November 16

Spot Test Titer Calculation 11/16/18

Rationale: After my first round of purification I need to calculate the titer of my lysate. However, since not enough TA is available to make multiple plaque assays I conducted a spot test to calculate my titer.

Procedure:

  1. Flooded plaque assay from Wednesday for 2 hours by pouring 8mL of phage buffer on the plate then putting it on a shaker.
  2. Filtered the phage buffer with a .22µm syringe filter.
  3. Diluted with phage buffer out to a dilution factor of 10^-8.
  4. Marked a plate to spot for dilutions of 10^0 to 10^-7.
  5. Made top agar with 2mL of LB broth, 2.5mL TA, 22.5µL of CaCl2, and 0.5mL of arthrobacter.
  6. Let the plate sit until solidified.
  7. Spotted each dilution in marked location with 4µL of corresponding lysate.
  8. Let spot dry then inverted and placed into incubator.

Observations:

The section with a dilution factor of 10^-7 had 16 plaques. From that I calculated that my lysate has a titer of 4e10. Since  the plaques had a radius of 0.75mm it will take 8µL of 10^-5 lysate to web  a plate.

Conclusions and Next Steps: Since I have a high titer I now have to make a couple webbed plates so I can collect enough lysate to run DNA extraction and then archiving.

Category: Sriram Avirneni | LEAVE A COMMENT
November 16

Calculating the Titer of Lysate 8

11/16/18

Rational:

To estimate the titer of lysate 8 in order to determine whether a new lysate need to be obtained or not.

Procedure:

  • Estimated titer from the spot titer using the 10^-7 dilution
  • Titer= (5 pfu/10 ML)(1000 ML/mL)(10^7 ML/mL)

Observations:

  • The control plate showed signs of contamination though the spot titer did not
  • Most of the spots were cleared
  • The spots for 10^-7 and 10^-8 were the only ones that were not cleared
  • The 10^-8 spot showed no signs of plaque
  • The arthro also seemed to be growing back over the cleared spots
  • Titer= 5*10^9 (high titer)

Conclusion:

Since the spot titer showed a high titer then I will do a plaque assay on the 10^-7 dilution in order to calculate the actual titer. I will also do a TEM with this lysate since it is a high titer.

Fig.1.8 – This figure shows the results of the spot titer. The image shows that all of the dilutions up to 10^-7 were cleared. It also shows that there were no plaque for the 10^-8 dilution.

Fig.2.8 – This image shows the contamination found on the control plate for this test.

Category: Emily Balint | LEAVE A COMMENT
November 16

November 16 2018 Lysate and Spot Titer Test- Soil C

Rationale:  The purpose of this lab is to collect lysate and perform a spot titer test to determine if the lysate is a high titer.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an ethanol burner was it.
  2. Lysate was harvested from the four flooded plates using a syringe and was filtered into a tube with a 0.22 um syringe filter. Approximately 25 ml were obtained.
  3. Serial dilutions were performed out to 10^-5 with the lysate collected(lysate 2) and lysate 1.
  4. Top agar was made for one plate, using 2 ml of LB broth, 22.5 ul of CaCl2, 2.5 ml of 2x TA, and 0.5 ml of arthrobacter. The top agar was poured onto a plate labeled LIP 11-16-18 Spot Titer Test. The plate was divided into 7 sections, one for the phage bufer test, and 3 for each of the lysates, 10^-3 to 10^-5.
  5. 10 ul of lysate was spotted into each of the six sections, and 10 ul of phage buffer was spotted into its section.
  6. The plate was allowed to sit for 15 minutes and then inverted and stored in the incubator until the next lab.
  7. The workstation was cleaned and the materials were properly stored or disposed of.

Observations:

  • Few bubbles seen on the plate
  • Top agar control was made on a second plate by a different group.

Interpretations/Next Steps:
The procedure was complete. The next step will be to determine the titer of the lysate collected and create a TEM grid.

 

Picture of Plates:

Category: Lucy Potts | LEAVE A COMMENT
November 16

Serial Dilution Spot Test Lysate 8

11/14/18

Rational:

To do a spot test titer from the lysate obtained from last lab’s serial dilution in order to calculate the new lysate’s titer.

