August 30

Spot Test 8.27.18

Spot Test 8.27.18

Rationale)

To conduct a spot test using enriched and direct isolation lysate from Soil Sample A in the hopes of achieving plaque that can then be picked and the phage that caused it isolated, and if no plaque is found determine the next steps in regards to conducting a plaque assay and potentially collecting another soil sample.

Scientific Question)

Does the presence of arthrobacter phage vary amongst oak tree species found on Baylor’s campus?

Procedures)

1. Set up an aseptic zone using CiDecon, 70% ethanol, and an ethanol flame.

2. Retrieve the 50mL conical vial of “Soil A Lysate Pt2 Enriched” from the refrigerator. Under aseptic conditions use a .22μ filter syringe apparatus and syringe, filter about 1.25mL of “Soil A Lysate Pt2 Enriched” into a micro test tube labeled, “8.27.18 NMN Lysate for Spot Test”, put the remaining unfiltered lysate back into “Soil A Lysate Pt2 Enriched”

3. Retrieve plate with base agar and divide/label in three sections: Direct Isolation, Enriched, and Negative Control, also label the plate as Spot Test NMN 8.27.18, retrieve another plate with base agar and label as the “Top Agar Control 8.27.18 EAG, NMN, SS”.

4. Retrieve 50mL conical vial and label 8.27.18 “Top Agar for Spot Test Control”.

5. For the “Top Agar for Spot Test Control” use M1V1=M2V2 to calculate for the total amount of 1M CaCl2 required for the Top Agar solution to have the CaCl2 be 4.5 mM, this number ends up being 42.75μL, use a micropipette under aseptic conditions to transfer this amount to the 50mL conical vial labeled “Top Agar for Spot Test Control”.

6. Using a 10mL serological pipette transfer 4.5mL of LB broth to the 50mL conical vial labeled “Top Agar for Spot Test Control”  under aseptic conditions, unfortunately, the cap of the vial “Top Agar for Spot Test Control”  was moved out of the aseptic zone in which this procedure was suppose to be conducted.

7. Using a 10mL serological pipette transfer 5mL of top agar from its container to the 50mL conical vial “Top Agar for Spot Test Control” under aseptic conditions and seal with the cap.

8. Mix 50mL conical vial “Top Agar for Spot Test Control” by inverting the vial repeatedly, after doing so for about 15 seconds pour into the plate labeled “Top Agar Control 8.27.18 EAG, NMN, SS” under aseptic conditions. Cover the plate and let rest for 15 minutes before placing in the incubator.

9. Retrieve a 50mL and label “NMN Top Agar Spot Test 8.27.18”

10. Retrieve LB broth and 4.5mL to “NMN Top Agar Spot Test 8.27.18”  under aseptic conditions using a 10mL serological pipette.

11. Retrieve the red-capped vial of .5mL Arthrobacter and pour into “NMN Top Agar Spot Test 8.27.18” under aseptic conditions.

12. Add .45μL of 1M CaCl2 to vial “NMN Top Agar Spot Test 8.27.18”, as was derived by M1V1=M2V2 in order to achieve a final molarity 4.5mM of CaCl2, using a 20-200μL micropipette under aseptic conditions.

13. Add 5.0mL of 2xTop Agar to “NMN Top Agar Spot Test 8.27.18” using a 10mL serological pipette under aseptic conditions, cover “NMN Top Agar Spot Test 8.27.18” with the cap and swirl to incorporate for 30 seconds.

14. Pour the 50mL conical vial labeled “NMN Top Agar Spot Test 8.27.18” into the plate labeled “Spot Test NMN 8.27.18” under aseptic conditions, cover the plate and let solidify for 15 minutes.

15. After 15 minutes has passed and the plate “Spot Test NMN 8.27.18” has solidified use a 0-10μL micropipette to transfer 6μL of  Direct Isolation from the vial labeled “NMN Soil A Direct Isolation 8.24.18” onto the corresponding zone of the divided and labeled plate “Spot Test NMN 8.27.18”, repeat this with “8.27.18 NMN Lysate for Spot Test” and Phage Buffer Solution. All of this is done under aseptic conditions.

