August 31

8-27-18 — Spot Test

Date: Monday, August 27th, 2018

Title: Spot Test

Rationale: The purpose of today’s lab is to set up a spot test on an agar plate with an ATC 21022 lawn in order to find a bacteriophage.

Procedure:

  1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
  2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
  3. I filtered 2 mL of my enriched isolation using a 22 micrometer filter into a 3 mL microcentrifuge tube.
  4. My group (Group 5) then received four agar plates. We each used one individually, then used the fourth plate as a top agar control to make sure our top agar wasn’t contaminated, thus invalidating any potential results.
  5. I labelled my plate with my initials (BJR) and divided the plate into three equal sections, labelled E, D, and B for Enriched isolation, Direct isolation, and Phage buffer respectively.
  6. We calculated how much of each material we would need for our plates: for my personal plate, I would use .5 mL arthrobacter ATC 21022, 4.5 mL LB Broth, 5 mL x2 top agar, and 45 microliters 1M CaCL2. For Group 5’s top agar control, we would use 4.5 mL LB broth, 5 mL x2 top agar, and 42.5 microliters 1M CaCl2.
  7. We used a P200 pipette to transfer 42.5 microliters 1M CaCl2 to a new 50 mL conical vial. This was to be used for Group 5’s top agar control.
  8. I used the same P200 pipette to transfer 45 microliters  1M CaCl2 to my own 50 mL conical vial.
  9. We used another pipette to transfer 4.5 mL LB broth into the group conical and 4.5 mL LB broth into my personal conical.
  10. I then added 5 mL of x2 top agar to my conical.
  11. I received .5 mL arthrobacter ATC 21022 to add my conical from the TA Lathan.
  12. I transferred the contents of my 50 mL conical to my agar plate then let it sit for  15 minutes. We also added the top agar to the control plate and let it sit for 10 minutes.
  13. I used a P10 pipette to drop 10 microliters of my enriched sample to the division of my plate marked “E,” 10 microliters of my direct sample to the division of my plate marked “D,” and 10 microliters of a phage buffer to the division of my plate marked “B.”
  14. We placed our plates into a room temperature incubator.

Observations:

  • Top agar solidifies quickly, so it’s better to add top agar last. I had added the top agar to my conical before adding arthrobacter, leading rushing and possible sloppy work.

Results:

  • This experiment yielded me a spot test set up to evaluate at a later date.

Next Steps:

  • My next step is to examine the plate in ~48 hours to see if there is a positive result.
Category: Brandon Reider | LEAVE A COMMENT
August 31

8-24-18 — Supernatant Filtration and Lysate Enrichment

Date: Friday, August 24th, 2018

Title: Supernatant Filtration and Lysate Enrichment

Rationale: The purpose of today’s lab was to filter my supernatant from my 50 mL conical vial to two new vials in order to examine them and use them for a Spot Test next week.

Procedure:

  1. I began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
  2. I lit a burner in the middle of the table in order to keep falling particles from contaminating my equipment or sample from above since the flame creates a circulating air current.
  3. I used a pipette in the aseptic zone to transfer my supernatant from the 50 mL conical to two 15 mL vials.
  4. The two smaller vials filled with supernatant were then spun for five minutes in a small centrifuge in order to separate any heavier particles that may have drifted up since 8-22-18 when I originally centrifuged my 50 mL conical.
  5. Under the fume hood, I used a pipette to transfer the supernatant from my two smaller vials to a 22 micrometer filter in order to separate potential bacteriophages from bacteria too large to fit through the filter.
  6. I transferred 10.5 mL of the filtered lysate to a 15 mL vial and kept 10 mL of filtered lysate in another 50 mL conical.
  7. I then added .5 mL arthrobacter (ATC 21022) to the Enriched sample (50 mL conical vial) in the aseptic zone.
  8. The direct sample was then refrigerated, while the enriched sample was incubated.

Observations:

  • Soil and other particles were present in the supernatant after the day spent out of the lab, meaning it’s important to filter out supernatant soon after being centrifuged in order to keep an uncontaminated sample as well as one that won’t clog the filter.

Results:

  • This experiment yielded me both an enriched and a direct isolation.

Next Step:

  • My next step is to set up a spot test next week to test for plaques and therefore bacteriophages.
Category: Brandon Reider | LEAVE A COMMENT
August 31

8/22/2018 Washing of Soil A

Objective:

  • Wash the soil sample (Soil A) to remove unnecessary material, such as dirt, rocks, and bacteria.
  • Isolate possible bacteriophage found in the soil, in lysate.

