August 31

Spotting Test results & Plaque Assay for Soil A 8.29.18

Research Question:

…………To find out how the presence of bacteriophages in the soil of oak trees would affect the pathological process of Oak Wilt Disease and if the variation in oak species has a correlation with the presence of bacteriophages.

Rationale:

…………To test if sample Soil A has the presence of bacteriophages, a spotting test will be administered to Soil A Direct Isolation & Enrichment. By adding drops of lysate from sample Soil A to a bacterial lawn on the agar plates, we can determine if bacteriophages are present in the lysate or not. A Plaque Assay also helps us determine if there is the presence of bacteriophages, by adding Arthrobacter directly to the lysate, we can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.

Experimental Procedure for Plaque Assay for Soil A:

……………………. 1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% …………………………Ethanol and let it evaporate)  on the workbench,  prepare:

…………………………………………………….(1) Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

………………………2. Checking the results of Spotting Test for sample Soil A

………………………3. Add 10µL of your lysate to a cultural tube w/0.5mL Arthrobacter, sit for 15 min.

………………………4. Add 8 ml LB Broth, 90 ul CaCl2 (aq) to a 50 ml conical tube.

………………………5. Add 10 ml 2x TA to the 50 ml conical tube, pipette to mix properly, take out 1 ml ………………………….and drop on the assigned section on the control plate.

………………………6. Take 4.5 ml of Top Agar from the 50 ml conical tube and pipette inside the ………………………….cultural tube to mix with lysate+Arthrobacter and add to a new agar plate, ………………………….slightly swirl to cover the plate evenly, set still for 10min before place in ………………………….incubator top-side down.

Observations, Results & Data:

……………………..

………………From the Spotting test, the TA Control appears to be uncontaminated as the gel still remain its original transparent yellow color. The Spotting plate is presented with several spots, proved to not be bubbles since the positions are inconsistent with the sketches drawn before. I hypothesize those may be either potential plaques or contamination An Interesting point worth noting is the “spots” are more prevalent in the Direct Isolation section, and not in the Enrichment section, which I originally presumed to have a higher probability to generate plaques due to theoretical higher concentrations of bacteriophage than Direct Isolation.

Interpretations & Conclusions:

……………….If I presume that all previous experiments for the Spotting test are not contaminated, then I would deduct the so-called “spots” might not be plaques due to the locations of the spots, more on the Direct Isolation section than the Enrichment section. The plate itself could potentially be contaminated before they were used since other unused plates also presented with similar conditions as described by the TA’s. I would wait for the Plaque Assay to yield its results to determine if sample Soil A contains bacteriophages or not.

Next Step:

………………If the Plaque Assay yields promising results I would continue trying to isolate the bacteriophages in sample Soil A, otherwise, I will search for new samples.

 

Category: Yang-En Yu | LEAVE A COMMENT
August 31

Spotting Test for Soil A 8.27.18

Research Question:

…………To find out how the presence of bacteriophages in the soil of oak trees would affect the pathological process of Oak Wilt Disease and if the variation in oak species has a correlation with the presence of bacteriophages.

Rationale:

………….To test if sample Soil A has the presence of bacteriophages, a spotting test will be administered to Soil A Direct Isolation & Enrichment. By adding drops of lysate from sample Soil A to a bacterial lawn on the agar plates, we can determine if bacteriophages are present in the lysate or not.

Experimental Procedure for Spotting Test for Soil A:

……………………. 1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% …………………………Ethanol and let it evaporate)  on the workbench,  prepare:

…………………………………………………….(1) Direct Isolation & Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

……………………..2. Add 5 ml LB Broth, 45 ul 4.5mM CaCl2 (ag) to a  new 50 ml conical tube (A) using …………………………Serological Tubes.

……………………………….4.5mM x 10ml = 1000mM x V, V=0.045ml=45ul

……………………..3. Add 5 ml of 2x TA to the solution in the new 50 ml conical tube (A) and pipette …………………………with Serological tube before pouring onto the agar plate, slightly swirl the plate …………………………to let it cover the whole plate evenly and set still. (labeled Group 5 Control …………………………8.27.18)

……………………..4. Add 13.5 mL LB Broth, 135 ul CaCl2 (aq) & 1.5 ml Arthrobacter to a different 50 …………………………mL conical tube (B).

