November 26

Lab Day 28: Serial Dilutions and Webbed Plate

Rationale

Created 10^-5 dilution to make a newly webbed plate in hope of flooding and creating a high titer.

Detailed Procedure

  1. 10 µL of “SJ 11/12/18 FS lysate 100” was added with 90 µL of phage buffer to create 10-1 dilution which was vortexed.
  2. 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
  3. 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
  4. 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
  5. 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
  6. Took 98 µL of 10-5 into 0.5 mL arthro. Set for 15 mins to infect.
  7. Took 4 mL of LB Broth, 5 mL of 2X TA, and 45 µL of 1M CaCl2 into 15 mL vial.
  8. Took 4.5 mL of top agar mixture and poured onto control plate.
  9. Took rest of top agar mixture and combined with arthro and 10-5 mixture.
  10. Took resulting solution and poured onto 10-5 plate.
  11. Allowed both plates to sit for 15 mins.

Conclusion/Next Steps

Since the calculations were 97.6 µL of 10-5 , only 98 µL were pipetted for easier pipetting. By next lab day, hopefully a webbed plate will be able to flood and make new plates to calculate the new titer. By Friday, hopefully a high titer will be calculated.

Category: Soo-Un Jeong | LEAVE A COMMENT
November 26

Comparing Plaques 11/26/18

Rationale: The plates ran last week turned out to not be webbed. They had two distinct plaque morphologies, so I will pick a plaque from each and run plaque assays to make sure this is the same phage making both types of plaque.

 

Procedure:

  1. Filled 2 microcentrifuge tubes with 100µL of 1X Phage Buffer labeled one alpha and the other beta.
  2. Picked a large plaque and placed in the tube labeled alpha. Pick a small small plaque and placed in the tube labeled beta.
  3. Diluted each sample out to the 10^-4 dilution factor.
  4. Enriched alpha dilutions in 0.5mL arthrobacter for 20 minutes.
  5. Made top agar with 10mL of LB broth, 12.5mL of 1X TA, and 112.5µL of CaCl2.
  6. Plated each mixture of alpha dilution and arthrobacter after pipetting in 4.5mL of top agar.
  7. Repeated steps 4-6 with the beta dilutions.
  8. Made a top agar control by plating 4.5mL of top agar.
  9. Let sit then inverted and placed in the incubator.

Observations:

The marked plaque on the top right is the plaque picked for the alpha samples. The plaque picked on the bottom left is the plaque picked for the beta samples.

The results show that both alpha and beta lysates produced both large and  small plaques. The top agar was contaminated, so when the  first two dilutions of alpha lysed the plate the contamination was able to grow back in place of arthrobacter.

 

Conclusions  and Next Steps: This means that  there is just one type of phage in my plate making the radically different plaques. Since I have a plate closed to being webbed and a lysed plate I will flood both so I can have enough lysate to perform DNA extraction. I’m flooding the lysed plate because the plate with a lower dilution factor also lysed, but the plate with a higher dilution factor nearly webbed so there is most likely some amplification that occurred.

Category: Uncategorized | LEAVE A COMMENT
November 24

11/19 ~ Creating Webbed Plates

Rationale: Created webbed plates from the team’s lysate (With titer strength of 2.2*10^11) to obtain enough lysate for all the members of the team.

 

Procedure:

  • Aseptic zone to prevent contamination from bacteria
  • Obtained the previous lysate and obtained six agar plates
  • Added in 10μL lysate into each 0.5mL arthrobacter vial (Six total)
  • Obtained a conical vial and pipetted in 14mL LB Broth, 17.5mL 2XTA and 157.5μL CaCl2
  • Immediately pipetted 4.5mL into each arthrobacter+lysate vial and then plated
  • Allowed to sit for 15 minutes and then moved to incubation

 

Observations:

The spot test for lysate #5; not the lysate used to web

The spot test for lysate #6; not the lysate used to web

 

Conclusion/Next Steps: Will be returning in 24 hours to flood the plates and then filter then to obtain a lysate of equal strength tot he 2.2*10^11

Category: Justin Yu | LEAVE A COMMENT
November 23

TEM Gridding

Results from Previous Experiment: Discovered our lysate to have a titer of 10^11.

Objectives: Complete TEM with the high titer lysate.

