September 21

9/17/18 ~ Plaque Picking, Serial Dilution, and Plaque Assay(s)

Rational:

Picking a plaque and performing a serial dilution, as well as perform plaque assays to isolate the bacteriophage

 

Procedure:

  • First, created an aseptic zone ensure no bacteria would contaminate the plates
  • Picked a plaque from the 9/12 plaque assay, and added it into 100μL Phage Buffer
    • Tube labeled as 10^0 dilution
  • Added 90μL of PB into two more micro-centrifuge tubes
    • For one tube, added 10μL of 10^0 into it, creating the 10^-1 dilution
    • For the second tube, added μL of 10^-1 into it, creating the 10^-2 dilution
  • Then added 10μL of each dilution to 0.5 mL of arthrobacter
  • Obtained a 50 mL conical vial and added 20mL LB Broth, 22.5 μL CaCl2, and 25 mL 2X TA
    • Created enough TA for 10 plates; three individual plates for the members in the group, and one more for the control plate
  • Immediately mixed the TA with the 10^0, 10^-1, and 10^-2 dilutions, and then plated
  • Let the plates sit for 15 minutes, and then incubated all 10 plates

 

Observations:

9/12 Plaque assay; can see the formed plaques

The 9/12 spot test

The 10^0, 10^-1, and 10^-2 plates

 

  • Justin was the only member in the team to have plaques in the spot test
  • For the plaque assay, there were many plaques, possibly indicating that the bacteriophage are lytic

 

Questions on the board:

  1. Although all members of group 4 had plaques on their plaque assays, only Justin had plaques on his spot test. This may have resulted from the fact that bacteriophage can go through two “reproduction” cycles: the lytic and lysogenic cycle(s). Justin’s bacteriophage may be lytic, and immediately puts the bacteria through lysis, while his group members’ bacteriophage maybe lysogenic. This could explain the spot test because the soil they pulled from were in the same general area, and they all have bacteriophage in their soil, as confirmed by the plaque assay.

  2. Lathan would need 40.2μL of 10^0 to web his plate

     

Next Steps:

Repeat the picking of plaque from the 10^0 plate if plaques appear, as well as run the plaque assays again. If plaques do not appear, pick another plaque from the 9/12 plaque assay and repeat.

 

Conclusion:

The lab proceeded without problem since Lathan explained the procedure of serial dilution and the process in the video lecture. Every member in my team worked together to finish the lab as swiftly as possible, while ensuring no contamination occurred.

Category: Justin Yu | LEAVE A COMMENT
September 21

09/19/18 Enrichment and Metadata

Rationale:

The agenda of lab today was to examine the plaque assay performed on Monday, clean and enrich the soil, and take the metadata for the soil composition, water percentage, and pH.

Materials:

  • LB Broth
  • 15-mL Conical Vial
  • 0.5-mL of Arthobacter
  • Soil Dispersion Liquid
  • Syringe Filter
  • Pipettes (Micropipette, Serological, squeeze pipettes)

Procedure:

  • Began by establishing an aseptic zone with CiDecon, 70% Ethanol, and a burner.
  • Removed the plaque assay and analyzed the results, plate was negative and control was contaminated yet again. Examined the 2X TA and the LB broth and determined that the broth was indeed contaminated.
  • After examination, the enrichment process began with adding soil up to the 2-mL mark of a 15-mL conical vial.
  • Aliquoted a new LB broth up to the 12-mL mark of the 15-mL conical vial.
  • Conical vial was sent to vortex for 15 minutes.
  • During vortex, water percentage of the plate was begun by first weighing an empty weighing plate.
  • After weighing the plate and recording, a small amount of soil was added to the weighing plate and then weighed.
  • After the vortex, the conical vial was then sent to the centrifuge for 10 minutes at 10,000 g.
  • During centrifuge, a small amount of soil was added to a vial and then filled with DI water. Shook vial for 10 seconds and left rest for 2 minutes.
  • Inserted pH paper into the soil mixture after 2 minutes for about 45 seconds. Removed after 45 seconds and recorded pH.
  • Tested soil composition after pH testing by adding 4-mL of soil, 8-mL of DI water, and 3 drops of soil dispersion liquid into a falcon tube.
  • Falcon tube was shaken for 30 seconds, and left to sit for 48 hours.
  • Once centrifuge ended, supernatant was filtered through a 2 micron syringe filter into another 15-mL conical vial. During the syringing process, approximately 2-mL of supernatant was spilled.
  • 2-mL of filtered lysate was separated into micro centrifuge tube for the direct isolation
  • The remaining 6.5-mL of lysate had 0.5-mL of Arthobacter added to it to form the enriched lysate.

