August 28

8.27.18- Spot Test

Rationale: A spot test was performed in order to display the results of Soil Sample A. The results will how whether or not a bacteriophage or bacteriophages are present through existence of plaques on a bacterial lawn.

Scientific Research Question: Does the species of oak tree cause any discrepancy in number or type of phages found in soil samples? **Yet to be confirmed**

Procedure Performed in Lab:

  1. Work site was cleaned using CiDecon (applied and wiped dry) and 70% ethanol (applied and wiped until half dry). An aseptic zone was established by setting up an ethanol burner near the center of the bench that the experiment and aseptic steps were completed close to.
  2. Enriched lysate labelled as “HMB Arthro Added 8/22” was obtained along with a syringe and 22 microliter filter for the end of the syringe.
  3. (Aseptic Step) 2 mL of enriched lysate was drawn into the syringe. 22 microliter filter was placed on end of syringe, then newly formed “Filter Sterilized Enriched Lysate” (abbreviated FSEL for labelling purposes) was created by moving the 2 mL of enriched lysate inside the syringe through the filter and into a microcentrifuge tube labelled “HMB FSEL 8/27/18” and “HMB” on lid. This step was done to remove existing bacteria from the enriched lysate while allowing possible bacteriophages to still pass through the filter and into the new tube.
  4. Plate with agar was obtained. On the bottom, it was divided into 3 sections (enriched, direct, and negative control) and labelled “HMB Spot Test 8/27”.
  5. One 50mL Conical Tube was obtained and labelled “HMB Top Agar 8/27/18”. A second Conical Tube was obtained and labelled as the negative control. Conical Tubes held components of Top Agar before the Top Agars (one with Arthro and one without) were poured out on experimental and control plates, respectively.
  6. Concentration of CaCl2 was obtained for Experimental Plate containing Arthro:
    1. (1M CaCl2)(Volume)=(4.5mM)(10mL)
    2. (1000mM CaCl2)(Volume)=(4.5mM)(10000 uL)
    3. Volume= 45 microliters of CaCl2
  7. Concentration of CaCl2 was obtained for Control Plate without Arthro:
    1. (1M CaCl2)(Volume)=(4.5mM)(9.5mL)
    2. (1000mM CaCl2)(Volume)=(4.5mM)(9500uL)
    3. Volume= 42.75 microliters of CaCl2
  8. (Aseptic Step) Experimental Plate Containing Arthro (labelled”HMB Top Agar 8/27/18″): 4.5mL of LB Broth was added to 50mL Conical Tube. 0.5mL of Arthro was added by Lathan Lucas to 50mL Conical Tube. 45uL of CaCl2 was added to 50mL Conical Tube.
  9. (Aseptic Step) Control Plate without Arthro: 4.5mL of LB Broth was added to 50mL Conical Tube. 45uL of CaCl2 was added to 50mL Conical Tube. No bacteria was added to this tube.
  10. (Aseptic Step) To finish the creation of Top Agar, 5mL of Top agar solution that had been warmed in an incubator was added to both the Experimental tube and Control tube. This step was done last because Top Agar solution hardens as it cools, so as soon as it had been added to both tubes and they had been gently swirled, the 50mL Conical Tube of LB Broth, Arthro, CaCl2, and Top Agar solution was poured over the plate labelled “HMB Spot Test 8/27” and the 50 mL Conical tube containing LB Broth, CaCl2, and Top Agar was added to the negative control plate.
  11. Waited 15 minutes to allow for complete solidification of Top Agar.
  12. (Aseptic Step) Micropipetted 10uL of Direct Isolate, Filter Sterilized Enriched Lysate, and phage buffer into their respective sections as labelled on the bottom of plate.
  13. Waited 15 minutes before placing plates into the incubator. Plates will be read on Wednesday to determine the presence of a phage or not.
  14. Lab station was cleaned by disposing waste products into correct locations and storing tubes/equipment in designated places. CiDecon and 70% ethanol were used to clean the bench in similar manners as they were at the beginning of the procedure.

Observations:

  • Filter Sterilized Enriched Lysate was yellow-gold in color after being moved through the 22uL filter.
  • Filter was said to cause resistance in pushing lysate out of syringe. However, during experiment, only slight resistance was felt during expulsion. If there are adverse results, this could be a reason why.
  • Top Agar was a dark golden color while kept in the Conical tube before being plated.
  • Drops of Direct Isolate, Filter Sterilized Enriched Lysate, and phage buffer momentarily stayed on surface of Top Agar before being absorbed. This caused Direct Isolate drop to slide to a location relatively close to the border between it and the next section, which should be observed and considered when taking results.