……………………..5. Add 5 ml of 2x TA to 50 ml conical tube (B) and pipette with serological tube …………………………before pouring 10 ml on three plates, then discovers an error occurred during …………………………step 3. 4. & 5. , discard plates and restart.

……………………..6. Add 4.5 ml LB Broth, 0.5 ml Arthrobacter & 45 ul CaCl2 (aq) to a 50 ml conical …………………………tube (C) using micropipette.

……………………..7. Section a new Agar Plate into 3 sections, each label  C (Phage Buffer as control), ………………………..(D) Direct Isolation & (E) Enrichment. (Plate labeled: JY5 Spotting test 8.27.18)

……………………..8. Add 5 ml of 2x TA to 50 ml conical tube (B) and pipette with serological tube …………………………before adding the solution on to a new Agar plate, slightly swirl the plate to let it …………………………cover the whole plate evenly and wait for 10 min.

……………………..9. Filter 2 ml Soil A Enrichment Lysate with filter paper (0.22um) to filter out ………………………….bacteria into an Eppendorf. (labeled: JY5 8.27.18)

…………………….10. Use micropipette to drop 1 ml of Phage Buffer, Direct Isolation Lysate & ………………………….Enrichment Lysate to the middle of each section and set still for 15 min before ………………………….placing in incubator.

Observations:

……………….During the adding of top agar, several bubbles formed on the plate, which is caused by adding the.solution too fast.

…………………As soon as 2x TA is added to the solution it starts to solidify, so right away when I tried to poke out the bubbles using pipette tips the bubbles wouldn’t pop, which I presume is due to the agar has already solidified.

Results & Data:

………………….The Spotting Test set up was successfully completed, after waiting for 48 hrs it will show whether there are bacteriophages in both Direct Isolation Lysate & Enrichment Lysate from sample Soil A.

…………………..The Spotting Test Plate is Labeled: JY5 Spotting Test 8.27.18.

Interpretations & Conclusions:

…………………..If preparing a solution more all at once, such as all in one conical tube for one group, ALWAYS double check the math before adding anything.

…………………..When adding the Top Agar to the Agar plate, always add at a moderate speed in case of any bubbles forming.

Next Step:

……………………If the spotting test on Soil A yields positive results than I am one step closer to the purification of bacteriophage. I will be doing Plaque Assay on sample Soil A in the next lab.

……………………In the next lab, I will need:

…………………………………………………….(1) Direct Isolation & Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

 

Category: Yang-En Yu | LEAVE A COMMENT
August 31

Soil Washing (Continue) + Direct Isolation & Enrichment for Sample Soil A 8.24.18

Research Question:

…………To find out how the presence of bacteriophages in the soil of oak trees would affect the pathological process of Oak Wilt Disease and if the variation in oak species has a correlation with the presence of bacteriophages.

Rationale:

…………To search for possible bacteriophages in the soil, this experiment takes Soil Sample A and go through the Washing process to collect potential bacteriophage-containing lysate for further analysis. By using LB Broth, centrifuge and Tube-top filters with vacuum suction, potential bacteriophage lysate can be collected. After collecting the lysate it will be divided into two tubes, one for Direct Isolation and the other for Enrichment. Direct.Isolation allows us to test in future experiments if the sample contains bacteriophage and Enrichment allows us.to let potential existing bacteriophage to multiply by manually adding Arthrobacter, letting us get more conclusive results in further experiments.

Experimental Procedure for Soil Washing(continue)                                                                                      Direct Isolation & Enrichment of sample Soil A:

…………..50 ml conical tube Soil A has been in the fridge for 44.5 Hrs.

…………..Prepare:

………………..(1) One Tube-top Filter

………………..(2) One 15 ml conical tube

………………..(3) Vacuum Suction (In the Hood)

………………..(4) 0.5 ml of Arthrobacter

………………..(5) One plastic dropper

…………..1. Attach Vacuum Suction to the Tube-top Filter, slowly drop the liquid onto the filter ……………..paper(.22um).

…………..2. Filtered for 30 min.

…………..3. Split the collected lysate and add 7.5 ml to a 15 ml conical tube for Direct Isolation, the ………………original 50ml conical tube is for Enrichment, which has 11 ml.

…………..4. Place the Directed Isolation lysate in the fridge.