Data/Analysis/Conclusions:

  • Plaque Assay was negative with the control plate scattered with contamination. The group began to examine the LB Broth and 2X TA that was used for each procedure and noticed the LB broth being extremely cloudy and containing precipitant. It was not nearly as clear as other LB broths. This lead to the conclusion that the LB broth was contaminated, and this appeared to be the case for several groups in class as well. Soil taken from the Burr Oak in North Village seems to be negative for phage and this very well could be attributed to the fact that the soil is garden soil and is not natural.

  • Contaminated LB broth (left) Uncontaminated (Right)

    Contaminated Control Plate

    Empty Plaque Assay

  • Soil pH appeared to be slightly acidic with a pH of approximately 6.5. This is interesting as the gardeners soil was basic with a pH of around 7.5. This difference of pH could possibly be the difference between phage presence or not.

Next Steps:

The next steps for this soil sample is to finish calculating the water percentage and the soil composition of the soil. In addition to this, both a plaque assay and a spot test need to be performed to determine the presence of phage in the soil.

 

 

Category: Gabriel Andino | LEAVE A COMMENT
September 21

9/19/18 Serial Dilution pt. 2

Cooper Johnson

Title: Serial Dilution pt. 2

Date: 19 September 2018

Rationale/Past Results: The 100 and 10-1 plaque assays from before yielded no plaques, however the 10-2 yielded one plaque-forming-unit. This could have been caused by a lytic/lysogenic cycle differential causing one phage to grow rapidly and another to not flourish as obviously. The TA control was also positive, signaling potential contamination among one or more of the plaque assay ingredients. The individual ingredients will be labelled to further isolate the cause.

Procedure:

Aseptic zone created by the following procedure:

  • 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.

A pipet tip was inserted into the selected plaque spot of the 10-2 plaque assay then transferred and mixed with 100 microliters of Phage Buffer solution. This solution is labelled “100” to represent a 100 serial dilution. Then, 2 plaque assays of the 100 lysate were made to further analyze the present phage(s).

The following recipe was used to make 7 plaque assays:

  • 14mL LB Broth
  • 5 microliters CaCl2
  • 5 mL 2X Top Agar

~4.5 mL pipetted into Arthrobacter culture tube containing 0.5 mL Arthrobacter and 10 microliters of 100 dilution lysate.

The 2 plaque assays were left to set for 10-15 min before placing into incubator for ~48 hours.

 

Conclusions: The plaque that was grown from the previous 10^-2 lysate has been plated for 2 plaque assays in hope of growing a plaque and eventually being able to identify, isolate, and amplify a singular phage for further analysis and answering the research question

Category: Cooper Johnson, Uncategorized | LEAVE A COMMENT
September 21

Results and Redoing of Plaque Assay of Soil B (09/17/18)

Results:

The plaque assay ran on Friday (9/14) was contaminated. However, there was no liquid in the plate like the spot test ran on Wednesday (9/12). Both the “KEA PA 9/14/18 enrich lysate” and the “KEA PA 9/14/18 control” plates looked similar to each other. The plates appear to look similar to each other and have a weird texture might be because the Top Agar started to solidify before the mixture was placed into the plates. The pictures below show these plates.