Results:
Test is not completed, so results have yet to be obtained. On Wednesday (after 48 hours have gone by), it will be possible to discern whether or not a phage is present on the plate by the presence or lack of plaques.

Next Steps: In the lab on Wednesday, a plaque assay will be performed in addition to analyzing the plates that were prepared by the spot test procedure. This will serve to confirm the results that will be obtained at the beginning of the lab session on Wednesday.

Category: Henry Burns | LEAVE A COMMENT
August 28

Sea Phages day 2: Spot Test

27 August 2018 ✷ Spot Test

A spot test is being performed to check for the presence of phage.

Scientific Question:

Which types of Oak tree does arthrobacter phage inhabit more frequently?

Procedure:

  • Workspace was cleaned with CiDecon and 70% Ethanol and an alcohol burner was lit to promote an aseptic work environment.
  • My petri dish was labeled with my initials and the date, in addition to 3 penny-sized circles (one labeled “negative control,” one labeled “enriched,” and the last labeled “direct.”
  • An additional petri dish was set up for my lab group; its purpose is to test the agar my group made.
  • In the process of making the plate, my group made 2 tubes: one for the control plate, and one with enough for our three plates (both used the same LB broth). SEE TABLE. The Top Agar was added last because it hardens as it cools and it was important to add the other components first as to be time efficient.

Components of Plate

control plate 

component volume concentration
2X Top Agar 5.0 mL 1.05X
LB Broth 4.5 mL –
1M Calcium Chloride 42.75 µL 4.5 µM
1000 µM*V=9500µL*4.5µM = 42.75 µL
2X*5.0 mL=M*9.5mL = 1.05 M

3 spot test plates

component volume concentration
2X Top Agar 3*5.0= 15 mL 1X
LB Broth 3*4.5= 13.5 mL –
1M Calcium Chloride 3*45= 135 µL 4.5 M
Arthro 3*0.5= 1.5 mL –
1000 µM*v=10000µL*4.5µM= 45µM CaCl2
15 mL TA/30 mL = .5(2) = 1X
  • The mixtures were poured into their respective plates and allowed to cool and harden.
  • The enriched solution was filtered through a 22 micron filter into a microcentrifuge tube in order to remove arthrobacter.
  • After plates hardened, a micropipette was used to place 4.4 µL of each sample into its respective circle on the plate (enriched, direct, negative control phage buffer).
  • The remaining direct isolation sample was refrigerated and the plates were placed in an incubator for 46 hours.
  • The lab space was then cleaned once again with CiDecon and 70% Ethanol Solution.

Observations/results/data:

  • After the enriched solution was filtered, it became paler yellow.
  • In the process of pouring my plate, my partner burned her finger on the burner flame and a small portion (less than 1 mL) of the agar solution spilled onto the table. Though this didn’t directly cause contamination to my plate, its possible there may be some issues with the data produced from my plate.
  • UPDATED: Upon returning to lab on Wednesday, August 29, I removed the petri dish from the incubator and found no plaques, only contamination. However, our agar control plate showed no signs of contamination, thus the error had to have occurred in the process of pouring and storing our plates because each member of my lab group had contamination on her plate (see images)
  • A likely source of contamination is that trying to maintain aseptic technique while having 3 people assist led to several moments where the open container we were working with left the aseptic zone and more precaution will need to be taken in future experiments.

Interpretation/Conclusion/Next Steps

  • The phage in each sample (if present) would infect the arthrobacter in the plate and kill it, leaving plaques that should be visible.
  • If phages are present, the spot test is a way to indicate their presence; however, it does not indicate concentration.
  • Instead, the use of a plaque assay next time will give further information in regards to the presence of phages from the soil sample.
  • UPDATED: Because no phage was detected in the spot test, only one plaque assay will be run (with the enriched lysate) just to practice the technique. At some point in the future, another soil sample will be collected to attempt to isolate a phage.
Category: Lily Goodman | LEAVE A COMMENT
August 28

8/27/18 Spot Test

Research Question:

Does the presence of Arthrobacteriophage appear more dominant in one oak tree species compared to another? If so, in this species, is there a correlation between the presence of Arthrobacteriophage and the presence of Oak Wilt Fungus growth?

