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.

Rational:

Today, we will be setting up and performing both a plaque assay and spot test for the enriched lysate of Soil B, as well as checking the results of the % water and % sand/silt/clay

Procedure:

• We first created an aseptic zone on our table using CiDecon and 70% ethanol, and lit an ethanol burner to create the aseptic zone
• I then massed my enriched lysate (21.59g) to find a partner for centrifusion. The enriched lysate was centrifuged at 3000 g’s for five minutes to pellet the arthrobacter
• While the lysate was being centrifuged, I checked the results of percent water and percent sand/silt/clay on my soil
• For percent water, I re-massed my soil+dish and got 11.72g, which meant the soil was 9.33g (Total dry weight – weight of dish)
  • I then took my dry soil weight and divided it by the wet soil weight (9.33/12.064), and got 77. I then subtracted 100 from this number to get 23% water
• For percent sand/silt/clay, I looked at my vial and determined where all the clay ended (Total 2 mL)
  • For sand, I determined it was 25% of the soil
  • For silt, I determined it was 50% of the soil
  • For clay, I determined it was 25% of the soil
• My table group decided that we will all be performing our spot test on one plate (1/4 per test), with the left over 1/4 being for our negative control (Phage buffer), as well as performing a plaque assay separately, while sharing one TA (Top agar) control plate
• For the spot test, we mixed together 2 mL LB Broth, 0.5 mL Arthrobacter, and 22.5 μL 1M CaCl2 into a 50 mL conical vial
  • The LB Broth was added with a cartwheel pipette with a 5mL tip, while the CaCl2 was added with the P200 pipette
• We then added in 2.5 mL 2xTA, and then immediately poured it onto our plate, and then let it solidify for 10 minutes
  • 2xTA added with the P200 pipette
• While waiting for the TA to solidify, we filtered our enriched and centrifuged lysate using a syringe and a 22μL filter
• We then spotted 10 μL of each of our enriched lysate on our separate sections of the dish, as well as spotted 10μL of phage buffer into our negative control 1/4
• We then moved the plate to an incubator
• For our plaque assay, we multiplied all our materials by four; we added 8 mL of LB Broth and 90 μL of CaCl2 into a 50 mL conical vial
• We then took our separate enriched lysates and added .5 μL arthrobacter into each one and let it sit for 10 minutes to infect
• We then added 10 mL 2xTA to our mixture, and immediately poured 5mL into our TA control dish (The TA will start solidifying after being added into the mixture)
• We then separately added 5mL of our TA mixture to each of our enriched lysate + arthrobacter mixture, and immediately added it to our plaque assay plates
• We let our plates sit for 15 minutes, and then moved the plates to incubation

Observations:

You can clearly see the separations of the dirt

My dried dirt, used to calculate % water

Our spot test dish; three of the quadrants are for the separate three members in our group, as well as 1/4 for our negative control

My Plaque Assay dish prior to anything added into it

My Plaque Assay dish with the procedure completed

• There was a significant color difference between the wet dirt and dry dirt
• The procedure for the spot test and plaque assay was performed more quickly than our first time

Next Steps:

Next time we come into lab, we will see if the spot test and plaque assay turn out negative or positive, and see if our controls worked

Rationale:

We will be washing and filtering our Soil B to created an enriched lysate. This is so we will be able to perform a spot test and plaque assay for the Soil B lysate.

Procedure:

• First, we created an aseptic zone on our table using CiDecon and 70% Ethanol. We also lit a ethanol burner to create the aseptic zone
• I then put 2mL of Soil B into a 15 mL conical vial, and added 10 mL of LB Broth
• I then shook the vial for ten minutes, with both my hand as well as the vortex machine
• My vial massed at 17.488 g and was then centrifuged at 10,000 g’s for five minutes
• While my vial was being centrifuged, I needed to begin the preparations for finding the percentage water,  percentage sand/silt/clay in my soil sample, and my soil pH
• To calculate the percentage water, I first obtained a weighing dish and massed it. It weighed 2.39 g
  • I then added soil into the dish, and massed it again. The dish + soil weighed 14.454 g, so the weight of the soil was 12.064 g
  • The dish with my soil sample in it was then placed into a hood to evaporate for 48 hours (Results in next post)
• To calculate percentage sand/silt/clay, I first obtained a 50 mL measurement vial, and added 4mL of soil into it
  • I then added water to the 10mL mark, as well as SDL (Soil Dispersion Liquid) to the vial, and shook for 30 seconds
  • After the 30 seconds, I placed the vial in the hood in a rack to let sit for 48 hours (Results in next post)
• To find my soil pH, i First took a pH vial and added a small amount of Soil B into it
  • I filled the rest of the vial with DI (Deionized) water, and then shook for 10 seconds
  • After letting the soil sit, I placed a strip of pH paper into the vial, and let it react for 45 seconds
  • The recorded pH of my soil was 6.5
• After my soil was centrifuged, I ran my liquid through a top filter (22 μL filter) to get my enriched lysate
• I had around 10 mL of enriched lysate in a 50 mL conical vial; I labeled my vial with my initials and the date, as well as “Soil B”
• I then added .5 mL of arthrobacter and incubated my tube

Data/Observations

My Enriched Lysate prior ro incubation

The soil-water solution with SDL in it; results in 48 hours

The pH vial and pH paper after measuring. My weighing dish can also be seen in the background

• My pH indicated that my soil is slightly acidic; it would be interesting to compare results to others in the lab
• The SDL had already started to work in the 50 mL measuring vial; I could see some distinct separation of materials in the vial

Next Steps:

My next steps would be to perform a spot test or plaque assay with the enriched lysate of Soil B. I will also need to check on the results of the percentage water and percentage sand/silt/clay next lab

Results:

On Friday, it was determined that my Plaque Assay for Soil A turned out negative without contamination.