Rationale:

In today’s lab, a plaque assay will be run with the same enriched lysate from soil B. From this plaque assay, the researcher hopes to receive a plate without contamination and be able to determine whether or not there are bacteriophages that specifically target Arthrobacter in the collected soil sample.

Procedure:

  1. Started off by cleaning the counter with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Next, 10 μL of enriched lysate from the soil sample B was added into a test tube which already had 0.5 mL of Arthrobacter.
  3. Then, the test tube was set aside for 15 minutes.
  4. The original recipe was quadrupled as shown below.

Calculations

Original Recipe

X4

X2*

2 mL LB Broth

8 mL LB Broth

4 mL LB Broth

2.5 mL 2X TA

10 mL 2X TA

5 mL 2X TA

22.5 μL CaCl2

90 μL CaCl2

45 μL CaCl2

*This calculation was used later since the amount of mixture in the control plate ended up with an excess amount.*

  1. Through the used a serological pipette to transfer 8 mL of LB Broth into a 50 mL conical vial.
    • This 50 mL conical vial was labeled “SA LEF KEA 9/17/18 TA control.”
  1. Then, 90 μL CaCl2 was transferredinto “SA LEF KEA 9/17/18 TA control” conical vial using a P200 micropipette.
  2. Obtained plates and labeled them “KEA 9/17/18 PA” and “SA LEF KEA 9/17/18 control.”
  3. Next, 10 mL of 2X TA was added and mixed into “SA LEF KEA 9/17/18 TA control” conical vial using a serological pipette.
  4. Then, 9.5 mL of the mixture was placed into the “SA LEF KEA TA control” plate.
  5. This left only enough for two more plates. The Arthrobacter with enriched lysate was poured and mixed into 5.0 mL of the TA mixture twice. Then, poured this into the corresponding plates.
  6. The steps 5-8 were performed the same way except with double the original recipe, and 5 mL were poured into both the control and the enriched lysate plate.
  7. Allowed 15 minutes for the plate to solidify.
  8. The plates were inverted and placed in the incubator at 48ºC for the next 48 hours.
  9. Cleaned lab counter with CiDecan and EtOH (70%).

Observations and Interpretations:

  • On the plate assay from Friday (9/14), most of the air bubbles which were created when the mixture started to solidify had disappeared.

Things to consider…

  1. Group 4 all had plaques on their plaque assays. Justin had the most and well-defined plaque (but all 3 got a plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot. What do I think is going on?

Justin’s group performed the procedures correctly. The plaques they had were extremely spread out. Since a plaque assay covers a whole plate, there is a better chance of receiving a plaque when compare to a smaller area where the plaque is dropped.

  1. Lathan checked a purified lysate by doing a plaque assay of 10-3 He counted 14 plaques. How many μL of Lathan’s 100lysate should he add to web a plate (75 mm in diameter) if his average plaque diameter is 1 mm?

Data:

  • Serial Dilution: 10-3
  • Number of Plaques Found on Plate: 14
  • Plate Diameter: 75 mm
  • Average Plaque Diameter: 1 mm

Calculations:

            Lathan should add 40.2 μL of 100lysate to web the plate.

Conclusions:

After participating in the lab for about a month, the researcher has gained more respect for individuals who work in labs such as for the Environmental Protection Agency (EPA) and Food Safety and Inspection Service (FSIS). Research can be frustrating since one might perform the same tasks a multitude of times and still have negative or contaminated results.

Next Steps:

On Wednesday, if the plaque assay is contaminated, the experiment will proceed by running another plaque assay. If the plaque assay is negative, the experiment will start all over with a new soil sample. If the plaque assay is positive, the experiment will proceed to perform purification processes.