16. Let the plate rest for about 6 minutes and then place into the incubator. Clean the area and leave.

Observations)

I observed some air bubbles in my top agar which could be mistaken as plaques if not careful. I also observed that there were minor instances in which contamination could have occurred and affected the results, additionally the top agar we produced was much more golden in appearance compared to the base agar.

                               

Conclusions/Next Steps)

The next time I do this I need to wait longer, closer to 15 minutes, for the samples to absorb onto the top agar instead of the 6 minutes I waited for. I also need to take greater care to avoid contamination of materials and equipment that should be kept in an aseptic zone as there were minor instances in which contamination could have occurred when I conducted the procedures. We also now have a spot test that can be used to determine if we have a potential phage we could isolate based on positive or negative plaque formation, which will be determined on 8.29.18. I will also conduct a plaque assay on 8.29.18 to provide further information about the presence or absence of phage in lysate derived from Soil Sample A.

Category: Nathan Newton | LEAVE A COMMENT
August 30

8.29.18- Plaque Assay

8.29.18- Plaque Assay

Results from Monday: After 48 hours in the incubator, the results from the Spot Test were ready to be analyzed. Upon analysis, the plate had no trace of plaque. The negative control plate properly displayed a bacterial lawn with no disruption, but neither enriched lysate nor direct isolate caused any formation of plaque on the experimental plate. The negative control section of the experimental plate also showed no plaque formation or disruption of bacterial lawn, which was to be expected because any other result would have been indicative of contamination.

Rationale: Plaque Assay test needed to be done to determine whether or not results from the Spot Test on Soil Sample A could be confirmed by presence or lack of a plaque (since the description above shows that Monday’s Spot Test showed negative results for presence of a bacteriophage, a result to confirm this would be a lack of plaque on the Plaque Assay). In conjunction, the Plaque Assay was performed to gain further comprehension of the procedure and how to perform each step.

Procedure:

  1. Lab space cleaned with CiDecon (sprayed on surface and wiped until dry) and 70% ethanol (sprayed on surface and wiped until 75% dry).
  2. Ethanol burner lit to establish aseptic zone. Each addition to tubes was done in this aseptic zone in order to prevent particles from falling into the samples and causing contamination.
  3. (In aseptic zone) Tube with 0.5mL of Arthro was obtained and labelled HMB Arthro 8/29/18.
  4. (In aseptic zone) 10μL of lysate added to tube with 0.5mL of Arthro. This mixture was set to the side to give the Arthro and lysate to interact while the rest of the experiment was set up.
  5. 50mL Conical tube was obtained.
  6. (In aseptic zone) 8mL LB Broth was added to the Conical Tube
  7. (In aseptic zone) 90μL CaCl2 added to the Conical Tube
  8. Plate with agar was obtained and labelled “HMB Plaque Assay 8/29/18”.
  9. (In aseptic zone) 10mL of 2X Top Agar was added to the Conical Tube with LB Broth and CaCl2
  10. 2X Top Agar, LB Broth, CaCl2 was mixed in Conical Tube (components of overlay solution) by gently shaking the tube back and forth.
  11. (In aseptic zone) 4.5mL of overlay solution was added into tube containing 0.5mL of Arthro and lysate. Mixture was swirled to mix.
  12. (In aseptic zone) Overlay solution was poured onto plate labelled “HMB Plaque Assay 8/29/18” and let settle.
  13. 1mL of overlay solution from Conical Tube added to Control plate shared by groups 3, 4, 7, and 8. This was done due to the shortage of plates in the lab because of contamination.
  14. Overlay on experimental plate was allowed to solidify and was stored in the incubator to be checked on Friday.

