • soil mostly mixed with LB broth
  • Created a milky color when mixed
  • After spun in centrifuge:
    • Yellow supernatant on top
    • Pallet on bottom, brown color
    • bubbles are gone
    • some soil and sticks floating at the top
    • layered colors of soil at bottom.
  • Filtering was slow due to items in the soil, causing the supernatant to require a second spin in the centrifuge.

Results:

The enriched sample was created. Approximately 12.5 mL were obtained of lysate. With the 0.5 mL of arthrobacter that was added to the sample, the enriched sample is 13.0 mL. The procedure to create both the enriched and direct lysate was not finished, but will be finished on Friday.

The sample will be stored in the laboratory until Monday at room temperature.

It is labeled: LIP 8-22-18 Enriched

Interpretations/Conclusions/Next Steps:

This procedure was not complete due to the sample not filtering completely.  In the future, trying to make sure there are less sticks and other material in the soil will allow this process to go more smoothly. The supernatant will need to be spun in the centrifuge again before collecting the direct lysate sample. The next step is to re-spin the supernatant and filter to create the direct lysate, which will be stored in the refrigerator.

Category: Lucy Potts | LEAVE A COMMENT
August 25

Soil Washing 8.22.18

Soil Washing 8.22.18

Rationale)

To wash the soil sample I had gathered, Soil Sample A, in order to produce a lysate that can then be both enriched and left as a direct isolation, for later use in conducting a plaque assay and spot test in order to determine the presence of bacteriophage in Soil Sample A.

Procedure)

1. Set up an aseptic zone using CiDecon and 70% Ethyl Alcohol to clean the workspace and lighting an ethanol flame.

2. Retrieve LB broth and the sample of soil that was collected, Soil Sample A, under aseptic conditions and with proper aseptic procedure open the LB broth and add enough LB broth until the 50mL conical vial containing the soil sample is filled to 35mL line.

3. Close both the LB broth and the now partly filled soil sample vial under aseptic conditions and return the broth to the storage area.

4. Shake the vial containing the LB broth and sample consistently for about 16 minutes, after which mass the vial and record the mass on the vial in order for it to be centrifuged properly.

5. Centrifuge the vial at 3000g for 5 minutes.

6. Retrieve a .22µm filter and set it up under a fume hood, connect the vacuum to it and proceed to collect the centrifuged soil sample A vial and a pipette. Using the =pipette transfer the supernatant from Soil Sample A to the top of the filter apparatus.

7. Wait for the supernatant to go through the filter, however, due to excess particulate in the supernatant only about 5mL of supernatant are able to be filtered into lysate.

8. Without removing the vial containing the lysate from the filter apparatus, pour the unfiltered supernatant equally by weight into two 15mL conical vials and label as Soil A, using a scale make sure the mass of each is only .1g apart and adjust the volume of supernatant in each vial as necessary to achieve a deviation of .1g.

9. After the excess unfiltered supernatant is taken care of open the package containing the lid for the vial of lysate, once done proceed to remove the filter apparatus from the lysate containing vial and quickly place the lid onto the vial. Label vial as Soil Lysate A.

10. Place all three samples into the fridge for continuation on 8.24.18.

Observations)

There was too much particulate in the supernatant for it to be filtered as apparently indicated by it being fairly cloudy and filled with larger particles compared to the clear golden colored lysate that was produced, the vial containing Lysate was labeled: NMN 8.22.18 Soil Lysate A, the two vials containing the unfiltered supernatant were labeled NMN 8.22.18 Soil A.

Conclusion/Next Steps)

For future soil washing, less particulate needs to be in the supernatant that is to be filtered which could be achieved by a greater amount time in the centrifuge or less shaking of the vial once it has been centrifuged, the experiment will continue on 8.24.18 wherein it will be determined if the Lysate contains bacteriophage by conducting a Plaque Assay and Spot Test. Overall the experiment and procedures worked relatively well in producing the desired result

 

Category: Nathan Newton | LEAVE A COMMENT
August 24

8/22/18 Soil Washing/Direct Isolation and Enrichment for Soil sample A

Rationale: Soil washing and enrichment on soil samples collected from Oak trees around Baylor University’s Campus. Without adding Arthrobacter, we had to create a direct lysate sample for the experiment from the soil. Our particular sample was found at 31*32’40 N 97*7’9” W near Waco Hall.