Category: Kathryn Adkins | LEAVE A COMMENT
September 21

9/17/18 Serial Dilutions

Cooper Johnson

Title:  Serial Dilution

Date: 17 September 2018

Rationale/Past Results: The spot test was negative, however, at least one plaque was found on the plaque assay. A lab partner grew many plaques on his plaque assay, perhaps the aforementioned phage underwent the lysogenic cycle, while the assays with less plaques experienced the lytic cycle, producing less phage. A serial dilution will be done to begin isolation of a phage.

Procedure:

              Aseptic zone created by the following procedure:

  • 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.

 

A pipet tip was inserted into the selected plaque spot and then transferred and mixed with 100 microliters of Phage Buffer solution. This solution is labelled “100” to represent a 100 serial dilution. Then, 10 microliters of the 100 solution was transferred to another vial of 90 microliters Phage Buffer and mixed to create a 10-1 solution. This process was repeated one more time to create a 10-2 solution. The three solutions are then plated on a plaque assay.

The following recipe was used to make 10 plaque assays:

  • 20 mL LB Broth
  • 225 microliters CaCl2
  • 25 mL 2X Top Agar

~4.5 mL pipetted into Arthrobacter culture tube containing 0.5 mL Arthrobacter and 10 microliters of dilution lysate.

One plaque assay done each of 100, 10-1, and 10-2 lysate. Plus, one TA Control.

Plates left to set for 10-15 minutes and then put into incubator for 48 hours.

 

Conclusions/Observations: The 100 lysate should yield the most plaque-forming-units, however all 3 are tested to eventually web and amplify.

 

  1. Group 4 got plaques on the plaque assays, but not on the spot test, also, another lab partner had much more defined plaques. This could result from a different phage discovery and a difference between lytic and lysogenic cycles.

 

  1. 14 pfu/10 microliters * 1000 microliters/1 mL = 1400 pfu/mL || 1400 pfu * 103 = 1.4 x 106 pfu/mL

 

(37.52)π/(0.52)π= 5625

5625/1.4 x 106 = .004 mL = 4.01 microliters. Lathan needs 4.01 microliters to web his plate.

 

Category: Uncategorized | LEAVE A COMMENT
September 21

09/17/18 Plaque Assay/Spot Test Results and Soil Gathering

Question on Board:

Lathan checked a purified lysate by doing a plaque assay with 10-μl of lysate at a 10^-3. He counted 14 plaques. How many μl of Lathan’s 10^0 lysate should he add to web a plate (75-mm in diameter) if his average plaque diameter is 1-mm?

Answer: 4.0178-μl

 

Rationale:

Today’s rationale was to analyze the plaque assay performed the week prior for the presence of phage. If there were any plaques, the next step would have been to create a serial dilution using the lysate. If there were no plaques, a second plaque assay was to be performed and new soil was to be gathered as well.

Materials:

  • Serological Pipette
  • Micropipette
  • 2.0 mL LB Broth
  • 2.5 mL 2X Top Agar
  • 0.5 mL Arthrobacter
  • 22.5 μl Calcium Chloride
  • 50 mL Conical Vial
  • Lysate

Procedure for Analyzing Plaque Assay and Spot Test:

  • Plates were removed from the incubator to analyze for spots.
  • Plate was held up to the light to check for any spots and plaques were empty with nothing to signify the presence of phage.
  • After plate was looked at, the control plaque was examined and contamination was present in the plate. After determining possible causes for contamination, a second plaque assay was begun for certainty.

Procedure for Plaque Assay:

  • An aseptic zone was established with CiDecon, 70% Ethanol, and a burner.
  • Initially began with creating a top agar for the whole group. Added 8-mL of LB broth and 90 μl of calcium chloride to the conical vial before realizing the concentration of the calcium chloride would be incorrect in the plates. It was decided to do a top agar independently with original measurements.
  • 2.0-mL of LB broth was added to a new 50-mL conical vials. Repeated for the control group as well.
  • 22.5-μl of calcium chloride was added to the LB broth by micropipette in both vials.
  • 0.5-mL of Arthobacter was then introduced to 10-μl of the lysate created for the previous plaque assay and left in a micro centrifuge tube for 15 minutes to infect.
  • After the 15 minutes had passed, 2.5-mL of the 2x TA solution was added to the 50-ml conical vials.
  • Immediately after the 2x TA was added, the lysate and broth were both mixed together in the 50-mL conical vial, swirled, and immediately poured onto the plate to solidify. Control agar was plated as well
  • After waiting 15 minutes, plates were left in the incubator for 48 hours.