   

Observation:

  • Plaque Assay plate had many bubbles and the surface appeared to be slightly bumpy. This could be a point to examine if problems occur.
  • Excess overlay solution was observed in Conical Tube. The mathematics seemed to excuse this observation (3 individuals used 4.5mL out of 18mL in tube resulting in 4.5mL leftover. 1 mL was used for the control plate, therefore allowing 3.5mL to be excess in the Conical Tube), but the discrepancy should be reported.
  • Overlay solution was a golden yellow, as seen before in the Spot Test.

Results from the Plaque Assay cannot be obtained until plate has had time for bacteria to grow and be influenced by any present phages. Therefore, results from plaque assay will be found on Friday during open lab session.

Conclusions drawn: Based on the results of the Spot Test, it is more than likely that the original sample obtained does not have bacteriophages. The lack of plaque shows that there is no death of cells, which is the trademark of a bacteriophage as they reproduce. However, the Plaque Assay may be able to overturn this conclusion if it does contain plaques.

Next Steps: Control and experimental plate will be observed on Friday to determine whether or not the results of the Spot Test are supported or not. If there are no plaques present, it is confirmed that the sample did not contain bacteriophage and another sample will be needed. If there are plaques present, they will be picked and processed further.

Update: Plate was observed and confirmed the negative result for presence of a plaque or bacteriophage. The bacterial lawn was not formed particularly well (as seen in picture below), but there was still no presence of a plaque. The next steps to take from this result will be obtaining a new Soil Sample that ties in with the overarching research question to aid in developing data to answer the question.

Category: Henry Burns | LEAVE A COMMENT
August 30

8/29/18 Plaque Assay with Spot Test Results

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:

From our Spot Test Monday 8/27/18, we were all hoping to find a plaque, meaning our Bacteriophage were present from our soil collection. Our group was the only full group (1/8)that possibly had plaque present. So the purpose of the Plaque Assay is to solidify our results form our spot test. It will confirm our results from our spot testing.

Materials:

  • Lysate
  • PY Broth
  • PY 2X TA
  • 1M Calcium Chloride
  • .5mL of Arthobacter host
  • Pipettes
  • Agar Plate

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.

We then started our Plaque Assay

  • We got our Spot Test Dishes from the incubator. We then checked to see if any plaque had formed. Dr. Adair and one of the TA’s said that our Spot Test could potentially have Bacteriophage.
  • Instead of doing two Plaque Assays (since we did have plaque growth form our Spot Test, we were supposed to perform two plaque assays), we did one Plaque Assay since we did not have petri dishes for the entire class. We created a Plaque Assay from our Enriched Lysate, to test if our Spot Test was accurate.
    • Formula “recipe” (figure 1).
      • .5mL Arthobacter + 10 microliter Lysate
      • 2.0 LB Broth
      • 2.5mL 2X TA
      • 22.5 microliter of Calcium Chloride
        • side note this formula x4 since we have three partners plus our group control plate, which the control was split four ways due to the fact we did not have enough petri dishes for the entire class.
  • We then got 0.5mL Arthobacter from the hood, and we then added 10 microliters of our Lysate that was stored in a cap, that was in the refrigerator from the Spot Test. We then let this Solution set for about 12 minutes +/- one minute.
  • We then started to make our agar solution for all of the plates, including the TA control.
  • Added 8mL LB Broth into our 50mL vial.
  • Added 10 microliters of Top Agar.
  • Added 4.5mL of this solution we made in our 50mL vial onto our individual dishes.
    • Side note: each transferred the solution using different pipettes, but this was done outside of the aseptic zone. Result being possible contaminations.
  • We then let the solution sit for about 10 minutes.
  • We then put our dishes in the incubator, upside down.
  • We cleaned our lab space, and one of the TA’s took our Spot Tests to let them grow.
    • Side note- Friday during open lab, we will know if both the spot test grew more plaque, and to see if our Plaque Assay results help support our spot test results.

Plaque Assay Steps (figure. 1)

Spot Test Results

TA Control for Four groups

 

Results:

We had a small plaque appear from the Spot Test, and hopefully the same from the Plaque assay that was done in the lab Wednesday 8/29/18. Results will be known Friday 8/31/18.