Procedure:

  • Allocated Petri Dish and Parafilm
  • Placed a strip of parafilm securely inside petri dish top
  • Placed a drop of lysate, 2 separate drops of water, and a drop of Uranyl less on the parafilm
  • Using precision tweezers, placed a copper grid, shiny side down, on the lysate for 5 minutes
  • Moved copper grid to the first drop of water for 2.5 minutes
  • Moved copper grid to the second drop of water for 2.5 minutes
  • Placed copper grid on uranyl less (UL) for 1 minute, and quickly blotted an excess UL once remove
  • Used tweezers to move copper grid into TEM Grid Holder

Future Plans: Complete TEM after thanksgiving break.

Category: Uncategorized | LEAVE A COMMENT
November 23

TEM Grids for Lysate 8

11/19/18

Rational:

To make an EM grid for lysate 8 in order to do a TEM and get an image of the phage in lysate 8.

Procedure:

  • Put 15 ML of lysate onto a plate
  • Put 2 20 ML drops of water onto the plate and 1 drop of uranyl acetate
  • Put the EM grid from A9 darks side down on the lysate and waited 5 min
  • Put the grid on the first water drop and waited 2.5 min and repeated for the second water drop
  • Put the grid on the uranyl acetate for 1 min
  • Put the EM grid in A9 after blotting excess moisture

Conclusion:

Next lab I will do TEM using the EM grid from this lab in order to get an image of the phage that are found in lysate 8. I will also start DNA extraction on lysate 8.

Category: Emily Balint | LEAVE A COMMENT
November 23

Plaque Assay 2 for Mel plaque 11/19/2018

Rationale: perform a second plaque assay for lysate collected from “mel plaque”, or the singular plaque found on the first plaque assay from Melissa’s soil

Control for first plaque assay of Melissa’s soil

First plaque assay for Melissa’s soil showing one plaque

Process:

  1. washed table and setup aseptic zone
  2. picked plaque and swirled it in 70 µL phage buffer
  3. Made LB agar media for 2 plates
    • 4 mL LB broth
    • 50 µL 1 M CaCl2
    • 5 mL 2X TA
  4. added 50 µL lysate (Mel plaque) to 0.5 mL arthro and let sit for 15 minutes
  5. Poured control plate with ~5mL from the LB agar media
  6. Added arthro lysate mixture to  LB agar media
  7. Poured plate from tube and let sit for ~15 minutes
  8. Incubated for 48 hours at 28 degrees Celsius

Next Steps: Another plaque assay but with alterations to get a higher titer.

Category: Rachel Melone | LEAVE A COMMENT
November 23

Preparation of TEM Grid (11/19/18)

Rationale: To prepare a TEM grid in order to view the phages under a transmission electron microscope

 

Procedures:

  • Using a petri dish, a strip of parafilm was set across the dish.
  • A drop of lysate (.15 µL) was set on the parafilm.
  • The, two drops of water (the same amount as lysate) was added.
  • After, one drop of uracil acetate was added onto the strip.
  • For five minutes, a copper filter was in the lysate.
  • After the five minutes, it was moved to the water for two and a half minutes.
  • The previous step was done again in the second drop of water.
  • After it has been in the water for its allotted time, the filter was placed in the uracil acetate for one minute.
  • The filter was removed and placed into its place.

 

Results and Analysis:

 

Conclusion:

To prepare a TEM grid, a drop of lysate was added to the strip of parafilm on the petri dish. Two drops of water and one drop of uracil acetate were then added. For five minutes, the copper filter was placed in the lysate then moved to the two drops of water for two and a half minutes each then moved to the uracil acetate for one minute.

 

Future Plans:

In order to complete a DNA extraction, 10 mL of lysate is needed. Therefore, a plaque assay will be made then flooded in order to get more lysate.

 

Category: Sabin Patel | LEAVE A COMMENT
November 23

11/16/18 Spot Titer Test

11/16/18 Spot Titer Test

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of calculating the titers of the lysates created during the last few labs by doing a spot titer test.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the 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 whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner

Materials for a Serial Dilution:

  • Phage Buffer
  • Microcentrifuge Tubes
  • Vortex Machine
  • Pipette

Materials for Lysate Filtering:

  • Syringe filter
  • 50 ml conical
  • Flooded webbed plates

Materials for a Spot Test:

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

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

Lysate 2 was collected and filtered

  1. A syringe was used suction lysate off of the flooded plates
  2. The lysate was filtered into a 50 ml conical

Then serial dilutions were performed on each lysate.