Procedure:

  • Cleaned lab desk
  • Filtered the flooded plates from last lab
  • Put 90 ML of PB into 8 microcentrifuge tubes
  • Diluted the lysate to get 10^1, 10^-2, … and 10^-8 dilutions
  • Put 2 mL of LB broth into a tube
  • Added 22.5 ML CaCl2, 2.5 mL TA, and .5 mL arthro
  • Poured on plate and waited 10 min
  • Put 2 mL of LB broth into a tube for the control
  • Added 22.5 ML CaCl2 and 2.5 mL TA
  • Poured on plate and waited 10 min
  • Spotted the dilutions and lysate onto the plate and waited 10 min
  • Put in incubator at 26 C at 4

Conclusion:

The plates from lysate 7 had been completly cleared after 24 hrs so they had been flooded the day before in order to prepare a new lysate. Next lab the titer of this lysate will be determined using the spot titer.

Category: Emily Balint | LEAVE A COMMENT
November 16

Serial Dilution Lysate 7

11/12/18

Rational:

To do a plaque assay for three dilutions in order to calculate the titer for lysate 7.

Procedure:

  • Cleaned lab desk
  • Put 90 ML of PB in to 3 microcentrifuge tubes
  • Added 10 ML of the lysate to the first tube (10^-1)
  • Added 10 ML of the 10^-1 lysate to the second (10^-2)
  • Added 10 ML of the 10^-2 lysate to the second (10^-3)
  • Added 10 ML of the lysate and the three dilutions to 4 tubes of .5 mL arthro and waited 10 min
  • Put 10 mL of LB broth into a tube
  • Added 112.5 ML CaCl2 and added 2 mL the TA mixture to each of the tubes of arthro
  • Added 2.5 mL of TA to the dilutions and control
  • Poured on plates and waited 15 min
  • Put in incubator at 26 C at 3:40

Conclusion:

Next lab the titer of lysate 7 will be determined. If it is a high titer then TEM will be done with the lysate if it is not a new lysate will be prepared in order to get a high titer lysate.

Category: Emily Balint | LEAVE A COMMENT
November 16

11-14-18 — Serial Dilutions and Spot Test with Positive Lysate

Date: Wednesday, November 14th, 2018

Title: Serial Dilutions and Spot Test with Positive Lysate

Rationale: The purpose of today’s lab is to make serial dilutions and then spot test them in order to evaluate which dilution will yield a plate that has a high titer and isn’t lysed fully.

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 and yielded negative results.
  3. Flooded lysate from positive plates with high titer was borrowed from a lab partner (Gabriel Andino) and 5 mL was transferred to a new conical vial.
  4. Nine microcentrifuge tubes were labelled PB for Phage Buffer and then 10^-1 through 10^-8.
  5. 1 mL of phage buffer was added to the tube labelled PB.
  6. 90 microliters of phage buffer was added to each other tube.
  7. 10 microliters of the flooded lysate were added to the 10^-1 tube and vortexed.
  8. 10 microliters from the 10^-1 tube were added to the 10^-2 tube and vortexed.
  9. Step 8 was repeated until 10 microliters from the 10^-7 tube were transferred to the 10^-8 tube and vortexed.
  10. Two plates were taken. One was labelled as TA control and the other was divided into nine sections, with each section being labelled after each of the microcentrifuge tubes.
  11. Top agar was made using the following recipe:
    1. 4 mL LB broth
    2. 45 microliters 1M CaCl2
    3. 5 mL 2x Top Agar
  12. 4.5 mL of the solution was added directly to the TA control plate.
  13. 4.5 mL of the solution was added to a culture tube containing .5 mL arthrobacter and pipetted to mix the solution.
  14. The culture tube was emptied into the spot test plate and left to harden for 15 minutes.
  15. 10 microliters from each microcentrifuge tube were added to the corresponding sections on the spot test plate and left for another 15 minutes to allow the samples to absorb into the agar.
  16. The plates were left in the incubator.

Observations: Some of the top agar and LB broth jars still seem to have contamination. From Gabriel’s experiments, the first few serial dilutions still lyse a plate fully. It could be beneficial to come in earlier after the plates have only sat in an incubator for a day so that a plate can be flooded before the plate is fully lysed.