Procedure:

  1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
  2. 50 mL tube was obtained that contained the sample of Soil A up to the 12 mL mark. LB broth was then poured into tube up to 35 mL mark.
  3. The mixture in the tube was shaken manually for 15 minutes.
  4. After mixing, the tube was massed in order to find another tube with a similar mass (+/- 0.1g) that it could be placed with in a centrifuge. The 50 mL tube was found to be 55.215 g.
  5. 50 mL tube containing mixture of Soil A and LB broth was centrifuged for 5 minutes.
  6. Liquid found at the top of the soil in the tube was then filtered using a top filter and vacuum. This was to remove the bacteria from the remaining soil. 10 mL of filtrate was obtained from the filter in a 50 mL tube.
  7. Half (5 mL) of the filtrate was put into a 15 mL conical vial and placed in the fridge. This was the direct lysate. Vial was labeled Direct Soil A.
  8. 0.5 mL of Arthrobacter was added to the remaining lysate (5 mL) in the 50 mL tube. This created the enriched lysate. 
  9. Enriched lysate was left to be shaken to be shaken until Monday (8/27).

Observations:

  • After the soil sample was centrifuged the liquid was still murky. This may have lead to difficulty during the filtering,
  • Filtering the lysate was difficult and took longer than other samples. Due to time, only 10 mL was obtained instead of all lysate that was available.

Results:

  • No results to report since test has not been completed.

Next Steps:

  • In the next lab, spot tests and plaque assays will be completed to see if bacteriophages are present in the soil sample, Soil A.

 

Category: Claire Wentzlaff | LEAVE A COMMENT
August 31

Plaque Assay 8/29/18

Rationale

Conduct a plaque assay to confirm the results from the previously conducted spot test. The formation of a plaque will indicate the possible presence of a phage.

Procedure

  1. Use the aseptic technique to clean the area. With gloves on, spray CiDecon on the lab table and wipe dry. Then wipe 70% EtOH on the surface and let the excess evaporate.
  2. Working near an ethanol flame, pipet 10 μL of the filtered enriched lysate created on 8/27/18 into a glass tube containing 0.5 mL of Arthrobacter. Wait 13 minutes.
  3. While the Arthrobacter and the filtered enriched lysate are sitting in the glass tube, begin to make your top agar solution.
  4. Using a 10 mL pipet and a suction device, pipet 8 mL of LB broth into a 50 mL conical tube labeled “EAG, NMN, SS 8/29/18 Top Agar for Plate Assay.”
  5. Using a micropipette, pipet 90 μL of calcium chloride into the 50 mL conical tube.
  6. Wait for the 13 minutes of the Arthrobacter and lysate sitting to be complete.
  7. After the 13 minutes as passed, place 10 mL of top agar into the 50 mL conical tube.
  8. Use a 10 mL pipet to dispense 5 mL of the solution in the 50 mL conical tube into the glass vial containing the Arthrobacter and lysate. Pour the contents of the vial onto a petri dish.
  9. Swirl the contents in the petri dish around to distribute evenly.
  10. Let the plate sit for 10 minutes before inverting and placing into the incubator for 48 hours.

Observation

There were possible plaques that appeared on the spot test conducted on 8/27/18, however they may be confused as air bubbles. When pouring the contents of the 50 mL conical vial onto the petri dish, the solution was no longer completely liquid and had assumed a slightly thicker texture.

Conclusion and Next Steps

The results of the plaque assay will be observed on Friday to determine if there are phages present in the soil collected.

Category: Emily Gaw | LEAVE A COMMENT
August 31

Spot Test 8/27/18

Rationale

Conduct a spot test with the enriched isolation produced on 8/22/18 in hopes of producing a plaque.