Objective: The goal of this procedure is to grow our “Bacteriophage” (if we have any). We are trying to see if there are bacteriophage present from our soil sample. From Monday’s experiment, we created an agar for the bacteriophage, to test if phage was present.

Procedures and Protocols:

Materials:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner
  • 18 ml LB Broth
  • Petri dishes
  • Lysate
  • PY 2X TA
  • Pipettes
  • Micropipettor
  • 1M Calcium Chloride
  • .5ml of Arthrobacter

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.

Procedure:

  • Got the enriched solution that was created Friday 8/24 from the tray my TA had.
    • Important side note- my lab partner made my enrichment solution Friday, and she forgot to label the container. She did not label hers as well, but from her notebook, her solution had 13mL of enriched solution. While examining both solutions, we could tell the two solutions were not labeled but had different volumes. She grabbed the one that had the 13mL solution.
  • Used a pipette to obtain 1.5mL of my enrichment solution.
  • Once the solution was in the pipette, I attached a filter to the bottom of my pipette, and this filter filtered out the “bacteriophage” from the other bacteria that were too big to fit in.
  • I set the solution that was just filtered into a small cap, and I sat it down after labeling it “ML Enrichment 8/27”
  • Then we got Petri dishes and labeled it with three circles (NC for our Negative Control, E for our test Enrichment, and D for Direct).
  • Then we got two orange vial tubes, one for our TA Control, and the other was for our agar solution.
  • We then measured out the LB Broth for both the TA Control and our top agar solution. 13.5mL in the top agar and 4.5mL in the TA control.
  • Calcium Chloride was then given to us after our math was complete (formula c1v1=c2v2. Calculations listed below). We used 42.75 for the TA control, and 135 for the top agar solution.
  • We then put 5mL of TA (top agar) onto our TA control dish from our vial tubes.
    • Note that the solution once poured onto the plate had many bubbles.

TA Control Before 8/27/18

TA Control After 8/29/18 (Note- Showing before and after since the group thought the bubbles would affect the outcome of our TA Control after 48 hours of making the solution)

 

  • We then got the TA (top agar) for our actual plates and added it to our vial tubes.
  • We got 1.5mL Arthobacter and mixed it with our solution that added up to 30mL.
  • We each poured 10mL onto each of the three plates (one for each lab partner)
  • We finished pouring the agar and we added 10 µL of enriched, direct, and phage buffer the appropriate sections.
  • Then we put it in the incubator.
  • We put in the incubator around 420.
    • We won’t get a full 48 hours but we will be close
      • Side note- the final three procedures were done by my lab partner.

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

C1V1=C2V2

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

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

V1=45 microliters

Results:

We will find out Wednesday to see if our spot test fails. This procedure helped to set the stage for Wednesday 8/29.

Analysis: 

The procedure was well organized, and it went smoothly. My lab partner did mess up with the LB broth/calculations/pipette, but overall we managed to do everything on time. My lab partners had to stay after the lab class in order to finish, and they were able to help me with my results. Next time, I will be sure not to touch the tip of the cap so that the bacteria on my fingertips do not fall into the cap, thus contaminating my experiment.

Future:

If the Spot test fails or succeeds, we would still have to do a plaque assay. The only difference is if there are spots from the spot tests, then you would have to do two plaque assays, one for the direct and enrichment.

 

Category: Michael Lum | LEAVE A COMMENT
August 27

8/24/18 Enrichment One Part Two

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 27

Washing (continued), Direct Isolation, and Enrichment of Soil A (08/24/18)

Rationale:

Finishing Wednesday’s part, more of the supernatant will filter through, so the steps of direct isolation of half of the lysate and enrichment of the other half can happen. By enriching the lysate, it allows for the determination of whether the bacteriophages in the sample target arthrobacter or not.

Procedure:

  1. The 50 mL conical vial containing the remaining supernatant was centrifuged.
  2. The remaining supernatant was filtered with a 0.22 μm top vacuum filter.
  3. The lysate samples from Wednesday (8/22) and Friday (8/24) were combined into one 50 mL conical vial to an approximate total of 10 mL.
  4. The rest of the lysate, from now on referred to as the direct isolation, was transferred into a 15 mL conical vial which was stored in the fridge for later use.
    • This vial was labeled “KEA 8/24/18 filter”
  1. To the 50 mL conical vial with approximately 10 mL of lysate, 0.5 mL of the arthrobacter was added under a cleanhood.
    • This vial was labeled “KEA 8/24/18 enrichment isolation”
  1. The 50 mL conical vial with the arthrobacter, from now on referred to as the enrichment isolation, was placed in the shaking incubator at room temperature.