Procedure for Soil Gathering:

  • A white oak (species unknown, possibly Burr Oak) was found near the Baylor Sciences Building.
  • Soil was extracted with a scoop by digging  and placed into a plastic bag.
  • Stored in a cold environment for the enrichment process during the next lab.

Data:

  • Plaque assay from previous lab was clearly contaminated. It was unsure if the contaminant was Arthobacter or external contamination from failure to aseptically perform procedures.
  • Plate was empty with no signs of phage present in neither spot test or plaque assay.
  • Tree seemed very healthy with no clear signs of disease. Soil was a very dark color with moderate amounts of moisture in it.

  • Control Group

    Plaque Assay

    Spot Test

 

 

 

 

 

 

 

Analysis/Conclusion:

  • The same LB broth and 2x TA were used from the last plaque assay and spot test to determine if they were the source of the contamination. If the plaque assay result comes out contaminated again, they will be examined for contamination.
  • Very high possibility that soil may just be negative for phage. This could be due to the fact that the soil that the tree was planted in is gardeners soil and not naturally occurring. The differences of minerals present in the soil could very well influence the presence of phage in the soil.

Next Steps:

  • The next steps are to enrich the new soil and examine the plaque assay for any contamination and presence of phage. Each member gathered soil from 3 different white oaks, so each sample will be tested for any phage concentrations present in the soil.
Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT
September 21

9/17/18 Plaque Assay of Soil #2 Enriched Lysate and Collection of Soil Sample #3

9/17/18 Plaque Assay of Soil #2 Enriched Lysate and Collection of Soil Sample #3

Objective:

The goal of these procedures is to use the previously created lysate to preform a plaque assay to test for phage presence in our previously collected soil (soil sample #2). In addition, during this lab period we also collected a third soil sample in case the plaque assay on soil sample two yielded a negative result. Our goal is to isolate a phage, and because our spot test yielded negative results, we did a plaque assay to confirm while also preparing to take next steps if soil sample 2 is negative for phage. We are also trying to avoid the contamination that occurred on our controls (see below). We are also seeking to address the following questions every lab:

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?

The question specific to my lab table is: Is the a difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine weather or not there are other factors that may determine phage presence.

In addiction to our guiding questions, for this lab we were asked to consider the following:

  1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?
    • After considering this question we determined that Justin’s sample likely had the highest titer and therefore resulted in more clearing and better defined spots. It is also possible that the way he cared for his sample was more conducive to phage survival (ether during soil transport or enrichment).
  2. Lathan checked a purified lysate by a plaque assay using 10 µL of a 10^-3 lysate. He got 14 plaques. How many µL of Lathan’s lysate should he add to web a plate (8 cm in diameter) if his average plaque diameter is 1 mm.
    • 4.6 µL of 10^0 lysate (work below)
Procedures and Protocols:

Materials for Aseptic Zone:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner

Materials for Plaque Assay:

  • .5 ml Arthrobacter
  • incubator
  • Pipette
  • Test tube stand
  • 50 ml tubes
  • Culture tube
  • LB Broth
  • 2X TA
  • 1M Calcium Chloride
  • Agar plate
  • Serological pipette

Materials for Soil Collection:

  • Sharpie
  • Tape Measure
  • Plastic Bags
  • Digging tools
  • 15 ml Tubes

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

  1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
  2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
  3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone

Then the plaque assay on the enriched lysate from soil sample #2 was preformed.