…………..5. Add 0.5 ml of Arthrobacter to the 50 ml conical tube for Enrichment and placed on ………………Shaker.

Observations:

  • After Filtration, the lysate in both tubes (15 ml & 50 ml conical tube) present a yellow, transparent color, which can deduct the particles in the lysate are extremely small and invisible to the naked eye.

Results & Data:

………The lysate filtered has a clear yellow color with no debris when examined by the naked eye.

………The 50 ml conical tube Enrichment is labeled: JY5 Soil A Enrich 8.24.18.

………The 15 ml conical tube Direct Isolation is labeled: Jy5 Soil A Direct 8.24.18.

Interpretations, Conclusions:

……….The filter time in the washing procedure may be slightly faster if the liquid is filtered through a filter with a larger hole before than filtering directly with the Tube-top Filter.

Next Step:

………..In the next lab, on 8.27.18, a Spotting Test will be administered on sample Soil A to determine if there are bacteriophages present in the lysate in both Direct Isolation and Enrichment.

………..For the coming experiment, I will need:

…………………………………………………….(1) Direct Isolation & Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

…………………………………………………….(9) Agar Plates

 

Category: Yang-En Yu | LEAVE A COMMENT
August 31

Soil Washing for Sample Soil A 8.22.18

Research Question:

…………To find out how the presence of bacteriophages in the soil of oak trees would affect the pathological process of Oak Wilt Disease and if the variation in oak species has a correlation with the presence of bacteriophages.

Rationale:

…………To search for possible bacteriophages in the soil, this experiment takes Soil Sample A and go through the Washing process to collect potential bacteriophage-containing lysate for further analysis. By using LB Broth, centrifuge and Tube-top filters with vacuum suction, potential bacteriophage lysate can be collected.

Experimental Procedure for Soil Washing of sample Soil A:

…………….1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% Ethanol ………………..and let it evaporate)  on the workbench,  prepare:

……………………….(1) Sample Soil A

……………………….(2) One 15 ml Conical Tube

……………………….(3) One 50 ml Conical Tube

……………………….(4) LB Broth

……………………….(5) Tube-top Filter

……………..2. Place sample Soil A to the 15 ml mark in the 50ml Conical Tube.

……………..3. Pour LB Broth to the 35 ml mark in the 50 ml Conical Tube Soil A.

……………………….(1) 50 ml Conical Tube Soil A weight: 51.20 g

……………..4. Vortex & Shake with Hand for 15 min to mobilize the soil.

……………..5. Set the 50 ml Conical Tube Soil A still for 15 min.

……………..6. To balance the weight in order to centrifuge,

…………………50 ml Conical Tube Soil A was added with water.

……………………….(1) 50 ml Conical Tube Soil A weight: 52.55 g

……………..7. Centrifuge at 3000 G for 5 min.

……………..8. Set the 50 ml Conical Tube Soil A still for 30 min.

……………..9. Filter the supernatant from the 50 ml Conical Tube Soil A with 1 microliter filter …………………paper.

……………10. Stored the Supernatant filtered from the filter paper into a new 50 ml Conical Tube …………………and stored in fridge.

……………11. Washing Procedure of sample Soil A is to be continued on 8.24.18.

……………12. Workbench was sterilized with Ci-Decon and 70% Ethanol for clean up.

Experiment Results and Observations:

…Observations:

  • The Soil Sample has a brown color and a moist texture.
  • After adding LB Broth and mobilizing the soil, the liquid is opaque and has a muddy color (On the left)
  • After the 50 ml conical tube was centrifuged there are sediments at the bottom of the tube, as the sketch shows (On the Right) the sediments lays slightly to the side, which assume is due to the position of the tube when it was in the centrifuge. The supernatant has a slightly translucent yellow color with a couple of floating debris at the surface of the liquid.

 

  • Going through a filter paper with a larger hole results in a faster filter time, yet the liquid still presents a translucent yellow color, indicating there are still particles of soil present in the liquid.

…Results:

………Due to the time limitations of lab hours and equipment, the washing procedure of sample Soil A has to be postponed to 8.24.18.

………The 50 ml conical tube is labeled: JY5 Soil A 8.22.18.