Procedure

  1. Use the aseptic technique to clean your lab area. Using gloves, spray CiDecon on the lab table and wipe dry. Then wipe the table with 70% EtOH and let the excess evaporate.
  2. Remove the 50 mL tube from the fridge containing the enriched isolation made on 8/22/18.
  3. While working near an ethanol flame, use a syringe to suction 2 mL of the enriched isolation up and attach a 0.22 μm filter to the end of the syringe. Push down on the syringe to filter the enriched isolation into a micro test tube labeled “EAG 8/27/18 Filtered Enriched Isolation.”
  4. Label a plate “EAG 8/27/18 Spot Test” and divide the plate into three quadrants labeled “direct”, “enriched”, and “negative control.”
  5. Label a 50 mL conical tube “EAG, NMN, SS 8/27/18 Control Top Agar for Spot Test.”
  6. Begin making the control top agar for the experiment by first adding 4.5 mLs of LB broth by using a 5 mL pipet attached to a suction device to the 50 mL conical tube.
  7. Add 42.45 μL of calcium chloride into the conical tube using a micropipette.
  8.  Quickly pipet 5.0 mL of 2X top agar into the 50 mL conical tube and swirl. Then pour the contents in the 5o mL conical tube labeled “EAG, NMN, SS 8/27/18 Control Top Agar for Spot Test” into a plate labeled “EAG, NMN, SS 8/27/18 Top Agar Control.” Let the plate sit for 15 minutes.
  9. While the plate is sitting, get another 50 mL conical tube and label it “EAG 8/27/18 Spot Test Agar.”
  10. In the 50 mL conical tube labeled “EAG 8/27/18 Spot Test Agar” pipet 4.5 mL of LB broth into the container.
  11. Using a micropipette, pipet 45 μL of calcium chloride into the 50 mL conical tube.
  12. Ask Lathan to add 0.5 mL of Arthrobacter into the tube. Then use a 10 mL pipet and a suction device to add 5 mL of 2X top agar to the 50 mL conical tube. Shake to invert.
  13. Pour the contents of the 50 mL conical tube labeled “EAG 8/27/18 Spot Test Agar” into the plate labeled “EAG 8/27/18 Spot Test.” Let the plate sit for 10 minutes.
  14. After 10 minutes, use a micropipette to pipet 5 μL of phage buffer into the negative control section on the plate. Pipet 5 μL of the direct isolation made on 8/22/18 onto the direct section of the plate. Pipet 5 μL of the filtered enriched isolation onto the enriched section of the plate.
  15. Let the plate sit for approximately 8 minutes and then place into the incubator for 48 hours.

Observation

While pouring the 50 mL conical tube labeled “EAG 8/27/18 Spot Test Agar” into the plate, air bubbles formed on the plate. This may be confused with possible plaques after 48 hours of incubation.

Conclusion and Next Steps

In the future, the results of the spot test will be observed and a plaque assay will be conducted with the enriched and direct isolation to confirm the presence of plaques.

Category: Emily Gaw | LEAVE A COMMENT
August 31

Soil Washing 8/22/18

Rationale

Wash the soil sample collected from section 3 of the Baylor campus quadrant map and produce a direct and enriched isolation from the lysate created.

Procedure

  1. Clean the work area by using the aseptic technique. While wearing gloves, spray CiDecon onto the lab table and wipe dry. Spray 70% EtOH onto the table afterwards, wipe, and allow the excess to evaporate.
  2. In the 50 mL conical tube labeled “EAG 8/22/18 Soil C” containing 15 mLs the soil sample collected, add approximately 20 mLs of LB broth or enough to fill the 35 mL line on the tube while working near an ethanol flame.
  3. Shake the 50 mL conical tube vigorously for 15 minutes.
  4. Measure the mass of the 50 mL conical tube by using an analytical balance.
  5. Using a centrifuge, spin the 50 mL conical tube for 5 minutes on a 3000xg rotations/minute setting.
  6. Filter the supernatant into another 50 mL conical tube labeled “EAG 8/22/18 Direct Lysate” by using a 0.22 μm top filter attached to a vacuum. (note: only 5 mLs of lysate was produced using the 0.22 μm top filter and an alternate method was then used to filter the remaining supernatant)
  7. Place the remaining unfiltered supernatant from the top filter in two 15 mL tubes. Make sure that the mass of the two tubes are equal.
  8. Spin the two 15 mL tubes in the centrifuge again with help of a TA for approximately 10 minutes.
  9. While working near an ethanol flame and using a syringe, suction 2 mLs of supernatant from one of the two 15 mL tubes and then attach a 0.22 μm filter to the end of the syringe.
  10. Manually push down on the syringe to filter the supernatant into the 50 mL conical tube renamed as “EAG 8/22/18 Enriched Isolation” until the volume on lysate in the tube reaches 10 mL.
  11. Pour 5 mL of the lysate from the 50 mL conical tube labeled “EAG 8/22/18 Enriched Isolation” into a 15 mL tube labeled “EAG 8/22/18 Direct Lysate.”
  12. In the 15 mL tube labeled “EAG 8/22/18 Direct Lysate,” pour in 0.5 mL of Arthrobacter. Give the tube to the TA’s.
  13. Place both tubes “EAG 8/22/18 Direct Lysate” and “8/22/18 Enriched Isolation” into the fridge.
  14. Clean the lab station using the aseptic technique (step 1).