Analysis:

This procedure was not hard at all after knowing what to expect from having done the Spot Test a few days before. It was a joy to see the small plaque and to know that group 6 all had successful results. The only thing that was hard for us was to ensure everything thing was done in an aseptic zone. This lab was better, but it could have been better regarding the usage of the pipettes in an aseptic zone.

Future:

We will use the data from the Plaque Assay to determine whether or not we actually have Phage present or not. If not, then we would just repeat the Plaque Assay, and see if our plaque from the Spot Test had grown or not. If not, then ultimately we would have to recollect soil and do this whole procedure again.

 

 

Category: Michael Lum | LEAVE A COMMENT
August 30

08/27/2018- Spot test

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

Spot Testing of Soil A (08/27/18)

Rationale:

By setting up and running a spot test on the “KEA 8/24/18 enriched,” it will become apparent whether or not there are bacteriophages in the lysate that go after arthrobacter.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Used aseptic technique (over a lite EtOH (100%) flame) to filter with a 3 mL syringe with a 0.22 μm tip the enrichment isolation from the 50 mL conical vial labeled “KEA 8/24/18 enriched” into a microcentrifuge tube.
    • This microcentrifuge tube was labeled “KEA 8/27/18 FS lysate Soil A.” FS stands for filtered sterile.
  1. Obtained two petri dishes (one for self and other to serve as a control Top Agar).
    • The petri dish for self was divided into thirds and sections were labeled: “Direct isolation Soil A,” “FS lysate Soil A,” and “Neg. Control.” The side of this petri dish was labeled “KEA 8/27/18 Spot Test.”
    • The petri dish that served as a control Top Agar was labeled “Team 2 8/27/18 Soil A TA Control.”
  1. The following calculations were conducted before proceeding to the next steps.

Calculations:

Conversion factors:

1,000 μL = 1 mL

1 M =1,000 mM

Equation:

C1V1 = C2V2, where C is concentration and V is volume

For “KEA 8/27/18 Spot Test,”

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

(1,000 mM) V1 = (4.5 mM) (10,000 μL)

V1 = (4.5 mM) (10,000 μL)/(1,000 mM)

V1= 45.0 μL

For “Team 2 8/27/18 Soil A TA Control,”

(1 M) V1= (4.5 mM) (9.5 mL)

(1,000 mM) V1 = (4.5 mM) (9,500 μL)

V1 = (4.5 mM) (9,500 μL)/(1,000 mM)

V1= 42.8 μL

  1. Used a P200 micropipette to transfer 45 μL of 1 M CaCl2into a new 50 mL conical vial.
    • This 50 mL conical vial was labeled “KEA 8/27/18 TA.”
  1. Used a P200 micropipette to transfer 42.8 μL of 1 M CaCl2into a new 50 mL conical vial.
    • This 50 mL conical vial was labeled “Team 2 8/27/18 TA.”
  1. Used a cartwheel pipette with a 5 mL tip to transfer 4.5 mL of LB Broth into both “KEA 8/27/18 TA” and “Team 2 8/27/18 TA” conical vials.
  2. Under a clean hood, added 0.5 mL of arthrobacter with a micropipette to “KEA 8/27/18 TA” conical vial.
  3. Used a cartwheel pipette with 10 mL tip to transfer 5 mL of 2X TA to both “KEA 8/27/18 TA” and “Team 2 8/27/18 TA” conical vials.
  4. Poured “KEA 8/27/18 TA” conical vial into “KEA 8/27/18 Spot Test” petri dish and “Team 2 8/27/18 TA” conical vial into “Team 2 8/27/18 Soil A TA Control” petri dish.
  5. Allowed 10 minutes to pass for Top Agar to solidify in both petri dishes.
  6. Used a P10 micropipette to add a 10 μL drop of direct isolation into the “Direct isolation Soil A” section on the “KEA 8/27/18 Spot Test” petri dish in an aseptic zone with EtOH (100%) flame.
  7. With a fresh micropipette tip, used a P10 micropipette to add a 10 μL drop of FS lysate into the “FS lysate Soil A” section on the “KEA 8/27/18 Spot Test” petri dish in an aseptic zone with EtOH (100%) flame.
  8. With a fresh micropipette tip, used a P10 micropipette to add a 10 μL drop of phage buffer into the “Neg. Control” section on the “KEA 8/27/18 Spot Test” petri dish in an aseptic zone with EtOH (100%) flame.
  9. Placed the petri dishes in incubator at room temperature.
  10. Cleaned lab counter with CiDecan and EtOH (70%).