Interpretations, conclusions, and next steps:

…..Interpretations and Conclusions:

……….When putting soil samples in the 50 ml conical tubes in the future, make sure sizable debris such as parts of grass and sticks are picked out so after centrifuge there won’t be any debris floating in the liquid causing.difficulties to conduct the washing procedure.

…..Next Steps:

……….In the next lab, on 8.24.18, I will be finishing sample Soil A’s Washing procedure, splitting the volume of the lysate in half into a 15 ml conical tube for Direct Isolation and 50 ml conical tube for Enrichment.

……….For the coming experiment, I will need:

……………………………………………………………(1) One Tune-top Filter

……………………………………………………………(2) One 15 ml Conical Tube

……………………………………………………………(3) 0.5 ml of Arthrobacter

……………………………………………………………(4) One plastic dropper

Category: Yang-En Yu | LEAVE A COMMENT
August 31

Sea Phages Day 3: Plaque Assay

29 August 2018 ✷ Plaque Assay

Because no plaques were present in my spot test (negative spot test), only 1 plaque assay will be run as to practice running this type of test (using filtered enriched lysate).

Procedure:

  • CiDecon and 70% Ethanol were used to wash the workspace and prepare the aseptic environment. An alcohol burner was lit to clean the air.
  • The plate was made–see table below for volume and concentration values. The LB broth was added first (4x the amount was made because the lab group consisted of 3 people with the addition of a control plate). Next, we added the calcium chloride. 2 mL of the mixture was removed for the test plate and put in a separate vial; 2.5 mL of top agar was added last so it wouldn’t harden while we were adding the other components. The tube was inverted to mix the components and then pipetted onto the dish.
    component volume concentration
    2X Top Agar 2.5 mL 1.05X
    LB Broth 2.0 mL –
    1M Calcium Chloride 22.5 µL 4.5 µM
    1000 µM*V=4500µL*4.5µM = 22.5 µL
    2X*2.5 mL=M*4.5mL = 1 M
  • 10 µL of filtered enriched lysate was added to a tube of arthrobacter and allowed to sit for 15 minutes.
  • The remaining 7.5 mL of TA was added to the shared vial and mixed; then 5 mL was transferred to a vial for Rachel and 5 mL was transferred to a vial for Lauren. After 15 minutes, the arthro mix was added to the TA vial, inverted to mix, and poured into the petri dish. After the plate solidified, it was inverted and placed in an incubator until 2 pm on Wednesday, September 5.

Observations, Results, Data:

  • Because the last plate I made was contaminated, I took extra care to ensure ALL work was done aseptically.
  • However, since the last test was negative, it is highly unlikely that the plaque assay performed today will produce any positive results.
  • Of the 24 people in the class, 9 had positive spot tests.
  • UPDATED: after sitting in an incubator for nearly a week, the assay yielded a negative result, meaning no phage was found. However, there didn’t seem to be any contamination, which is an improvement from last week’s experiment.

Interpretations, conclusion, next steps:

  • Because the bacteria (and potentially a phage) are mixed before entering the plate, the chance of infection is higher on a plaque assay than on a spot test, thus this test may be more telling of the presence of a phage in a sample. However, a control plate is necessary to ensure the agar isn’t contaminated because the phage may cover the entire plate rather than just a small circle as in the spot test.
  • Though the chances for my particular plate are low, there is still a possibility my plaque assay may yield a positive result (phage present).
  • The negative result from the spot test will be used in answering the class’s overarching scientific question regarding the presence of phages in relation to the types of oak trees on Baylor’s campus.
  • In the future, more soil will need to be collected from another tree in hopes of isolating a new phage to continue research on.
Category: Lily Goodman | LEAVE A COMMENT
August 31

8/29 Spot Test Results & Plaque Assay

Rationale: Today, I will be checking the results of our spot tests conducted on 8/27, as well as start the procedure of a plaque assay. The objective of today is to correctly run a plaque assay.

Scientific 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?