Observations

After LB broth was added to the initial soil sample in the tube labeled “EAG 8/22/18 Soil C,” the tube had a mass of 53.51 grams. Despite being spun in the centrifuge for 5 minutes on the 3000xg setting, the supernatant that was produced in the 50 ml conical vial was still foggy and dark colored. After being put in the centrifuge a second time, the supernatant was significantly lighter in color and was easier to filter.

Conclusion and Next Steps

Since I was able to make a direct and enriched isolation, coming in on 8/24/18 was not needed. On 8/27/18, the enriched isolation will be used to make a spot test.

 

Category: Emily Gaw | LEAVE A COMMENT
August 31

8-22-18 — Soil Washing

Date: Wednesday, August 22, 2018

Title: Soil Washing

Rationale: The purpose of today’s lab was to isolate potential bacteriophages from dirt, bacterium, and other particles in the soil sample I collected (Soil A*) by filtering the sample into a direct and enriched lysate.

Procedure:

  1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
  2. We lit a burner in the middle of the table in order to keep falling particles from contaminating our equipment or samples from above since the flame creates a circulating air current.
  3. I transferred the sample of Soil A from a plastic storage bag to a 50 mL conical vial in the aseptic zone until the soil reached the 15 mL mark.
  4. I transferred LB broth to the 50 mL conical vial in the aseptic zone until Soil A and the LB broth together reached the 35 mL mark.
  5. I shook the 50 mL conical vial with LB broth and Soil A for 15 minutes (15:11-15:26). This was done in intervals switching between my hand and a vortex machine.
  6. During the mixing process, I weighed my 50 mL conical vial, finding it to be 52.01 g. The weight of the vials are important so that the centrifuge can be balanced without a risk to the rotor being posed by uneven weights.
  7. My 50 mL conical vial was placed in the centrifuge along with other students’ samples at 3,000 revolutions per minute for five minutes. This was done to separate possible bacteriophages from heavier particles such as dirt.
  8. Some students at this point filtered their supernatant to isolate their lysate; however, due to time constraints, my centrifuged 50 mL conical was placed in the fridge to be worked on later in the week.

Observations:

  • The mixed solution of LB broth and Soil A was dark and larger particles were unevenly distributed throughout.
  • After being centrifuged, the dirt was all at the bottom of the 50 mL conical. It was tightly packed and smoothed over.
  • The resulting supernatant was mid-to-dark yellow in coloration after being centrifuged, similar to a slightly dehydrated urine sample.

Results:

  • This experiment yielded me a 50 mL conical with a separated supernatant to be filtered at a later date.

Next Steps:

  • My next step is to filter my supernatant before examining my lysate for bacteriophages.

*Soil A is my first sample of soil, found near a red oak with a few dying branches outside of the Dawson residence hall at Baylor University.

Category: Brandon Reider | LEAVE A COMMENT
August 31

Date: August 22nd 2018 

Title : Soil Sample Washing  

Rationale : We washed soil samples that we collected from our designated area in hopes of isolating a possible bacteriophage that maybe present in the soil.  

Procedure:  

Preventing Contamination:  

  • In hopes of preventing contamination we cleaned the table with Cidecon and wiped the table dry. Then we cleaned the table with 70% ethanol and let it air dry.  
  • We created an aseptic zone with an ethanol flame, so that while transferring materials we can avoid further contamination 

Creating the Soil Sample:  

