Yours

4.2: Phage/Plaque Assay Procedure

HGTC BIO 225: Microbiology Lab Manual · LibreTexts

0%

Contents

    Text

    Size
    100%
    Theme
    Typeface
    Line length
    Spacing

    From LibreTexts, under CC BY-SA, as the source states it; its equations are written in TeX, which the PDF shows typeset. This page at LibreTexts.

     

    Plaque Assay 

     

    Name: _____________________________________________________ 

    Course Section: ______________________________________________

    The following activity will teach students how to complete a plaque assay and calculate an original virus titer. Read the following procedure carefully before starting the activity. 

     Materials 
    • 100 uL T4 Bacteriophage
    • 1.5 mL of E. coli liquid culture
    • 10 mL of soft agar (Melted at 50°C)
    • 4 nutrient agar plates
    • 7 mL of filtered/sterile water
    • 10 sterile test tubes
    • 50°C incubator/microwave (something to melt the soft agar)
    • P200 pipette and tips
    • P1000 pipette andtips
    • Tape (For labeling test tubes)

    Procedure 

    Step 1: Preparing the soft agar

    1. Remove melted agar from the 50°C incubator or melt the agar in a microwave for 25 seconds.
    2. Grab four culture tubes, label each with a dilution (10-3 to 10-6), and pipette 2.5 mL of soft agar into each of the tubes.
    3. Place the tubes with the soft agar back into the 50°C incubator.

    Step 2: Serial diluting the T4 bacteriophage

    1. Take 4 nutrient agar plates, label with your initials, a dilution (10-3 to 10-6), the date, and your section and place these in the 37°C incubator to warm.
    2. Label 6 test tubes with dilutions 10-1 to 10-6. Measure 900 uL of filtered/sterile water into each of the tubes.
    3. Add 100 uL of T4 bacteriophage into the first tube labeled 10-1 and mix thoroughly by pipetting up and down.
    4. Pipette 100 uL of 10-1 dilution into the 10-2 labeled tube and mix thoroughly.
    5. Pipette 100 uL of 10-2 dilution into the 10-3 labeled tube and mix thoroughly.
    6. Pipette 100 uL of 10-3 dilution into the 10-4 labeled tube and mix thoroughly.
    7. Pipette 100 uL of 10-4 dilution into the 10-5 labeled tube and mix thoroughly.
    8. Pipette 100 uL of 10-5 dilution into the 10-6 labeled tube and mix thoroughly.

     

    Step 3: Plating the bacteriophage using a bacterial lawn

    1. Remove the test tube of melted soft agar labeled 10-3 from the 50°C incubator. Then add 300 uL of E. coli liquid culture and 100 uL of the 10-3 dilution of the T4 phage to the test tube.
    2. Roll the tube between your hands to mix and then pour the contents on top of your nutrient agar plate labeled 10-3. Swirl the plate around to evenly distribute the liquid over the plate.
    3. Repeat the previous two steps, but with the 10-4, 10-5, 10-6 dilutions of the T4 phage. (Make sure for each labeled plate you use the SAME dilution of phage.)
    4. Let all the plates sit for 20 minutes to harden. Then flip plates and incubate at 37°C overnight.
    5. Count plaques on the plate that has a range of 30-300 plaques and calculate the original virus concentration.

    Results    

     

      10-3 Dilution Plate 10-4 Dilution Plate  10-5 Dilution Plate  10-6  Dilution Plate

     

    Number of Plaques

     

     

     

     

    		      

     Note: If there are too many plaques to count, just denote the phage number as TNTC (Too numerous to count). 

    General Questions 

    1. Define bacteriophages and describe their two different lifecycles. 

     

     

     

     

    2. How do microbiologist culture bacteriophages? Can bacteriophages grow without a host?

     

     

     

     

    3. What is a plaque? Why can plaques be used to calculate the original concentration of a phage sample (original virus titer)?

     

     

     

     

    4. Which of your dilutions plates should be used to calculate the original T4 virus titer? Why?

     

     

     

     

    5. Using the total number of plaques counted from your selected dilution plate, calculate the original virus titer of the T4 phage. 

     

     

     

     

    6. How can bacteriophages be applied to medicine to help combat antibiotic resistance?

     

     

     

     

    7. Provide an example of how bacteriophages are used to limit bacterial growth in ready-to-eat meats.

     

    Attributions

    "Lab 10: Plaque Assay and Biochemical Tests (Day 1)" by Nazzy Pakpour & Sharon Horgan, LibreTexts Biology is licensed under CC BY-SA 4.0

    "Microbiology Chapter 6.2: The Viral Life Cycle" by Parker et al., Openstax, Digital: ISBN-13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Microbiology Chapter 6.3: Isolation, Culture, and Identification of Viruses" by Parker et al., Openstax, Digital: ISBN-13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Microbiology Chapter 9.1: How Microbes Grow" by Parker et al., Openstax, Digital: ISBN-13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Raven Biology 12th Edition Chapter 26.3: Bacteriophage - Bacterial Viruses" by LibreText Biology is licensed under CC BY 4.0

    "Biology 2e Chapter 21.3: Prevention and Treatment of Viral Infections" by Clark, Douglas, and Choi, Digital: ISBN-13: 978-1-947172-52-4, Openstax is licensed under CC BY 4.0

    4.2: Phage/Plaque Assay Procedure is shared under a CC BY-SA license and was authored, remixed, and/or curated by LibreTexts.