Procedure:

  • First, we cleaned our lab table/area with CiDecon and 70% ethanol and placed an alcohol burner in the area to create an aseptic zone
  • We then obtained three agar plates, one for each of us to run our plaque assay on, as well as one plate where four of our groups (Groups 5, 2, 1, and 6) would run our control
    • My plate was labeled “Plaque Assay”, along with my initials, the date, and “Enriched Lysate Soil A”
  • We first measured out 22.5μL of 1M CaCl2 with the P200 micropipette [Min. 20μL and Max 200μL] (Labeled with a yellow sticker) and transferred it into our TA 50 mL conical vial
  • We then added 2mL of LB Broth to out TA conical vial via a cartwheel pipette with a 5 mL tip
  • This is the point in our experiment where we realized that we forgot to multiply the materials by 4 since there are three of us in our group (Plus the TA needed for our control), so we then multiplied our materials (Originally 0.5mL LB Broth, 2.5 mL 2X TA, 22.5mL 1MCaCl2) by four (My personal plaque assay plate, Kathryn’s, Emily’s, and our team’s control), resulting in our group obtaining 8mL LB Broth, 10mL 2X TA, and 90mL 1M CaCl2
  • We then added another 67.5 μL of 1M CaCl2 to our TA conical vial via the P200 micropipette [Min. 20μL and Max 200μL] (Labeled with a yellow sticker)
  • Similarly, we added another 6mL of LB Broth to our TA conical vial via a cartwheel pipette, with a 10 mL tip
  • I then added 10μL of my enriched lysate to 0.5 mL of Arthrobacteria via the white pipette (The P10 pipette) [Min. 1μL and Max 10μL] and let it sit for 15 minutes
    • We do this to allow time for any bacteriophage in the lysate to infect the Arthrobacteria
  • After waiting 15 minutes, we then added 10 mL of 2x TA to our TA vial with a cartwheel pipette with a 10 mL tip, and then immediately added 1mL to our control dish (Quadrant shared)
    • We have to immediately add it to the dish because as soon as the TA is added into the mixture, the solution will start to solidify
  • I then added 5mL of the TA to my arthrobacteria + enriched lysate vial, and mixed it with the pipette (Cartwheel pipette with a 5 mL tip)
  • I then added the mixture onto my plate, and let it sit for around 10 minutes to solidify
  • The plate was then transferred to an incubator set at room temperature
  • We cleaned up our work area with CiDecon and 70% ethanol

Results:

  • So my spot test turned out negative, and our team’s control TA dish was contaminated. This was a minor setback, but with the start of the plaque assay, we are hoping that we will get positive results that our lysate(s) have bacteriophage

Observations:

  • The procedure of the plaque assay was very similar to the spot test, however the only change in procedure was that the lysate and arthrobacter were introduced together first so if there are any bacteriophage in the lysate, it would be able to infect the lysate

  • The control plate for our four groups; notice the quadrants drawn on the bottom of the plate

    The plate for my plaque assay, prior to adding the TA or arthro + lysate mixture

    The negative results of the spot test

    The contaminated control plate of Group 2’s spot test

     

  • Next Steps: We will be waiting for the results of the plaque assay. If it turns out negative, then we will need to obtain another sample soil different from where we got our first sample (Sample A). If positive, then I think a retesting of a spot test may be wise to double check the lysate.
Category: Justin Yu | LEAVE A COMMENT
August 31

Plaque Assay 8/29/18

Scientific Question:

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

Rationale:

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

Materials Used: 

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

Procedure: 

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

Observations/Results/Data:

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

Conclusions/Next Steps:

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

 

Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT
August 31

Plaque Assay for Phage Discovery

Date: August 29, 2018

Title: Plaque Assay for Phage Isolation

Rationale: Regardless of spot test result, a plaque assay is done to obtain another measure of phage presence.

Materials: Arthrobacter, LB Broth, 2X Top Agar, 1M CaCl2, Enriched lysate, pipet, micropipette, plate, vials

Procedure: To create an aseptic zone, the following was done:

  • Counter washed and wiped with CiDecon
  • Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
  • Ethanol lamp lit to create rising heat and a current that protests samples from falling contamina

 

  • mL Arthrobacter and 10 microliters of filtered enriched lysate (filtered at spot test)

In 50 mL tube the following was added (for 3 plaque assays and 1 group control):

  • 8 mL LB
  • 10 mL 2X Top Agar
  • 90 microliters CaCl2

~18 mL of solution

  • Once the solution was finished, 4.5 mL was added to the small vial of Arthrobacter + lysate (enriched, filtered) and poured onto the plate. (NOTE: One vial was broken in the transition to the plate. The counter was re-sanitized, and another vial was prepared)