Non-contaminated Control Plate

The negative plaque assay

Rationale:

Collect a new sample of soil, as well as come up with our scientific question

Scientific Question:

Each table section came up with their own question, and ours is, “Is there a difference in Arthrobacterphage in live oak trees at Baylor versus planted trees in developed areas?”

Procedure:

Our table groups (Groups 3 &4) came up with our scientific question, and then went out to collect soil (In a 15-mL vial and bag). My group (Group 4) was tasked with gathering soil from planted trees, so we went to Teal Residential College and looked at trees there. My tree is on the northern side, and closer to the building than the rest of the trees. I gathered soil two feet from the base of the tree, as well as took leaf samples for identification.

Observations:

• My tree is not an oak tree, and I have yet to find out the specific type it is (I will have to look through a tree category book)
• Although my tree had lots of brown leaves on it, most of it looked healthy and green
• While digging through the soil, I found a grub of some sort

Next Steps:

We will be washing and filtering my soil on Monday, and will be creating a direct and enriched lysate

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.

Rationale: After obtaining my lysate, I will be performing a spot test to see if there are any bacteriophage present in my lysate that will infect arthrobacter.

Procedure:

• First, we cleaned our lab area/station with CiDecon and 70% ethanol to create an aseptic area where we can work with our samples, minimizing the chance of contamination
• I then isolated my lysate via a 3 mL syringe and filtering; I ran the lysate through the filter and into a microcentrifugetube (Labled JY 8/27/18 FS lysate) [FS stands for filtered sterile]
  • The filter was 22μL in size
• My group then got 4 petri dishes; one singular dish for each member, as well as a dish for our team that we will use as our top agar control
  • Throughout this blog entry (and later entries), I will abbreviate top agar as TA
  • For my personal plate, I drew on the bottom and split the plate into three separate thirds and labeled them as “Negative Control, FS Lysate Soil A (Enriched), and FS Lysate Soil A (Enriched)
    • When I created my enriched lysate, I was only able to filter out 10 mL, so I wasn’t able to create an direct isolation
    • The plate was also labeled Spot Test Soil A
  • Our team plate was labeled “Team 2 8/27/18 Soil A TA Control”
• We then did calculations to see how much of the following materials we needed for our TA
  • For my personal TA, I need 0.5mL Arthrobacter, 4.5 mL LB Broth, 5mL 2x TA, and 45μL 1M CaCl2
  • For our teams TA, we needed 4.5mL LB Broth, 5mL 2x TA, and 42.8μL 1M CaCl2
• We used a blue pipette w/ a yellow sticker (The P200 pipette) [Min. 20μL and Max 200μL] to transfer 42.5μL of 1M CaCL2 to a 50 mL conical vial
  • This vial became our Team 2 TA vial
• I used the same pipette to transfer 45μL of 1M CaCL2 to a 50 mL conical vial
  • This vial became my personal TA vial, and was labeled accordingly with my initials, date and TA description
• We then used a cartwheel pipette with a 5mL tip to transfer 4.5 mL of LB Broth into both our team vial and my personal TA 50 mL vial
• I then took my vial to Lathan (Our TA) to have 0.5mL Arthrobacter added into my 50 mL TA vial
• We then added 5mL of 2X TA (Via a cartwheel pipette with a 10 mL tip) into both our team vial and my personal TA 50 m vial
• The contents of my vial was then immediately poured into my petri dish, and allowed to sit for 10 minutes for the TA to solidify
  • The contents of the team’s control TA vial was also poured into the team’s petri dish
• I then used a white pipette (The P10 pipette) [Min. 1μL and Max 10μL] to drop 10μL of my FS Lysate to both sections on my plate
• I also dropped 10μL of phage buffer into the Negative Control area of my petri dish
• All our dishes were placed into incubators at room temperature
• We cleaned and sprayed down our work area with CiDecon and 70% ethanol

Observations:

• In one of my thirds labeled “FS Lysate”, there were bubbles from when the TA was added
• Using the cartwheel pipette proved harder than anticipated, since it is based on how fast you release the liquid

Image of my plate after the drop of lysate was added

The circled areas are where we noticed bubbles in the TA

Next Steps:

• The next steps would be to see if the drop test turned out positive or negatively, and regardless of the outcome, proceed with a plaque assay, which will also give us information if there is bacteriophage in my lysate

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

Procedure:

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

Observations:

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

Results:

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

Next Steps:

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