Observations and Interpretations:

  • The supernatant centrifuge faster than it did previously. This might be because the particles slowly started to combine back into the supernatant while waiting for others to complete the filtering on Wednesday.
  • I forgot to use an EtOH (100%) lamp when transferring over some of the Friday’s sample into the Wednesday’s sample to have approximately 10 mL in the enrichment isolation conical vial. However, the lysate was transferred under the clean hood.

Next Steps:

On Monday, the enrichment isolation will be used in the spot test to determine whether there are any bacteriophages that target arthrobacter in the lysate.

Category: Kathryn Adkins | LEAVE A COMMENT
August 27

Washing of Soil A (08/22/18)

Research Question Discussion:

After discussing with fellow BEARS in the SEA members, I have developed a prototype research question that expresses both something not too narrow and that can be applicable to a real-life situation. The dependent variable in this study is whether or not the Arthrobacterphage is present or the positive signs appear from either a spot test an/or the plaque assay. We considered many variables that could potentially affect the results of the lab. These variables included: soil pH, soil type, distance from water resource, temperature, weather, elevation, climate, and tree species. From here we were able to narrow down the factors. Since we will be working on Baylor University campus, the area for collecting samples is small enough that variables such as temperature, climate, elevation, and the weather should be relatively the same. As far as soil type, we don’t know if there is a sufficient amount of soil type samples to test. If we studied the tree height, it would be difficult to measure it accurately. Same as the distance away from a water source. After all, what exactly do we mean by water source? Is the water source a sprinkler, creek, or the Brazos River? The class felt like tree species would be a good variable to test. Specifically, the independent variable would be the different types of oak trees, so we could relate it to the real-world problem of oak wilt disease. The following question has two parts: one as a broad, generalized question and one as a specific applied real-life scenario. We wrote the question in this format so we can have a testable hypothesis. Based on the observation from the first spot test, finding a bacteriophage is not easy. So ideally, if we do find a sufficient amount of bacteriophage in the next few weeks, we can continue to answer the second part of the question, and thus, apply the scenario to a real-life situation. If not, we can simply drop the second part.

 

Purposed Prototype Research Question:

Does the presence of Arthobacterphage appear more dominant one oak tree species than the others? If so, in this species is there a correlation between the presence of Arthrobacterphage and the present of oak wilt fungus growth?

If this prototype research question is used, we will need to define what we mean by “oak wilt fungus growth.” Also, we will need to define where we will be collecting the sample.

As far as metadata, if we used the prototype research question above, we would need to identify the oak tree species.

 

Rationale:

Through washing the soil, the bacteriophages will become separated from the dirt and bacteria. The addition of the arthrobacter will allow for the determination of whether the bacteriophages present attack arthrobacter specifically or not.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Added approximately 15 mL of soil sample (collected on Tuesday from Area 2) into a 50 mL conical vial and labeled it “KEA 8/21/18 Soil A.”
  3. Through aseptic technique (performed the following over an EtOH (100%) lamp and being extremely cautious by not allowing the conical vial or cap to touch anything), poured the LB Broth into the 50 mL conical vial with about 15 mL of the soil sample to the 35 mL mark.
  4. Used an analytical balance to measure the mass of the conical vial.
  5. Mixed the LB Broth into the soil by hand-shaking for approximately 15 minutes.
  6. The vial was then centrifuged at 3000xg for 5 minutes.
  7. The supernatant was then pipetted into a 0.22 μm top vacuum filter to separate the lysate.
  8. The lab counter was cleaned with CiDecan and EtOH (70%).

Observations:

  • With the addition of the LB Broth, the conical vial weighted 53.18 grams.
  • Before the conical vial was centrifuged, the hand-shaken soil and the LB Broth appeared to look like chocolate milk.
  • After the conical vial was centrifuged, the supernatant appeared to be light colored and murky with a few dark particles floating at the top. The pellet had streaks of dark and light brown throughout. The following image shows the pellet in the conical vial.

  • After approximately 20 minutes of filtering out the supernatant, only a little less than 10 mL of the lysate formed. The lysate was a light shade of yellow.

Next Steps:

On Friday (8/24), the supernatant will finish filtering. It will be spilt into the direct isolated lysate and the enriched isolated lysate. Next, 0.5mL of arthrobacter will be added to the enriched lysate. After finishing these steps, the enriched lysate should be prepared for Monday’s spot test.