  1. Four agar plates were labeled. An agar plate was labeled with initials, date, and description for each group member, and one agar plate was labeled with data and “TA control”
  2. The remaining enriched lysate from last procedure (spot test), stored in a pipette cap, was gathered (like cap picture below)
  3. 10 µL of the remaining enriched lysate was aseptically transferred into a culture tube containing .5 ml of Arthrobacter using a Serological pipette
  4. The culture tube was capped and set aside for 15 minutes. This process was repeated twice more (once for each group member).

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

  1. The agar was prepared according to the following recipe (makes four plates):
  2. Under aseptic conditions, 8.o ml of LB broth was transfered into a 50 ml tube.
  3. Under aseptic conditions, 90 µL of 1 M CaCl2 was transferred into the same 50 ml tube. *Note: the first time we attempted to pipette the calcium, we accidentally drew too much out so we had to discard the pipette tip full of calcium and try again*
  4. Under aseptic conditions,  5.o ml of 2X TA was transferred into the same 50 ml tube
  5. The mixture was pipetted several times to mix it

When agar preparations were finished the bacteria and lysate had been allowed to sit for 15 minutes

  1. 4.5 ml of the contents in the 50 ml tube was transferred into the culture tube containing lysate and bacteria
  2. The mixture was pipetted several times to mix it
  3. Then the mixture was poured from the culture tube into the agar plate labeled with initials, date, and description
  4. The plate was capped and set aside for 10 minutes to allow agar to solidify. This procedure was repeated twice more, once for each group member.
  5. The remaining contents in the 50 ml tube was poured onto the TA control agar plate
  6. The TA control plate was allowed to sit for about 10 minutes before being placed into the incubator
  7. Once the labeled plaque assay had solidified, the plate was inverted and placed in the incubator
  8. Plates were left to incubate until nest class

Soil collection was preformed as follows:

  1. The species of tree that soil should be collected from was identified *Note: in this case we choose live oak as we did in soil sample 2*
  2. The provided bags were labeled with name, date, and description
  3. A tree of this species was loacted on campus
  4. The GPS coordinates were noted
  5. The tree’s trunk diameter was measured and recorder
  6. The tree’s height was measured and recorded using shadow length of a known height and then measuring the shadow of the tree and using trig
  7. The tree’s average canopy diameter was measured and recorded by taking measurement of the shortest and longest diameters and averaging them together (results in the table in results column)
  8. A digging instrument was used to clear debris and dig down several centimeters into the soil
  9. The provided plastic bags were filled with soil and a tree leaf (for confirmation of species identification)
  10. The bags were brought back to lab
  11. Refrigerate Tube and bag until next class
Results:

The results immediately gathered from these procedures are seen in the table above. The results of the plaque assay on soil #2 will be recorder here when available. It will not be possible to ascertain weather or not phage are present in soil sample #3 until the soil can be washed and then assays and spot tests can be preformed.

Update:

There are visible plaques on all three plates (see images below for my plates), but their is also contamination in the control. We are unable to determine how this contamination came to be, but we are operating under the assumption that it is not the cause of plaques forming. We will test this assumption in the future.

Analysis:

Based on the appearance of the tree and the measurements we took, we would assert that the tree is most likely healthy. Should we find phage in this soil and in the soil of trees nearby, this information could become important in determining if tree health is a factor for phage presence. It is also important to note that this was a live oak tree and it could become clear that live oak trees do not readily have phage in their soil after further sailing and analysis of soil sample #2’s plaque assay. The opposite could also be true and we will be more equipped to analyse our data once we test for phages.

Update:

Based on the results of visible plaques we can assert that there is likely phage present in the soil from sample #2. However, because the control plate was contaminated we cannot be certain. The results of further testing of plaques should reveal if there are in fact phages in soil sample #2, or if my group simply managed to contaminate everything

.

Future:

If necessary I will wash the that soil was collected (soil #3) and create lysates in order to run spot tests and plaque assays.