Results: This experiment yielded a spot test with 8 different serial dilution samples as well as a top agar control.

Next Step: The next step is to choose a dilution based off the results of the spot test that has a high titer but will not lyse a plate fully. Then, a plaque assay will be made and flooded if it is webbed.

Category: Brandon Reider | LEAVE A COMMENT
November 16

Plaque Assay Using Known, Positive Lysate (11/12/18)

Rationale: To make a plaque assay with the positive lysate to receive a high titer

 

Procedure:

  • The tables were thoroughly wiped down with CiDecon and ethanol and an aseptic zone was also set up to prevent contamination.
  • 125 µL of positive lysate and 0.5 mL Arthrobacter were placed in a new tube and left alone for 10 minutes to allow for infection.
  • Next, 22.5 µL CaCl2 was combined with 2.0 mL of LB broth.
  • After 10 minutes, lysate and Arthrobacter were added with LB broth and CaCl2.
  • Then, 2.5 mL 2X Top Agar was added to the LB broth and CaCl2.
  • The mixture was poured over the plate and then left alone to solidify before being placed in the incubator.

Results and Analysis:

Conclusion:

Due to the contamination of the negative control, a plaque assay was done. First, lysate and Arthrobacter were combined and left alone for 10 minutes to infect. Then, LB broth was added along with the calcium chloride. After the allotted time, the lysate and Arthrobacter were added to the LB broth and calcium chloride. 2X Top Agar was then added to the solution and poured onto the agar plate. For 15 minutes, the plates remained near an aseptic zone to solidify. After, the plates were placed in an incubator.

Future Plans:

When a plate is high titered with no contamination, the plate will be flooded with phage buffer and filtered to perform dilutions.

Category: Sabin Patel | LEAVE A COMMENT
November 16

Serial Dilutions with Lysate from Flooded Plate (11/14/18)

Rationale: After flooding the plate, the lysate will be filtered then used to perform serial dilutions to conduct a spot test

 

Procedure:

  • The lysate from the flooded plate was taken using a syringe then filtered.
  • Into eight tubes, 90 µL of phage buffer was added to each.
  • From the 10^0 dilution, 10 µL of lysate was added to the tube containing phage buffer.
  • From the 10^-1 dilution, 10 µL was taken and added to the 10^-2 tube and so on.
  • After completing the dilutions, a spot test was made by combining 2.0 mL of LB Broth, 22.5 µL of calcium chloride, and 0.5 mL of Arthrobacter
  • Then, 2.5 mL of 1X Top Agar was added then poured onto the plate.
  • After 15 minutes of allowing the top agar to solidify, 0.7 µL of each dilution was added to their respective areas within the plate.
  • The plate was left alone to allow for the spot dilutions to set in the agar.

 

Results and Analysis:

While removing the lysate from the flooded plate, the top agar tore.

 

Conclusion:

After setting up the correct measures to prevent contamination, eight small tubes were filled with 90 µL each. From the 10^0 lysate, 10 µL was transferred to another tube, creating the 10^-1 dilution. From the 10^-1 dilution,  10 µL was transferred to the 10^-2 tube to create the dilution and so on. Next, the spot test was made by combining LB Broth, 1X Top Agar, calcium chloride, and Arthobacter then poured onto a labeled plate.  After 15 minutes, the lysate was dropped in drops of 7 µL in their designated areas. The plates were left alone for 15 minutes to solidify then placed inverted in the incubator.

 

Future Plans:

Identify the dilution with the highest titer but not lysed then use that lysate to make a plaque assay to determine the titer of the plate, If it has a high titer, flood the plate.