Category: Kathryn Adkins | LEAVE A COMMENT
August 27

8/22/18 Enrichment One Part One

8/22/18 Enrichment One Part One

Objective:

The goal of this procedure was to preform 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.

Procedures and Protocols:

Materials:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner
  • 1 50 ml tube
  • ~ 20 ml LB Broth
  • Centrifuge

In order to accomplish this goal two procedures had to occur. The first was the creation of an aseptic zone.

  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

The second procedure was soil washing and enrichment (see photo below).

  1. Collect soil from designated oak tree by digging a few centimeters below the grass
  2. Place soil in a 50 ml tube up to the 15 ml line
  3. Add LB broth up to the 35 ml line under aseptic conditions
  4. Shake the tube for 15 minutes (during this 15 minutes the tube was dropped on the floor, potentially disturbing the soil and broth, but then the shaking resumed)
  5. Place tube in centrifuge for 5 minutes on 3,000 X g in order to separate the soil (pellet) and supernatant
  6. Place tube in the fridge to complete the procedure on a later date

Results:

This procedure yielded a tube of centrifuge separated supernatant and pellet as seen in the image. The solids separated by mass in the centrifuge with the most dense on the bottom. The centrifuge was spun on a low enough power to leave any existing phages in the supernatant.

Analysis:

The procedure had to be stopped halfway through so the results were incomplete and will be detailed in the next entry. Based on the results I do have I can make the assumption that the centrifuge didn’t fully separate my sample because it appears to be murky and there is debris on the top of the supernatant.

Future:

My plans for the future are to complete the procedure I started during Friday’s open lab in order to have an enriched sample and direct sample.

Category: Lucy FIsher | LEAVE A COMMENT
August 27

Spot Test 8/27/18

Rationale: We are spot testing in order to see if we found a bacteriophage in our soil sample B from tree A that can infect arthrobacter.

Procedure:

  1. I started out by cleaning off the table with CiDecon and then letting 70% ethanol dry onit. Once complete I lit a burner to create an aseptic zone.
  2. I then filled a syringe with 2mL of my enriched lysate from the previous lab whileholding both the syringe and my tube within the aseptic zone.
  3. I used a 22μm filter to filter out 1.5mL of lysate into a small tubule.
  4. In order to start making top agar I first placed 18mL of LB broth in a 50mL tube. Thenpipetted out 4.5mL into a different 50mL tube so that I can make some TA to use as a

    control.

  5. Added 135μL of CaCl2 to the tube with 13.5mL of LB broth and 42.75μL to the tubewith 4.5mL of LB Broth.
  6. Added 1.5mL of arthrobacter to the non-control tube then added 15mL of 2X Top Agarto the tube and pipetted up and down. Afterwards I pipetted 10mL into my plate.
  7. Poured 5mL of 2X Top Agar into the control tube and shook then poured into plate.Note: I wasn’t supposed to shake the tube, but it shouldn’t affect the control much other

    than leaving bubbles in the plate.

  8. After waiting 10 minutes for top agar to dry I pipetted 10μL of my enriched lysate ontothe plate on the designated enriched zone which I marked with sharpie on then back. Note: The drop I pipetted onto the plate rolled from the spot I dropped it down to the middle.
  9. Then I pipetted 10μL of my direct solution from the previous lab, which was stored in the fridge, onto the marked area.
  10. Lastly, I pipetted 10μL of phage buffer onto the negative control marked area.
  11. Let sit for 12 minutes, then stored in the incubator for 48 hours.

Observations:
I found a small plaque in the center where my enriched lysate had rolled down to. Everyone in my group had found a plaque and we all took our sample from the same place. 9 of the 24 students found a plaque.

My Plate:

Control Plate:

Interpretations and Next Steps:
This could possibly mean that my sample may have contained a bacteriophage able to infect arthrobacter. Since our tree seemed to be a sick red oak and only 9 of the 24 students got a plaque and all three members of my group, who all took a sample from the same tree, got a plaque there may be a correlation between the presence of a bacteriophage and trees getting sick. Further tests would need to be done to see if I truly did find a bacteriophage. Also, I would need to test more trees to see if the correlation is truly a correlation and not a fluke of some sort.

Category: Sriram Avirneni | LEAVE A COMMENT
August 27

8/22/18 – Washing and Enrichment

Rationale: Washing of the soil will get rid of the unwanted particles such as dirt and other debris, and will leave us with the bacteriophage to become our lysate which we can use.