  • I added the LB Broth until the 35 mL mark to the soil sample of 12 mL I had collected the previous day. Therefore, it was a total of 22.5 mL of LB broth  
  • Made sure while transferring the broth we stayed near the aseptic zone.  
  • One mistake that I made was placed the cap on the table while focusing on the LB Broth, so will have to see in the future if that has any effects on my results  
  • After I added the LB broth to the soil we shook the tube for 20 minutes.  
  • I weighed the mass of the entire tube and it turned out to be 53.23 grams.  
  • After finding the mass of our soil and LB broth solution we found a partner who had a mass within the range of plus or minus 0.1 grams. This was important for when we were using the centrifuge. 
  • In order for accurate results out of the centrifuge we had to make sure that there had to be a equal balance of weight on either side of the centrifuge.
  • After the 20 minutes of shaking, we placed the test tubes into a centrifuge.  
  • The use of the centrifuge is to separate the liquid and the heavier particles

Creating the Enriched and Direct Lysate Samples 

  • After our test tubes came out of the centrifuge we extracted the supernatant, in other words the liquid part at the top half of the test tube, using a dropper.  
  • We put the supernatant in another vacuum filter, which can filter out some of the smallest particles, but leaves bacteriophages behind.  
  • While using the dropper, I avoided any big particles left behind such as small pieces of bark from my sample 
  • I also left behind the pellet, which is the heavier material at the bottom of the test tube 
  • After extracting most of the supernatant into the vacuum we waited as it filtered through  
  • Since the filter was taking a long time, I filtered 10 mL which I put in a separate test tube and labeled as my enriched sample  
  • Then I poured the rest of the unfiltered sample into another test tube and labeled it as an unfiltered direct sample.  
  • I stored both my enriched and direct sample in the fridge.  

Observations/ Results/ Data : 

  • I observed that as I shook my sample for 20 minutes the color turned to be a chocolatey color and started foaming up
  • I will have to keep an eye on whether any contamination occurred because I placed the cap on the table  
  • There were no results or data that were collected this time, as we just washed our soil sample and tried to separate possible phages with the rest of the soil particles 

Conclusions/ Next Step  

  • Next time we come to lab, I will need to filter my direct sample to use it for testing on the spot test.  
  • I will also be performing a spot testing to check if our sample has phages, and if not I might have to retake a soil sample and retry hoping to find a phage in the new sample. 

 

Date: August 27nd 2018 

Title : Spot Testing of Soil Sample

Rationale :  We are performing a a spot test using the direct, enriched and control soil samples on a petri dish in hopes of finding a plaque which shows us that there are phages in our sample  

Procedure:  

First we followed the same cleaning procedure as last time for lowering the chances of contamination, and also set up a ethanol flame.  

Creating the Top Agar Solution for Control  

  • I brought the enriched sample and direct sample from the fridge and placed on my lab table  
  • We also got syringes, four 50 mL test tubes, and four petri dishes  
  • I took 2 mL of the enriched sample using a syringe and put it into micro test tubes to use for the lab.  
  • I did this in an aseptic zone  
  • After using the syringe, I disposed the syringe  
  • I then labeled my petri dish. I divided it into 3 different sections on the bottom and labeled them control, enriched, and direct.  
  • My lab group and I decided to do the control first, so we worked on that first  
  • We first added 4.5 mL of the LB broth using pipettes into a larger test tube (50 mL)  
  • Using the formula C1V1 = C2V2 we found the required amount of CaCl2 is 42.75 ul.  
  • So, then we added 42.75 ul of CaCl2 into the control test tube.  
  • We used the pipette marked 20-200 ul.  
  • We also made sure to change the pipette tips between uses.  
  • Next, we added the Top Agar into the LB broth and CaCl2 solution using a pipette, since Top Agar sets quickly and therefore would have needed to work fast.  
  • After adding the top Agar our solution was a total of 10 mL.  
  • We then shook the solution a few times to make sure it was an even mixture.  
  • Lastly, we poured the solution into a petri dish and let is sit to solidify.  
  • While adding and mixing the solutions we made sure that we were near an aseptic zone.  

Creating Individual Spot Testing  

  • My lab group decided to create a big batch, so we tripled all the measurements  
  • We first obtained a new pipette tip and used the leftover LB Broth at our tables to pipette 4.5 mL of LB broth into each of our tubes  
  • After changing pipettes, we added 45 ul of CaCl2 in each tube as well.  
  • After adding these two ingredients to make our individual plates, we obtained 0.5 mL of Arthro from the hood, almost completing our solution. 
  • We then finally added the 5.0 mL of Top Agar using a glass pipette and pipette bulb. 
  • Also while adding all the solutions we made sure that we were in the aseptic zone. 
  • I ended up with 10.0 mL of my Top Agar solution and let it sit for 10 minutes so that it will solidify  
  • An additional step I did was to filter the unfiltered direct solution which I did while waiting for my Top Agar to set.  
    • Since the solution was untouched for a few days, the solution had separated the heavier parts and the liquid remained at the top  
    • Using a syringe I extracted 2mL of the liquid part of the direct solution and put it into a mini test tube basically filtering it a little more  
  • Using a pipette I added 5 microliters of the enriched, direct and buffer, which was provided by the professor, into its respective parts on the petri plate.  
  • I closed it and let it sit for about 10 minutes so that there was enough time for the agar to soak up each of the solutions. 
  • Then lastly I placed the petri dish in the incubator.  