Based on the update, this will not be immediately necessary. On Wednesday I will pick a plaque and preform serial dilutions. Based on the results of that I will likely try to web a plate or be forced to begin testing soil sample #3.

Category: Lucy FIsher | LEAVE A COMMENT
September 21

9/19/18 Plaque Picking and Serial Dilution

9/19/18 Plaque Picking and Serial Dilution

Objective:

The goal of this procedure is to use one of the plaques discovered after Monday’s procedure to preform plaque assays at different dilutions. This will help confirm the presence of phage and will contribute to isolating them. Our goal is to isolate a phage. We are focusing on avoiding the contamination that occurred on our controls of both our spot test and plaque assay. We are also seeking to address the following questions every lab:

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?

The question specific to my lab table is: Is the a difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine weather or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for Aseptic Zone:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner

Materials for Plaque Assay:

  • .5 ml Arthrobacter
  • incubator
  • Pipette
  • Test tube stand
  • 50 ml tubes
  • Culture tube
  • LB Broth
  • 2X TA
  • 1M Calcium Chloride
  • Agar plate
  • Serological pipette

Materials for Serial Dilution:

  • Microcentrifuge tubes
  • Phage Buffer
  • A lysate that requires diluting (10^0)

Materials for Phage Picking:

  • Agar plates with plaques of interest
  • Micropipette tip
  • Phage buffer
  • Microcentrifuge tubes (incorrectly referred to as pipette caps in previous entries)

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

  1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
  2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
  3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone

Then a phage was picked *Note: Each group member picked one plaque from their respective plates for a total of 3 picked plaques*

  1. 100 µL of phage buffer was transferred into a microcentrifuge tube labeled with initials, date and the description “10^0”
  2. A pipette tip was used to stab the center of the chosen plaque on each plate (the chosen plaque is indicated my the red arrow on the image below) *Note: My hands shake and it is possible I contaminated my pipette tip with the surrounding agar when I tried to stab my plaque* 
  3. The (hopefully) phage-infected tip was swirled in the phage buffer and then the solution was vortexed and set aside.

Then the serial dilutions were preformed.

  1. Three levels of dilution were created: 10^0, 10^-1, 10^-2
  2. The microcentrifuge tube labeled “10^0” created in the procedure above served as the first dilution
  3. Two more microcentrifuge tubes were gathered and labeled with initials, date, and the descriptions of “10^-1” or “10^-2” respectively
  4. Each of these tubes was then filled with 90 µL of phage buffer
  5. 10 µL of solution was taken the 10^0 tube and transferred to the tube labeled 10^-1
  6. It was vortexed to mix
  7. 10 µL of solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
  8. It was vortexed to mix and set aside

Then the multitude of plaque assays were preformed.

  1. Ten agar plates were labeled. An agar plate was labeled with initials, date, and description of dilution (10^0, 10^-1, or 10^-2) for each group member, and one agar plate was labeled with data and “TA control”
  2. The dilutions that were created in the last procedure were gathered
  3. 10 µL of each dilution was aseptically transferred into a culture tube containing .5 ml of Arthrobacter using a Serological pipette
  4. The culture tube was capped and set aside for 15 minutes. This process was repeated eight more times

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

  1. The agar was prepared according to the following recipe (makes ten plates):
  2. Under aseptic conditions, 20.o ml of LB broth was transferred into a 50 ml tube.
  3. Under aseptic conditions, 225.0 µL of 1 M CaCl2 was transferred into the same 50 ml tube.
  4. Under aseptic conditions,  25.o ml of 2X TA was transferred into the same 50 ml tube
  5. The mixture was pipetted several times to mix it

When agar preparations were finished the bacteria and lysate had been allowed to sit for 15 minutes