 

Category: Sabin Patel | LEAVE A COMMENT
November 16

NOVEMBER 9TH, 12TH, 14TH-Labs

  • NOVEMBER 9TH, 2018
    • OBJECTIVE:
      • Create a lysate from the new soil sample collected 
    • PROCEDURE:
        • Soil was filled to the 2mL mark of a test tube 
        • Then 10mL of LB broth was added to the tube 
        • It was shaken fro 15 minutes, before then bing placed into the centrifuge for 10 minutes where it was then spun at 3,000G 
        • Once the tube being centrifuged was done, a top filter was then used to filter out the supernatant 
        • Then .5mL of Arthrobactor was added to the filtered supernatant 
        • The tube was then left in the incubator 
    • RESULTS: 
      • No results to report 
    • CONCLUSION: 
      • No results to discuss
    • NEXT STEPS:
      • Run a plaque assay 
  • NOVEMBER 12TH, 2018
    • OBJECTIVE:
      • To plate the plaque assay with no contamination 
    • PROCEDURE: 
      • Tables were cleaned, lamps were lit
      • A test tube was filled with: (2X TA was added last to prevent solidifying) 
        • 4mL LB broth
        • 45𝝁L  CaCl2
        • 5mL 2X TA
      • 15 𝝁L of lysate was pipetted into test tube containing arthro and was left to sit for 15 minutes 
      • After the 15 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the  lysate and arthro 
      • The test tubes were then poured into the plates, which were then given 10 minutes to solidify
      • Plates were then inverted and placed in the incubator 
    • RESULTS: 
      • As seen in Figure 21, the control and test plate were free of contaminants (control is top plate, bottom is test plate)
    • CONCLUSION: 
      • Its very difficult to view in Figure 21, but there were 5 small clear circles which are believed to be bacteriophage, and will be tested
    • NEXT STEPS: 
      • Pick plaques 
  • NOVEMBER 14TH, 2018
    • OBJECTIVE: 
      • To pick what is believed to be plaques, and plate them without contamination 
    • PROCDURE:
      • Tables were cleaned and lamps were lit
      • The plaques were circled and labeled 
      • 5 micro centrifuge tubes were filled with 100 𝝁L of phage buffer 
        • A compound microscope was used to view the plaques, which were then “picked” by gently tapping the plaque with the end of a micro pipet 
        • The pipet is then placed into the micro centrifuge tube containing 100 𝝁L phage buffer 
        • The tube was then vortexed for 2 seconds 
      • The steps above were repeated for every plaque 
        • A test tube was filled with: (2X TA was added last to prevent solidifying) 
          • 12mL LB broth
          • 135𝝁L  CaCl2
          • 15mL 2X TA
        • 15 𝝁L of lysate was pipetted into test tube containing arthro and was left to sit for 15 minutes 
        • After the 15 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the lysate and arthro 
        • The test tubes were then poured into the plates, which were then given 10 minutes to solidify
        • Plates were then inverted and placed in the incubator
    • RESULTS: 
      • The results can be seen in Figure 22, 23, and 24
      • As it can be seen the control was contaminated, and samples P1, P2, and P5 were all negative for phage presence 
      • Samples P3 and P4 were positive for phage presence, as seen in Figure 23, and 24
    • CONCLUSION: 
      • Samples P4 and P3 both contain phage, while the other samples appear to be negative. The source of contamination on the control is unknown, and may be due to exposure to bacteria during plating. 
    • NEXT STEPS:
      • Begin serial dilution, then flood plate
Category: Lauren Foley | LEAVE A COMMENT
November 16

Flooding and Dilution Spot Test for Sample (Gabe) 11/14/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A spot test for the diluted samples would be a fast way to determine the best sample volume to web and flood the plate and get the most phages possible.

Experimental Procedure for Spotting Test for Soil (Gabe):

(1) Lysate

(2) LB Broth

(3) 2x TA

(4) 1M CaCl2

(5) Arthrobacter host

(8) Phage Buffer

1. The plate was flooded and then lysate filtered.

2. The lysate was diluted into 10^0, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8.

3. The top agar was prepared with 2.0 ml LB broth, 0.5 ml Arthrobacter, 4.5 mM CaCl2 and 2.5 ml 2X TA.

4. The top agar was poured onto the plate and set still to solidify for 15 min.

5. The lysate dilutions were then spotted onto their respective sections on the plate and set still 15 min

6. The plate was then placed in incubator for culture.

Observations, Results & Data:

The plate was almost completely lysed, only traces of colonies can be found on the sample plate.

Interpretations & Conclusions:

Due to the plate being almost completely lysed, the plate could then be flooded and collect the lysate.

Next Step:

The results of the spot test would help determine the dilution optimal for webbing a plate.

Category: Yang-En Yu | LEAVE A COMMENT