  1. Five levels of dilution were created for each lysate (called lysate 1 and 2): 10^-1, 10^-2, 10^-3, 10^-4, 10^-5
  2. Ten microcentrifuge tubes were filled with 90 µL of phage buffer
  3. 10 µL of the previously created lysate 1 (10^0) were transferred to one of the 10^-1 vials
  4. The tube was vortexed to mix
  5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
  6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-5 dilution for lysate 1
  7. 10 µL of the previously created lysate 2 (10^0) were transferred into the other 10^-1 vial
  8. The tube was vortexed to mix
  9. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
  10. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-5 dilution for lysate 2

Then the spot titer test on the new lysates was performed.

  1. One agar plates was labeled as shown:
  2. Agar was prepared according to the following recipe (makes one plate):
  3. 4.5 ml of the agar was transferred to the labeled plate
  4. The plate was swirled and set aside to allow the agar to solidify
  5. When agar was solidified, 10 µL of phage buffer as well as 10^-3, 10^-4, and 10^-5 dilutions for each lysate were transferred to their appropriate spot on the plate
Results:

As can  be seen from the image above, our lysate spots traveled. When calculating it appeared that lysate 1 had a 10^8 titer and lysate two had a 10^7 titer.

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay or spot test. We have been amplifying the titer of our lysates, so this spot test should reveal a high titer lysate.

Future:

Because neither lysate produced quite a high enough titer, Lucy and I will need to web more plates with the hopes of creating a higher titer lysate to use TEM.

Category: Lucy FIsher | LEAVE A COMMENT
November 23

11-19-18 — High Titer Plaque Assay

Date: Monday, November 19th, 2018

Title: High Titer Plaque Assay

Rationale: The purpose of today’s lab is to make a plaque assay using a dilution that will form plaques without lysing a plate completely.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

  1. An aseptic zone was set up.
  2. Plates from last lab were evaluated.
  3. The 10^-8 dilution was the only spot that had formed plaques that were countable, so that dilution was selected.
  4. Top agar for a plaque assay was setup using the following formula:
    1. 4 mL LB Broth
    2. 5 mL 2x Top Agar
    3. 45 microliters 1M CaCl2
  5. A culture tube with .5 mL arthrobacter was mixed with 10 microliters of 10^-8 lysate and left to infect for 15 minutes.
  6. 4.5 mL of the top agar solution was added to a top agar control plate and left to sit for 15 minutes.
  7. 4.5 mL of the top agar solution was added to the culture tube, pipetted, and added to a plate.
  8. The plates were inverted and left to incubate over night.

Observations: The majority of the dilutions tested on the spot test were too high of a titer to make individual plaques, so the 10^-8 lysate was used in order to make a plate that a titer calculation can be made with. There was contamination on the top agar control from last experiment.

 

 

Results: This experiment yielded a plaque assay and a top agar control that can be evaluated in the future.

Next Steps: The next step is to calculate the titer and volume needed to web a plate using the new plaque assay.

Category: Brandon Reider | LEAVE A COMMENT
November 23

Plate Webbing To Create a High Titer Lysate 11/19/18

Rationale

Today we will calculate the titer of our lysate from previously performed spot titer test. After calculating the titer, we will web 5 plates.

Procedure

  • Established an aseptic zone.
  • The calculated titer was used to determine the amount of lysate needed to web a plate.
  • 26.7 µL of 10^-2 lysate was added to 0.5 mL of Arthrobacter. This process was repeated 5 times and each mixture was set aside for 10 minutes.
  • 12 mL of LB broth, 135 µL of CaCl2, and 15 mL of TA was added to another vial.
  • 4.5 mL of the mixture in the second tube was combined with each vial and was poured onto a plate. Five plates were produced along with a control.
  • The plates were set aside for 10 minutes to solidify. After 10 minutes, the plates were placed inside an incubator.

Observations

High titer calculations:

Spot Titer Test:

The 10^-4 lysate dilution was used in calculations due to the 10^-3, 10^-2, 10^-1 lysate dilutions creating webbed or lysed spots.

Conclusions/Next Steps

Next, we will flood our hopefully webbed plates to produce a high titer lysate.

Category: Emily Gaw | LEAVE A COMMENT