Observations/ Results/ Data: 

  • Some observations were as I was pouring in the Top Agar I realized there was this bubble on the side of the plate, which did not pop.  
  • Some of the other observations in this experiment was making sure that we made the correct mixture for the solution .  
  • Another observation we had to make was which pipette to use for what and in general the max and min pipetting values for each 
  • Hopefully the result will be that there is a plaque, showing to us that there is a phage in the sample.  
  • There was no data or results for this part of the experiment   

Conclusions/ Next Step  

  • The next step is that we will look at our spot test and see if we found a plaque or not. Depending on the results we will proceed with a plaque assay  
  • If there is plaque on the spot test, then we will perform an enriched and direct petri dish for the plaque assay  
  • If there is no plaque on the spot test, then we will only perform an enriched plaque assay  
  • Some conclusions that we can get from this test is whether we found a phage or not.  
  • The control can show us if there is any source of contamination from the Top Agar solution

 

Date: August 29nd 2018 

Title : Plaque Assay  of Soil Sample

Rationale :  We are performing a plaque assay, to either confirm that we have a phage or to retry to see if the phage was missed in the spot test.  

Procedure:  

Making the Top Agar solution for Plaque Assay:  

  • After cleaning up the table with Cidecon and 70% ethanol, we set up an ethanol flame for a aseptic zone.  
  • Obtained the spot test from the incubator and inspected the results  
  • Decided to perform an enriched plaque assay since I think that my spot test had plaque in the enriched portion of the petri dish  
  • I obtained the enriched lysate from the fridge.
  • I then obtained the 0.5 ul Arthro and labeled it with name and information. 
  • Then I used a 0-10 ul pipette to add our lysate of 10 ul to our 0.5 ul Arthro near the aseptic zone  
  • Then we were instructed to waited for 15 minutes, but in the meantime, we decided to make our Top Agar solution  
  • I took the 8 ml of the LB broth and added it to our 50 ml tube using the pipette  
  • We took 90 ul of the CaCl2, which was passed out in class, and pipetted it into our solution in the 50 mL tube.  
  • My lab group and I decided to wait some more time before we added the Top Agar into the solution so that the solution doesn’t solidify fast, since by this time only 7 minutes had passed.  
  • We proceeded with our experiment at the 14 minutes mark  
  • Lastly, we added 10 mL of Top Agar to our solution using our glass pipette and bulb  
  • We then stirred the solution to make sure that its evenly mixed and proceeded by pouring 5 mL of the entire solution into each of our plates with the Top Agar and the Arthro  
  • There was one bubble which showed up which could later be mistaken for a plaque, so I noted it. 
  • I waited 10 minutes before placing the solution into the incubator upside down.  
  • In the meantime, I wrapped up my spot test and kept it in a different place to keep the test for plaque in case one doesn’t show up in the plaque assay  

Observations/ Results/ Data: 

  • Some observations from the results of the spot test:  
  • Enriched had a spot which looks like a plaque.  
  • Direct – sort of had a clearing but not completely sure if plaque  
  • Buffer – had very little clearing kind of looked hazy but probably not plaque 
  • In my plaque testing plate there was one bubble while pouring the Top Agar 

    Spot Test – Can see a possible plaque on the enriched portion

  • Plaque Essay – noticed a bubble

Possible Question:  

  • Does different tree species affect the Arthrobacter phage we are trying to find?  