Observations:

  • When the Top Agar solidified, air bubbles formed. In the “KEA 8/27/18 Spot Test” plate, there were many bubbles in the “FS lysate Soil A” section and a few in the “Direct isolation Soil A” section. The picture below displays these air bubbles.

  • When the 10 μL drop of FS lysate was placed with the P10 micropipette on the “KEA 8/27/18 Spot Test” plate, the drop hydroplaned over to be on top on the line separating the “FS lysate Soil A” and “Direct isolation Soil A” sections.

Next Steps:

On Wednesday, the spot test will be analyzed to see whether or not it contained a bacteriophage that specifically targets arthrobacter. If the plates are positive, the experiment will continue with a plaque assay. If the plates test negative, the experiment might either try a plaque assay or decide to start all over with a new sample of soil.

Category: Kathryn Adkins | LEAVE A COMMENT
August 30

8/29/2018- Plaque Assay Soil A

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 29

August 27, 2018 Spot Test Soil A

Rationale: To perform a spot test to determine if there are any arthrobacter bacteriophages in the enriched lysate sample.

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

Description of Procedure:

  1. The first step in this procedure was to clean the lab station. This was done with CiDecon and 70% Ethanol. An aseptic zone was created with a 100% Ethanol burner being lit.
  2. First the enriched lyste was filtered using a sterile 0.22 uL filter to filter the added arthrobacter out of the sample. 2 mL were removed from the enriched lysate, and 1.5 mL was filtered into a 1.5 mL micro centrifuge tube, using a sterile syringe. The tube was labeled EF, which stands for filtered sterilized enriched lysate.
  3. 4 petri dishes were then obtained, one for each person in Group 4 and one for the Top Agar control. My individual plate was labeled LIP 8-27-18 SP1 (Spot Test 1), and three sections were drawn onto the bottom of the plate, one for the enriched, direct, and negative control spot tests. The Control plate was labeled Group 4 8-27-18 Control TA.
  4. Next, the top agar was made in a 50 mL tube. The tube was labeled LIP 8-27-18 TA1 (Top Agar 1). The below calculation was done to determine the amount of CaCl2 needed.
  6. The below calculation was done to determine the amount of CaCl2 needed for the control plate.
  8. 4.5 mL of LB Broth was added first in an aseptic zone with a sterile pipette, to both the experimental and control tubes.
  9. Next, 0.5 mL of arthrobacter was added to the experimental tube, but not the control tube.
  10. 45 uL of CaCl2 were added to the experimental tube, while 42.75 uL of CaCl2 were added to the control tube.
  11. Lastly, the 2x TA was added to both the control and experimental top agar solutions and gently mixed with the micro pipette.
  12. The Top Agar solutions were then poured onto the correct labeled petri dishes.
  13. The plates were then allowed to sit for 10 minutes until the Top Agar solidified.
  14. After the plates set, 10 uL of the enriched and direct lysates, as well as the phage buffer for the negative control, were spotted into their correct areas on the petri dishes. Nothing was added to the control TA.
  15. The liquids were then allowed to absorb into the agar for 15 minutes.
  16. The plates were then incubated until Wednesday. This process was complete at 4:35.
  17. The  lab station was cleaned before leaving. This was done with CiDecon and 70% Ethanol. All used materials were properly disposed of.

Continue reading

Category: Lucy Potts | LEAVE A COMMENT
August 29

August 29th 2018 Plaque Assay Soil A

Rationale: To visually confirm or deny the presence of plaques in the soil, after negative results on a spot test.

