August 31

8/29/18 Plaque Assay of Enriched Lysate

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8/29/18 Plaque Assay of Enriched Lysate

Objective:

The goal of this procedure is to determine weather or not bacteria phages are present in the soil collected last week. Based on the results of the spot test it is likely that there are phages present (see spot test results below). In this procedure a plaque assay on the enriched lysate will be conducted to confirm the results of the spot test.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

Procedures and Protocols:

Materials:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner
  • .5 ml Arthrobacter
  • refrigerator
  • Pipette
  • Test tube stand
  • 50 ml tubes
  • LB Broth
  • 2X TA
  • 1M Calcium Chloride
  • Agar plate
  • Serological pipette

In order to complete the procedure an aseptic zone was created.

  1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
  2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
  3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

Then the plaque assay on the enriched lysate was preformed.

  1. Divide the bottom of an Agar plate into four sections and label them Groups 1, 2, 5, and 6 in order to create a TA control. Set aside.
  2. Label a second agar plate with initials, date, and description (plaque assay will be preformed in this plate).
  3. Gather the remaining enriched lysate from last procedure (found in the pipette cap seen below)
  4. Using a Serological pipette, aseptically transfer 10 µL of the remaining enriched lysate into a culture tube containing .5 ml of Arthrobacter
  5. Reseal tube and recap pipette cap
  6. Allow the lysate and bacteria solution to sit for 15 minutes while the agar is prepared

While the lysate and bacteria are allowed to sit in the culture tube prepare the agar *Note:this was a collaborative effort so the agar was prepared for three full agar plates and a control:

  1. Prepare the agar according to the following recipe (use 4 plate recipe):
  2. Under aseptic conditions, pipette 8.o ml of LB broth into a 50 ml tube. Cap the tube.
  3. Under aseptic conditions, pipette 90 µL of 1 M CaCl2 into the same 50 ml tube. Cap the tube.
  4. Under aseptic conditions, pipette 10.o ml of 2X TA into the same 50 ml tube
  5. Pipette the mixture several times to mix it

When agar preparations are finished the bacteria and lysate have been allowed to sit for 15 minutes

  1. Pipette 4.5 ml of the contents in the 50 ml tube into the lysate and bacteria culture tube
  2. Pipette the mixture several times to mix it *Note: in the process of pipetting the mixture and air bubble caused some of the mixture to spill out of the culture tube, potentially upsetting the ratios in place*
  3. Pour the mixture in the culture tube into the agar plate labeled with initials, date, and description
  4. Cap the plate and allow the plaque assay agar to solidify for 10 minutes
  5. Aseptically Pipette 1 ml of the contents in the 50 ml tube onto the control TA plate in the section labeled group 6
  6. Allow the TA control plate to sit for about 10 minutes before being placed into the incubator
  7. Once the labeled plaque assay has solidified, invert the plate and place it in the incubator
  8. Leave to incubate until next class
Results:

The results of this procedure will not be immediately clear until Friday’s open lab or Wednesday’s normal lab, but once they are available they will be included here.

Update: The plates appear to be inconclusive based on cloudy appearance and due to a lack of positive or negative controls, one cannot make any assertions about phage presence.

Analysis:

It is hard to analyse the results of this lab because the results themselves are unclear. However, it can be concluded that when the agar plates are redone thicker agar lawns should be used and i would be beneficial to include positive and negative controls as a means of comparison. It is also possible that samples were contaminated, but it is hard to confirm this.

Future:

Due to the inconclusive nature of the plaque assays, I will need to redo my plaque assay likely with positive and negative controls in order to confirm the results of my spot test.

Category: Lucy FIsher, Uncategorized | LEAVE A COMMENT
August 31

Plaque Assay 8/29/18

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Scientific Question:

Does the presence of Arthrobacter phage vary in oak species? Is there one species where they are more dominant?

Rationale:

In today’s lab, the goal was to successfully perform a plaque assay assessment to test the presence of phage in soil samples. Although the results of the spot test yielded negative results for the presence of phage, plaque assays have the ability to yield different results.