Materials:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner
  • .5mL Arthrobacter
  • 15mL conical vial
  • Pipette
  • 50mL tube

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.

Description of Procedure: 

  • As we were utilizing the aseptic technique, my lab partner opened my 50mL vial (that had the soil), and he placed the top on the table away from the aseptic zone for about five seconds before picking it back up.
    • note possible contamination.
  • I filled the tube with LB broth that Dr. Adair had provided us. As I filled it up to the required mL, Ramen closed my cap, and he sat the tube down over the carriages.
  • I then brought my tube over to the weight, in which it read 51.64g.
  • I then started to shake my tube for the next 15 or so minutes. After the class spinning, we had to find a partner that had a +/- .1mL mass of our broth.
  • I didn’t find anyone, but someone around the range. So I had to add water to my solution. The new mass of my solution read 52.56g.
  • After this, one of the TAs took my solution, and he gave it to Dr. Adair, where she put the many solutions in a centrifuge at 3000g for three minutes.
  • After the three minutes, we went back to the lab with our solutions, where we were told to come back Friday 8/24/18 to finish our findings.
  • Friday- I filtered lysate came out to 16ml Lucy pipetted 6ml into the small 15ml vial for the direct sample and added the Arthrobacter  to the remaining 10ml

Observations:

  • Transferring the first set of LB broth was not done in an aseptic zone.
  • The addition of water before the centrifuge spin was not done in an aseptic zone.

Results: 

The procedure form Wednesday was completed with the direct lysate sample having now been obtained.

Analysis:

The process was difficult, from making sure everything was done in an Aseptic zone (not everything but as in, the opening of our vials, to transferring the LB broth) to the precise measurements, it was a lot the first day. Time aspect played a big part since I was unable to actually finish this step, due to the fact I had a class after. My lab partner did this step and the extraction. Note, after the enriched solution was gathered from Friday, 8/24, my lab partner did not label my solution, but she did record the volumes of the solutions.

Interpretations/Conclusions/Next Steps: 

The procedure was now complete. Friday I filtered lysate came out to 16ml. I will need to be sure that I use the Aseptic technique, and not let my lab partners do my lab in a non-aseptic environment. Next step Monday 8/27/18, I will prep the dish/dishes that I will need in order to run a spot test, and hopefully, I will have bacteriophage.

 

 

 

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
August 23

Welcome to BEARS in the SEA 2018

Welcome to your brand new blog at blogs.baylor.edu.

BEARS in the SEA stands for Biology Education and Research Students in the Science Education Alliance!  You are part of the SEA PHAGES program, which stands for Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science.  So many letters for a great educational experience! The purpose of this blog is to provide you with an online notebook.

Lab notebooks are an essential part of learning authentic scientific skills.  Notebook are the official and legal documents for all scientific discoveries.  You will need to keep a hand-written account of all that you do in lab, using a bound notebook (not a spiral or loose-leaf paper or paper towel or post-it…) that is dedicated to only the lab.  It should be written with pen, not pencil, and pages should never be removed.  This hand-written notebook is your official lab notebook and you are responsible for keeping up with it.

The blog is a public record of what is in your notebook, with the added function of including your pictures and images and comments from other team members or people that have read your blog.  It should be written clearly (never post an image of what you have written), and in a professional manner so that someone could replicate what you did in your research. The reflection on and the recording of what you did in lab is a productive activity and this process often reveals mistakes and new ideas.

The blog will be due each week as a pdf download that you submit to a Canvas assignment. You should also make a folder on your computer to archive your blog for your own personal academic portfolio.

Category: Dr. Adair | 1 Comment on Welcome to BEARS in the SEA 2018