Procedure:

  • Firstly, we had to create an aseptic zone using CiDecon and Ethanol (70%). This zone is necessary when experimenting to ensure that no other microorganisms may contaminate the experiment.
  • We sprayed down our workspace with CiDecon and wiped dry. We then sprayed the Ethanol and wiped, letting the ethanol evaporate.
  • We then used a burner in the middle of our work space to ensure that no microparticles or microorganisms would land in our open vials (The open flame creates an area of low pressure and creates a circling air current)
  • LB broth was added to my vial of soil sample until it measured 35mL. The addition and pouring of the LB broth was done within the aseptic zone
    • When pouring LB broth into my vial of soil, we over-poured and ended up with 37mL of solution.
  • The solution was then mixed for 15 minutes. This was done with hand shaking as well as through the use of a vortex machine.
  • During the mixing process, I weighed my sample and found that it was 53.89 g. The reasoning of finding the mass of the sample was for centrifugation, where another sample of similar weight (±0.1g) was needed to balance the machine.
    • When finding another sample that was similar to my weight, I needed to add 1.29g of water to ensure my sample was within ±.1g of my partners.
  •  After mixing was complete, the sample was centrifuged at 3,000 revolutions per minute for five minutes to separate the more dense particles in the solution from the smaller micromolecules and microorganisms.
  • After centrifugation, I was left with a conical vial separated between the supernatant (Smaller particles in the solution, hopefully with bacteriophage) and the solid, more dense materials at the bottom of the vial.
  • The supernatant was then run through a .22 micrometer filter to get rid of any remaining particles, resulting in 10 mL of lysate
  • I only had enough lysate to create an enriched sample, which was done through the addition of .5 mL arthrobacter to the lysate, and was stored; the addition of arthrobacter to the lysate was done in an aseptic zone.

Observations:

  • When mixing my sample, my vial resembled the consistency and coloration of chocolate milk.
  • After centrifugation, the contents were separated into visually noticeable “sections” where the more dense particles were at the bottom of the vial, and was darker in color, while the top “sections” were more clear.
  • The supernatant was yellowish-clear in coloration.

Results:

  • The experiment resulted a usable vial of lysate which will be used in the next step of my experiment.

Next Steps:

  • The next step would be to examine the lysate to see if there are any bacteriophage present via the spot test technique/procedure.

 

Category: Justin Yu | LEAVE A COMMENT
August 27

Sea Phages Lab Day 1: Cleaning the Soil

22 August, 2018 ✷ Cleaning the Soil

After collection of soil from an oak tree in Zone 8 (near Gordon Teal Residential College), the soil was cleaned in order to isolate possible bacteriophage samples.

Procedure

  • 20 mL of soil was removed from the collection vial and stored in a refrigerator for future tests. 15 mL of the original sample remained in the vial.
  • The table was washed with Cidecon and 70% Ethanol solutions to promote an aseptic environment. An alcohol burner lamp was lit to clean the air surrounding the workspace.
  • 20 mL of LB broth was added to the soil sample; the soil/LB broth mixture was then shaken by hand for 15 minutes to mix. At the end of 15 minutes, the vialwas massed and found to be 52.398 g.

  • The vial was centrifuged at 3000 x g for several minutes. The mixture separated into 3 layers: heavier soil, a milky-yellow liquid, and some less dense wood chips at the top.

  • Using a vacuum filter, the milky-yellow liquid from the center layer was filtered through a 20 micron filter. The resulting filtrate was paler yellow in color and more transparent.
  • 5 mL of the lysate (filtrate) was poured into a 15 mL vial and refrigerated, and the remaining 10 mL were combined with 5 mL Arthrobacter (ATCC 21022). This sample was then stored in a shaking incubator for 5 days.

Observations/results

  • As the lysate was cleaned and filtered, its color became lighter and more transparent.

Interpretations, Conclusion, Next Steps

  • The purpose of this portion of the experiment was to purify the soil sample and isolate any bacteriophages in the soil sample. Thus, if any were present, they would be isolated in the enriched and direct isolation samples resulting from vacuum filtration.
  • The next step is plaque assay and spot test–the potentially infected arthrobacter will be introduced to a nutrient-rich bacterial lawn and the presence of bacteriophages will be seen if plaques are present (places where the phage killed bacteria at the end of its lytic cycle).
Category: Lily Goodman | LEAVE A COMMENT