Results/Observations: The plate and the solution were yellowish and was put into the incubator to grow over the weekend. If the result is positive, I would expect to see growth on the plaque assay, and a negative result on the control. A negative result would constitute contamination on the control, or no growth on the plaque assay. Assuming a positive result, the next step would be to begin isolating the discovered phage. (See attached picture of resulting plaque assay plate before incubation)

Plaque Assay (Pre-incubation)

Category: Cooper Johnson | LEAVE A COMMENT
August 31

Spot Test for Lysate

Date: Monday, August 27, 2018

Title: Spot Test for Lysate

Rationale: By conducting a spot test, the phage (if present) will be given an opportunity to grow on a bacterial lawn and establish a plaque for further research.

Materials: Arthrobacter culture, LB Broth, CaCl2, 2X Top Agar, 50 mL test tubes, micro centrifuge tubes, pipettes, micropipettes, 22-micron filter, plates, phage buffer

Preparation: To create an aseptic zone, the following was done:

  • Counter washed and wiped with CiDecon
  • Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
  • Ethanol lamp lit to create rising heat and a current that protests samples from falling contamina

Procedure:

  • Enriched lysate filtered through 22-micron filter into micro centrifuge tube to prepare for spotting.
  • With a new 50 mL vial, the following was added:
    • 5 mL ArthrobacterI (pipetted by TA)
    • 5 mL LB Broth
    • 0 mL 2X Top Agar
    • 45 microliters CaCl2 (via micropipette)

 

~ 10 mL added to plate to solidify for 15 minutes

 

  • After the plate solidified, 10 micro liters of filtered lysate was spotted onto the section labelled “E” for enriched. 10 microliters of direct lysate were spotted onto the section labelled “D” for direct. There was a third section labelled “C” that is designated as a negative control area. The plate was incubated for 48 hours to discover if a phage was present.

Results/Observations: After 48 hours, the spot test showed a potential phage growth from the direct isolation spot, and was put into a walk-in fridge to further incubate in case of a negative plaque assay. (See pictures)

 

Potential Plaque from Spot Test

Potential Plaque from Spot Test

Potential Plaque from Spot Test (View from Bottom)

Category: Cooper Johnson | LEAVE A COMMENT
August 31

SEAPHAGES Soil Washing and Lysate // August 22, 2018

Date: Wednesday, August 22, 2018

Title: Soil Washing and Lysate Preparation

Rationale: By washing the collected soil and preparing two different kinds of lysate from a centrifuged supernatant, the process of isolating and discovering a phage will begin.

 

Materials Used: CiDecon, 70% EtOH, 50 mL test tubes, 15 mL test tubes, top filter, LB Broth, Arthrobacter culture, centrifuge

 

Preparation: To create an aseptic zone, the following was done:

  • Counter washed and wiped with CiDecon
  • Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
  • Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

Procedure: With the soil that was collected from an oak tree, the following was added to a test tube:

  • LB Broth (poured to 35mL mark on tube) (Broth creates a favorable environment for bacteriophages to grow in)

Then, the tube was shaken for 15 minutes

Observation – The shaken tube was much lighter with the addition of LB Broth, turning the dark brown dirt to a lighter, coffee color uniform throughout the whole tube.

 

The tube was then inserted into a centrifuge and spun for 15 minutes to separate the supernatant and the solid soil.

 

After the tube was centrifuged for 15 minutes, the supernatant and the soil were separated clearly, with a dark solid at the bottom of the tube and a yellow supernatant filling the rest of the tube.

 

The newly acquired supernatant was then poured through a 22-micrometer top filter to filter out any bacteria (phages are just barely small enough to pass through.)

The supernatant was poured into the top of the filter and turned on via a vacuum to activate the filter.

The filtered supernatant, now lysate, was split into 2 different isolations

  • A direct isolation, where pure lysate was poured into a 15 mL tube and refrigerated until Monday
  • An enriched isolation, where lysate and 0.5 mL of Arthrobacter ATCC 21022 were combined and incubated until the following Monday

Conclusion/Observations: With two different isolations, different experiments and tests can be run to try and discover a phage. In the case of no growth, the process can be repeated until a phage is found. Within the enriched lysate, Arthrobacter, LB Broth, and the supernatant are all in one suspension, providing a favorable environment for growth and, hopefully, infection.

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