 

Conclusions/ Next Step  

  • There seems to be phage in my soil sample from the spot test in the enriched section of the plate.
  • Just to confirm it we performed a plaque assay, which we will observe next time.
  • If there is some clearing in the plaque assay, then we can use the plaque to continue with the next portion of our experiment.  
  • If there is no plaque on the plaque assay plate, then we will use the plaque that we found on the spot testing to continue with our experiment.  
Category: Sona Subramanian | LEAVE A COMMENT
August 31

8/29/18 Plaque Assay of Enriched Lysate

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

Analyzing Spot Test and Conducting Plaque Assay for Soil A (08/29/18)

Rationale:

Based off the results from the plates that had been spot tested on Monday (8/27), the experiment will proceed to perform a plaque assay to determine whether or not there are bacteriophages present in the soil sample that target arthrobacter.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Analyzed plates from Monday and decided to continue with a plaque assay.
  3. Performed the following calculations to determine how much of the materials was required.

Calculations

Original Recipe

 X4 X3*

2 mL LB Broth

 8 mL LB Broth

6 mL LB Broth

2.5 mL 2X TA

10 mL 2X TA

N/A

22.5 μL CaCl2

 90 μL CaCl2

67.5 μL CaCl2

*This calculation was performed after step 9, when the lab group recognized that only the original recipe amount was added.*

  1. Obtained two plates (one for self and another one to serve as a control).
    • Labeled plate for self as “plaque assay enriched lysate Soil A.”
    • The control plate was split into four sections to be shared with four different groups due to a shortage of plates.
  1. Through aseptic technique (over an EtOH (100%) flame), used a P200 micropipette to add 22.5 μL CaCl2into a new 50 mL conical vial.
    • Labeled 50 mL conical vial “Team 2 TA 8/29/18.”
  1. Used a cartwheel pipette with a 5 mL tip to add 2.0 mL of LB Broth to “Team 2 TA 8/29/18” conical vial.
  2. Unfortunately, this vial spilled, so lab group started again with a new 50 mL conical vial.
    • Labeled 50 mL conical vial “Team 2 TA 8/29/18.”
  1. Through aseptic technique (over an EtOH (100%) flame), used a P200 micropipette to add 22.5 mL CaCl2into “Team 2 TA 8/29/18” conical vial.
  2. Used a cartwheel pipette with a 5 mL tip to add 2.0 mL of LB Broth to “Team 2 TA 8/29/18” conical vial.
  3. Used a P200 micropipette to add 67.5 μL CaCl2into “Team 2 TA 8/29/18” conical vial to make a total of 90 μL CaCl2.
  4. Used a cartwheel pipette with a 10 mL tip to add 6 mL of LB Broth to “Team 2 TA 8/29/18” conical vial to make a total of 8 mL of LB Broth.
  5. Used a cartwheel pipette with a 10 mL tip to add and mix 10 μL of enriched isolated lysate into a test tube with 0.5 mL arthrobacter in it.
    • Labeled test tube “KEA 8/29/18 Arthrobacter with Enriched Lysate.”
  6. Allowed 15 minutes for the “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube to set.
  7. Used a cartwheel pipette with a 10 mL tip to add and mix 1 mL of 2X TA into the “Team 2 TA 8/29/18” conical vial.
  8. Used cartwheel pipette with a 10 mL tip to drop 1 mL of the Top Agar onto the control plate.
  9. Used a cartwheel pipette with a 5 mL tip to add and mix 5 mL of 2X TA into the “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube.
  10. Used a cartwheel pipette with a 5 mL tip to transfer over “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube contents onto “ERB 8/29/18 plaque assay Soil A” plate.
    • Crossed out “ERB” and labeled it with “KEA.”
  11. Placed plates in incubator at room temperature.
  12. Cleaned lab counter with CiDecan and EtOH (70%).

Results from the Spot Test of Soil A:

-On “Team 2 8/27/18 Soil A TA Control” plate, there were yellow spots indicating that the Top Agar was contaminated. The picture below shows this plate.

-On “KEA 8/27/18 Spot Test” plate, there were air bubbles that looked like plaques and yellow spots indicating contamination. The pictures below shows this plate.

Observations:

-When transferring over “KEA 8/29/18 Arthrobacter with Enriched Lysate” test tube contents, air bubbles formed and solidified on the plate. The picture below shows this plate.

Next Steps:

On Friday, check results of Plaque Assay. From these results, different things might happen. If it is contaminated, we might try running either a spot test or plaque assay with the same soil sample, or maybe filter the soil sample again. If it is negative, we will start all over with a new soil sample. If it is positive, we will celebrate and continue the experiment on the next lab day.

Category: Kathryn Adkins | LEAVE A COMMENT