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

Description of Procedure:

  1. The workstation was cleaned with CiDecon and 70% Ethanol before the procedure was started. A 100% Ethanol burner was lit to form an aseptic zone.
  2. To perform a plaque assay with the enriched lysate, the first step was to obtain 0.5 mL of arthrobacter. To this 10 uL of enriched lysate was added. This tube was labeled LIP 8-29-18 AEPA (Arthro Enriched Plaque Assay). This was allowed to sit for 15 minutes.
  3. While the arthrobacter and enriched lysate were allowed to sit, The Top Agar solution was created. 8.0 mL of LB Broth was added to a 50 mL tube. The Top Agar for Group 4 was made in one tube, as well as the control top agar. This tube was labeled Group 4 8-29-18 TA Plaque Assay.
  4. Next 90 uL of CaCl2 was added to the LB Broth. Because the solution contained 5 mL total, the amount of CaCl2 for each person was 22.5 mL, but since this TA was being used for three group members and a control plate, this number was multiplied by 4, to get 90 uL total.
  5. 10 mL of 2X TA was then added to the LB Broth and CaCl2.
  6. After, 4.5 mL of the TA solution was added to the tube containing arthrobacter and enriched lysate for each member of Group 4. It was mixed and then immediately poured on to a petri dish for each group member. My plate was labeled LIP 8-29-18 PA1 (Plaque Assay 1).
  7. For the control TA plate, 1 mL of the TA solution was added to a petri dish. This control plate was shared by groups 3, 4, 7, and 8, so it was divided into 4 sections. Group 4’s 1 mL was put only into the quadrant labeled Group 4.
  8. The plate was then allowed to sit for 10 minutes to solidify and was then stored in an incubator inverted until Friday, 8-31-18, at 2:00 pm, approximately 46 hours from the time it was finished. The time the procedure was complete was 3:40 pm.
  9. The workstation was the cleaned with CiDecon and 70% Ethanol. All used materials were properly disposed of.

Observations/Results/Data:

Observations:

  • The top agar solidified much faster with the plaque assay than with the spot test.
  • Due to limited plates, 4 control top agar solutions were done on one plate for groups 3, 4, 7, and 8.
  • There were some bubbles on the set plate when the procedure was completed.

Results: The procedure was completed on time and the plates will be stored in an incubator inverted until Friday 8-31-18.

Interpretations/Next Steps/Conclusions:
The procedure was complete on time. It went smoothly and was done correctly. The plate will now be allowed to grow until 8-31-18 at 2:00 pm. If there are plaques, then they will be picked. If there are no plaques, a new soil sample will need to be found.

Category: Lucy Potts | LEAVE A COMMENT
August 29

Plaque Assay 8/29/18

Rationale: I am conducting a plaque assay to confirm whether or not the plaque I found in my spot test done on soil sample B from tree A was truly due to the presence of a bacteriophage.

Procedure:

  1. Mixed diluted enriched lysate made in the previous procedure with 0.5mL of arthrobacter

    and let sit. Totaled about 12 minutes of letting sit before being poured in the plate.

  2. Used aseptic technique to create an aseptic zone.
  3. Made top agar by mixing 8mL of LB broth, 90μL of CaCl2 and 10mL of 2X top agar.
  4. I pipetted 4.5mL of the solution into my tube of arthrobacter and enriched lysate and

    pipetted up and down to mix.

  5. I then poured the mixture onto the plate and let dry for 10 minutes.
  6. Due to a lack of plates we split a plate in four and poured 1mL of our top agar without

    any arthrobacter into one of the quadrants to use as a TA control.

  7. I stored my plate in the incubator for 48 hours.

Observations:
Plaque assay returned negative.

Next steps:
Due to the negative results of the plaque assay I will have to go look for more soil and test once again.

Category: Sriram Avirneni | LEAVE A COMMENT
August 28

Saabye Blog Post 1!

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.

Category: Uncategorized | LEAVE A COMMENT