  1. 4.5 ml of the contents in the 50 ml tube was transferred into the TA Control plate, capped, and set aside
  2. 4.5 ml of the contents in the 50 ml tube was transferred into the culture tube containing lysate and bacteria
  3. The mixture was pipetted or occasionally vortexed several times to mix it
  4. Then the mixture was poured from the culture tube into the agar plate labeled with initials, date, and description of the appropriate dilution
  5. The plate was capped and set aside for 10 minutes to allow agar to solidify. This procedure was repeated eight more times, once for each group member and each dilution.
  6. Once the labeled plaque assay had solidified, the plate was inverted and placed in the incubator *Note: One of my plates refused to solidify so I put it in the incubator not inverted*
  7. Plates were left to incubate until nest class

 

Results:

There are no immediate results from this procedure. The results will be made clear on Monday and recorded appropriately. It will be important to note whether or not contamination occurred in our control plate though, because if it did this can still cast potential doubt about the existence of phage in our soil sample #2. All results will be recorded after Monday’s lab.

Analysis:

Based on the appearance of what we assume to be plaques on our plaque assay, it seems reasonable to assert that there are phage in our soil sample #2. This will be ether confirmed or denied by the results of our dilutions. In addition, we were extremely careful in conducting our procedures during this lab in order to avoid contamination, so if it appears, we will need to take more serious action. I will be able to assert a lot more once I see the results of my dilution assays.

Future:

Depending on the results of the dilution assays, I will ether move forward to try to web a plate and further explore my phage, or I will have to start over. If necessary I will wash the that soil that was collected (soil sample #3) and create lysates in order to run spot tests and plaque assays.

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September 20

Lab Day 8: Enrichment + Soil Metadata

Rationale

Complete enrichment process of new collected soil and results from soil metadata from new soil. Label group number on LB Broth and 2X TA jars to prevent future contamination.

Detailed Procedure: Soil Metadata

  1. Filled soil up to 10 mL mark in a 50 mL vial and filled rest with D.I water up to 30 mL mark.
  2. Shook for 10 secs and added soil dispenser solution.
  3. Left to rest on rack for 48 hours.
  4. Pinched soil into pH vial and filled rest with D.I water and shook for 10 secs
  5. Left to rest for 2 mins
  6. Pinched off 1 inch of pH paper and dipped into pH vial for 45 secs.
  7. Recorded pH of soil

Detailed Procedure: Enrichment

  1. Filled LB Broth into vial from Lab Day 7 up to 12 mL mark in aseptic zone
  2. Shook using vortex for 15 mins.
  3. Weighed vials and centrifuged for 5 mins
  4. Syringe filtered supernatant (8 mL into 50 mL vial and 1 mL into 15 mL vial)
  5. Poured 0.5 mL Arthro into 50 mL vial

Results

  • weight of plate + soil after 48 hours= 6.48 g
  • ph of soil= 6
  • weight of vial before centrifuge= 19.75 g

Conclusions

After 48 hours, I will be able to determine what type of soil was collected. After incubation of enriched lysate, group 6 will be able to do spot test and plaque assay of the new soil and check to see if there are any presence of plaque.

 

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September 20

Lab Day 7: Results

Rationale

Check to see if any plaque remains. If not, why/how does our results mean to the group question. Find new soil. Control plate from Lab day 6 was contaminated, but showed negative results from plaque assay plate. Other group members did spt test and had negative results as well.

Detailed Procedure

  1. Dug up new soil from a red oak in between the BSB and business building.
  2. Filled 15 mL vial up with soil up to 2 mL mark
  3. Filled bag with soil
  4. Began soil metadata by weighing plate and soil

Results

  • weight of plate= 2.34 g
  • weight of plate + soil= 6.68 g

Group question

  1. Lathan checked a purified lysate by doing a plaque assay (10 microliters lysate) of a 10^-3 lysate. He wanted 14 plaques. How many microliters of Lathan’s lysate?

Conclusion

New soil’s metadata must be done by next lab day to determine which type of soil we collected. Soil enrichment will also be done by next lab day.

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