Materials Used: 

  • 10 μL Lysate
  • PY Broth
  • PY 2X TA
  • 40% Dextrose
  • 1M Calcium Chloride
  • 0.5 mL of Arthrobacter host
  • Agar plate
  • Pipettes (Micropipettes and serological pipettes)

Procedure: 

  1. I began this procedure by setting up an aseptic zone on my workbench.
  2. Next, I gathered the necessary materials to create the top agar. Each group member grabbed their own plate for their plaque assay. I labeled mine with a sharpie and began to make the top agar.
  3. Because the top agar made was supposed to be split 4 ways, the measurements for the top agar solution varied slightly from the spot test. For this procedure the top agar consisted of:
    • 8 mL of LB Broth
    • 10 mL of PY 2x Top Agar
    • 90 μL of Calcium Chloride
  4. I added the 8 mL of LB broth into our 50 mL conical tube by using the serological pipette.
  5. After the LB Broth was added, I used a 20-200 μl micropipette to transfer 90 μL of the calcium chloride to the conical tube.
  6. Then I took the filtered lysate from the spot test procedure in the micro centrifuge tube and I used a 1-10 μL micropipette to transfer 10μL of the lysate to the vial containing the 0.5 mL of arthrobacter. I allowed this mixture to sit for approximately 15 minutes to let any possible phage present infect the bacterial hosts.
  7. After 15 minutes had past, 5 mL of the top agar was extracted from the tube to add to the arthrobacter solution, the vial was mixed gently, and then plated immediately onto my plaque and let it sit for 15 minutes.
  8. Unfortunately, while transferring the top agar to my partner’s vial, we noticed the vial had broken and was leaking my partner’s solution all over the table, so before advancing any further, an aseptic zone was reestablished. We then continued with the transfer of top agar to the remaining vials.
  9. We then took the leftover agar and added it to the control plate, leaving the agar to solidify and add to the incubator for 48 hours.

Observations/Results/Data:

  • The top agar this time looked very similar to the top agar created for the spot test. There was no contamination of the top agar as everything was performed aseptically.
  • The color of the top agar stayed relatively dark yellow, very clear once poured and solidified.

Conclusions/Next Steps:

  • The top agar has yet to be examined, but if the results for phage are positive there will be empty lawns throughout the plate where the phage has killed its bacterial host.
  • If no spots are examined, then the soil sample will be confirmed negative for phage and I will have to collect more soil samples from different plots on campus.

 

Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT
August 30

Spot Test 8/27/18

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Rationale: Today’s goal was to correctly perform a spot test to determine the presence of phage in the soil samples that we had extracted in the week prior to this one. Hopefully, if done correctly with no contamination, spots will appear in our plaque after 48 hours of incubation.

Materials:

  • Lysate
  • PY Broth
  • PY 2X TA
  • 40% Dextrose
  • 1M Calcium Chloride
  • 0.5 mL of Arthrobacter host
  • Agar plate
  • Pipettes (Micropipettes and serological pipettes)

Procedure:

  1. This procedure started with establishing an aseptic zone to decontaminate the work bench. The bench was sprayed and wiped down with CiDecon, then followed up with 70% Ethanol, which was spread on the table allowed to evaporate. Following this, I grabbed our burner to finish setting up the aseptic zone.
  2. Next, I gathered the materials necessary to create the top agar that is needed for the spot test. I grabbed 4 plates (one for each group member and one for control), 2 serological pipette tips,a 50 mL conical tube, and I also set out four micro centrifuge tubes which will be used later in the lab. I then marked my plate with a sharpie to show where I would spot my direct isolation, my enriched isolation, and my phage buffer.
  3. Next, I began to create the top agar, I measured out 4.5 milliliters of LB broth to add to the conical tube using a serological pipette. My pipette was having issues, and could not go past 4 milliliters of liquid, so I had to use the serological pipette for the 4 milliliters, then I had to use a micropipette to gather 500 micro-liters to add to my broth to equal 4.5 milliliters to add to my 50 mL conical tube.
  4. After the LB broth was added, I needed to add calcium chloride to our top agar. Initially, the quantity of calcium chloride needed was unknown, but what was known was that we needed the molarity of calcium chloride to equate to 4.5 mM or millimolar. To solve for the volume needed, there is a simple equation that I used to get a final value of 45 microliters of calcium chloride
  6. Following the calculation, the calcium chloride was added to our broth mixture using a 20-200 microliter pipette.
  7. Instead of adding arthrobacter next, I filtered my enrichment from the previous lab through a 22 micron filter by syringe. I pulled out approximately 2 milliliters and pushed it gently through the filter into a micro-centrifuge tube.
  8. After the filtration process, 0.5 ml of arthrobacter was added to the broth mixture.
  9. Followed up immediately by pipetting 5.0 milliliters of PY 2x Top Agar with serological pipette and allowed the top agar to mix.
  10. Then I very quickly plated my top agar and the group’s control plate, swirled the plate around, and waited 15 minutes for the plate to solidify.
  11. After 15 minutes, I added 10 microliters of my direct, enriched, and phage buffer to their designated spots and put the tray to incubate for approximately 45 hours.
  12. After 45 hours I removed the tray to analyze my results.

Observations/Results/Data:

  • After examining the plate, it was concluded that there was no phage present in the lysate that I had extracted from my soil sample. The plate looked the same as it did when it was put in the incubator, no spotting whatsoever. This indicates a lack of phage as the arthobacter had not been cleared from the plate. There was no contamination as the agar looked exactly like the control.
  • Plate had the same color, but had a liquid forming on the top. Unsure what exactly caused that. 

Interpretations/Conclusions:

  • The results of the spot test indicate that there is no phage present in the soil sample that I collected the week before. This can be deduced by the lack of any clearing or any indication of bacterial death on the plate. If phage were to be present, there should be minor bacterial clearing in any part of the plate.

Next Steps:

  • There may still be hope for the soil sample. The next step is to perform a plaque assay to view the results of that experiment, as plaque assays and spot test have the ability to yield different results. The plaque assay will be performed the next time I am in lab, and if the results are negative that means I have to collect more soil samples to test for phage.
Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT
August 30

08/27/2018- Spot test

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Date: 8/29/2018

Plaque Assay Soil A

Objectives:

  • perform the spot test

Materials Required: Filtered Enriched lysate, serological pipettes, micropipettes, micropipette tubes, LB broth, Top Agar 2X, 50 ml                                               conical vials, 1M CaCl2 stock solution

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

M1V1=M2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Procedure:

  1.  First the aseptic zone was set up: pour Cidecon on the desk and wiping it till the desk dry. then pour ethanol and wipe it all over the table and let it evaporate.
  2. Light the ethanol lamp to create an air current near which the samples can be opened to prevent things from getting into the samples.
  3. Retrieved the LB broth, a 50 ml conical tube and a serological pipette
  4. While in the aseptic zone, transfer 4.5 ml of LB broth to the 50 ml conical vial.
  5. now retrive the 1 M CaCl2 stock solution.
  6.  Using the micropipette, i transfered 45 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
  7. retrieved 0.5 ml of arthrobacter from Lathan ( Teaching Assistant)
  8. add the arthrobacter to the 50 ml conical tube.
  9. add 5 ml of top agar to the 50 ml conical tube.
  10. pour the contents of the 50 ml conical tube onto the agar plate.
  11. to let the top agar solidify, i waited for 10 minutes.
  12. collect the enriched sample tube, a syringe and a filter of 22 microns.
  13. take a sample from the enriched tube using the syringe.
  14. attach filter to the syringe and pour the lysate out slowly into a microcentrifuge tube
  15. collect the direct isolation sample from the fridge.
  16. collect a phage buffer from the instructor
  17. mark the agar plate with three spots , one for the enriched, one for the direct and one for the phage buffer.
  18. spot 10 microliters each of the phage buffer, enriched sample and the direct isolation sample.
  19. let the sample rest for 10 minutes
  20. i put the plate in the incubator, where it will remain for 48 hours.

Analysis:

there was no event that may have caused contamination to the sample. the aseptic zone was properly maintained. the procedure was properly followed.

Future notes:

read the instructions carefully and work faster so as to finish on time and prevent mistakes.

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
August 30

8/29/2018- Plaque Assay Soil A

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Date: 8/29/2018

Plaque Assay Soil A

Objectives:

  • Analyse the results of the spot test from Monday (08/27/2018)
  • Make a plaque assay with the enriched lysate of soil sample A that was filtered on Monday

Results from Spot Test:

  • No plaques have formed on my Spot test for soil A. According to these results, there are no bacteriophages in my soil sample.
  • I have also made a major error. I forgot my filtered enriched sample on the table on Monday and this may affect the results of the plaque test today.
  • There was also some moving liquid inside my spot test dish. May be just some of the TA that had no solidifies before incubation.

Adjustments:

Due to a lack of an adequate number of Agar plates, adjustments were made to the experiment for today. Everyone had to do an enriched sample plaque Assay and the following adjustments were made to the measurements of the ingredients required to make the top agar. the top agar was made for the entire group in one conical vial.

2 ml LB broth

2.5 ml 2X  Top Agar

22.5 microliters 1 M CaCl2.

These values were then multiplied by 4 to account for the fact that it was for the entire group. the measurements used for the group:

8 ml LB broth

10 ml 2X Top Agar

90 microliter 1 M Cacl2

5 ml of this mixture for the top agar was poured onto each plate.

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

M1V1=M2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Materials Required: Filtered Enriched lysate, serological pipettes, micropipettes, micropipette tubes, LB broth, Top Agar 2X, 50 ml conical vials, 15 ml conical vials

Procedure:

  1.  First the aseptic zone was set up: pour Cidecon on the desk and wiping it till the desk dry. then pour ethanol and wipe it all over the table and let it evaporate.
  2. Light the ethanol lamp to create an air current near which the samples can be opened to prevent things from getting into the samples.
  3. Retrieved the LB broth, the same one I used on monday, a 50 ml conical tube and a serological pipette
  4. While in the aseptic zone, transfer 8 ml of LB broth to the 50 ml conical vial.
  5. now retrive the 1 M CaCl2 stock solution.
  6.  Using the micropipette, i transfered 90 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
  7. Set this vial in the rack.
  8. retrieve 0.5 ml of arthrobacter.
  9. using the micropipette, i transfered 10 microliters of my filtered enriched lysate to the arthrobacter.
  10. i let the testtube rest for 10 minutes in its rack.
  11. after the 10 minutes are up, add 10 ml of top agar to the conical vial.
  12. using a serological pipette, transfer 5 ml of the top agar mixture to the test tube with the arthrobacter and the lysate.
  13. My test tube cracked and spilled onto the table. i had to restart the process with the measurements for one sample.
  14. i added 10 microliters of the filtered enriched lysate to another 0.5 ml culture of arthrobacter. let it rest for 10 minutes
  15. Into a 15 ml conical vial in the aseptic zone, add 2 ml of LB broth, 22.5 microliters of 1M CaCl2 and 2.5 ml of 2X top agar, using the same methods as before.
  16. Transfer the contents of the 15 ml conical vial to the test tube of arthrobacter and lysate after the 10 minute rest.
  17. pour the contents of the test tube into the agar plate.
  18. to let the top agar solidify, i waited for 10 minutes.
  19. i put the plate, upside down in the incubator, where it will remain for 48 hours.
  20. pour a part of the top agar mixture prepared into the top agar control plate for the 4 lab groups.

Analysis:

there was no event that may have caused contamination to the sample. the aseptic zone was properly maintained. the procedure was properly followed.

Future notes:

take extra care while handling lab equipment to prevent future damage that could lead to more delays. We have also decided on a research question : Does the presence of arthrobacter appear more dominant in the soil of one oak species than the others? Is there a correlation between the presence of Arthrobacterphage and the presence of oak wilt fungus?

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
August 28

Saabye Blog Post 1!

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This post is for the first week and the first day of the second week.

 

So this week we started our research into SEA Phages, and let me tell you, its quite the difference from walking around the Lab, to jumping across the sewer to get the average crown width. Of course, that didn’t make any of this less fun.

The first week started off pretty easy, nothing too difficult, and realistically none of this is going to be super difficult. We have our procedures, and even when we are manipulating variables, I assume the lab protocols will keep us from getting too far off track.

However, that assumes the student-researchers know what they’re doing. It would be accurate to say that we all know mostly what we’re doing, but little things here and there require practice. I would not be surprised if there were some rookie mistakes outlined in my lab journals that prevented me from being able to accurately take data, without having to backtrack a few days. But, these can all be fixed within a lab period, maybe quicker if done on a Friday when many people are busy and cannot make it to open lab.

I’m excited to see what my next steps will be for my lab, whether that means going back and retrying my work from the previous day until its correct, or progressing onto the next steps.

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August 27

8/24/18 Enrichment One Part Two

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8/24/18 Enrichment One Part Two

Objective:

The goal of this procedure was to finish preforming soil washing and enrichment on soil samples collected from oak trees around Baylor University’s campus in order to isolate bacteria-phages. The particular sample used in this procedure was found at 31*32’40” N 97*7’9″ W near Waco Hall. This is a continuation of 8/22/18 lab journal.

Procedures and Protocols:

Materials:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner
  • Top filter with 50 ml tube attached
  • Fume hood with a vacuum tube
  • .5 ml Arthrobacter
  • 15 ml conical vial
  • refrigerator
  • Pipette
  • Test tube stand
  • 50 ml tube

In order to complete the procedure an aseptic zone was created.

  1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
  2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
  3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

Then the soil washing and enrichment procedure was complete (see photo below for full procedure and see last entry for previous steps).

  1. Remove the refrigerated sample (in a 50 ml tube) from the fridege
  2. Open the sealed top filter under the fume hood and attach to the vacuum tube
  3. Turn the vacuum on
  4. Use a small pipette to transfer the supernatant from the 50 ml tube to the top filter
  5. Wait for the supernatant to filter to a minimum of 10-15 ml (completely is preferred and 18 ml were collected in this instance)
  6. Once the desired about of lysate has been obtained discard the filter in the bio-hazard container and seal the 50 ml tube with the filtered lysate
  7. Under aseptic conditions pipette 5 ml of lysate into a 15 ml conical vile, label, and set aside
  8. Add .5 ml of Arthrobacter to the remaining lysate in the 50 ml tube *note that this tube should have been labeled but I forgot to do so* and seal the tube
  9. Place tube into shaking device and let it set until Monday

Important to note:

During this lab I was responsible for completing two separate soil washings, my own and my lab partners. I have only detailed my soil washing in the procedure above.

Results:

This procedure yielded a direct isolation (5 ml) and an enriched isolation (13 ml) of the supernatant. Both appeared of these isolations were yellow in color and could contain phages.

Analysis:

The procedure was more difficult than I initially assumed, and filter times were longer than expected. One possible way to improve upon the design of this lab could potentially be to centrifuge the soil and LB broth for a little bit longer in order to have better separation. It is possible, although unlikely that the results could have been contaminated during the course of this procedure and that may affect results. Assuming that this is not the case then future procedures will reveal weather or not phages are present.

Future:

My plans for the future are to use these two isolations to preform a spot test during Monday’s lab period. The results of this spot test will allow me to discover if there are phages present in the soil sample I collected.

Category: Lucy FIsher, Uncategorized | LEAVE A COMMENT
August 25

8/22/2018- Washing

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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

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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 24

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

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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