22: VIRAL PLAQUE ASSAY
- Page ID
- 157078
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Perform a viral plaque assay using serial dilutions to quantify the number of infectious virus particles in a sample, and accurately calculate the plaque-forming units per milliliter (PFU/mL).
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Explain the relationship between bacteriophage replication cycles (lytic and lysogenic) and plaque formation, and evaluate the impact of bacterial growth phase and experimental technique on assay outcomes.
BACKGROUND
Viruses are not classified as living organisms because they lack the ability to reproduce independently. Instead, they must infect a host cell (a living cell that supports viral replication) and utilize its internal machinery to carry out the processes necessary for viral reproduction. Structurally, a virus consists of genetic material—either DNA or RNA—enclosed within a protein coat. Upon infection, the virus takes over the host cell’s metabolic processes (the chemical reactions essential to sustaining life) to produce new viral particles.
In this laboratory exercise, we will be working with a bacteriophage, (a type of virus that specifically infects bacterial cells). In order to propagate a bacteriophage, we must provide it with actively growing bacterial cells and the appropriate nutrient conditions required for bacterial growth.
To accomplish this, a small volume of liquid culture containing millions of rapidly dividing bacterial cells will be spread onto the surface of a nutrient agar plate. Unlike experiments designed to isolate individual colonies, this method aims to produce a bacterial lawn (continuous, uniform layer of bacterial growth). While individual bacterial cells are not visible to the naked eye, their collective growth creates a hazy, opaque surface that resembles a grassy lawn.
Following the preparation of the bacterial lawn, bacteriophage is added to the plate. When a virus particle encounters a susceptible bacterial cell, it attaches to the cell surface and injects its DNA into the host. This initiates the bacteriophage lytic cycle (a viral replication process in which the bacteriophage commandeers the host’s cellular machinery to synthesize and assemble new virus particles. Once the replication process is complete, the host cell undergoes lysis (rupturing of the cell membrane), releasing newly formed viruses into the surrounding environment. These viruses then infect neighboring cells, perpetuating the cycle for as long as the bacterial population remains in the log phase (the period of rapid cell division when resources are abundant).
Each successful infection initiated by a single virus particle produces a plaque (visible clearing in the bacterial lawn). These clear zones represent areas where host cells have been lysed by the virus. If the viral sample is viable and evenly distributed, individual plaques will be uniform in size and shape. However, if the sample contains an excessive number of viruses, the entire bacterial lawn may be destroyed, preventing proper plaque visualization.
Some bacteriophages are also capable of an alternative reproductive strategy known as the lysogenic cycle. In this pathway, the viral DNA is incorporated into the bacterial chromosome, forming a prophage (viral DNA integrated into the host genome). The infected bacterial cell, now referred to as a lysogen, continues to grow and divide without producing new viruses or undergoing lysis. The prophage is stably maintained and passed on to daughter cells during bacterial replication. Additionally, lysogenized cells typically become immune to reinfection by the same type of bacteriophage.
Under certain environmental stress conditions—such as exposure to ultraviolet (UV) light) or chemical agents—the lysogenic cycle can be disrupted. The prophage may be excised from the bacterial chromosome, initiating the lytic cycle and leading to viral production and eventual cell lysis. This reactivation results in plaque formation similar to a standard lytic infection.
To quantify the concentration of infectious virus particles in a sample, a plaque assay is performed. If a sample is overly dilute, plaques may be absent or too few to count accurately. Conversely, if the viral concentration is too high, the host cells may be entirely lysed, resulting in no visible lawn or plaques may merging together preventing accurate count. To achieve a countable range of plaques, a series of serial dilutions (systematic reductions in concentration) is prepared. A well-planned dilution series allows for accurate estimation of the number of viable virus particles present in the original sample
MATERIALS
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1 5 mL of bacteriophage (obtain from instructor)
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1 5 mL log phase bacterial culture (obtain from instructor)
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6 Nutrient agar plates, (3 per student)
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6 sterile transfer pipets
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3 Nutrient broth dilution tubes, 9.0 ml each
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1 Bent glass rod
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1 Alcohol jar
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1 Turntable
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6 10 mL Serological sterile pipette
METHODS/PROCEDURES
Gather and label your materials
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Number the nutrient broth dilution tubes 1, 2, and 3 then make sure all three tubes have equal volume of broth. If the volume is not equal use a transfer pipette the transfer broth between tubes until equal
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Each student should label their 3 plates with the required information along with A, B, and C along with the appropriate dilution factor.
2. Performing serial dilutions
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Obtain the bacteriophage from your instructor then gently swirl the nutrient broth tube to distribute the phage in the medium
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Using a sterile transfer pipette, transfer 1.0 mL of the virus sample into the first dilution tube then distribute the virus by gently by tapping or swirling the tube. Discard the used pipet
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Using a fresh sterile pipet, transfer 1.0 mL from tube 1 into tube 2. Mix gently and discard the pipet
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Using another fresh sterile pipette, transfer 1.0 mL from tube 2 into tube 3. Mix gently and discard the pipet
Be sure to use a new pipet for each transfer to prevent cross-contamination
3. Preparing the viral plaque assay plates
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Obtain the host bacterial culture from your instructor
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Gently mix the host bacterial culture to resuspend cells
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Using a sterile transfer pipet, dispense 3–4 drops of the bacterial suspension onto the surface of plate A then immediately add 0.1ml of the first viral dilution tube
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Use the alcohol jar, turntable and cell spreader cell to spread the virus and bacteria on the agar plate to form a lawn
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Flame the glass rod by dipping it in alcohol and briefly passing it through a flame
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Spread the mixture of bacteria and virus evenly across the surface of the agar
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Repeat steps b-f for each plate. Be sure to re-sterilize the rod between each plate
4. Absorption and incubation
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Allow the plates to sit at room temperature, agar-side down, for at least 15 minutes to permit full absorption of the liquid into the agar.
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After 15 minutes invert the plates (agar side up) and place them in the incubator as directed by your instructor.
Print a hard copy to bring to lab (PDF).
👉 If you are filling this out on a digital iPad or tablet please note put your name here and take a screen shot.
You are also welcome to print the PDF and turn in a physical copy of the following.
Exercise 11:
NAME ______________________
EXPECTATIONS
Which plates do you think will have the highest number of viral plaques? Why?
RESULTS
Choose one of your plates that has clear plaques and draw it below. Remember to label fully.
Name of the bacterial host culture______________________ Virus sample ID____________________
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Number of plaques |
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Plate A |
Plate B |
Plate C |
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Student 1 plates |
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Student 2 plates |
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Calculate the concentration of plaque forming units per mL in the original sample (PFU)
Viral concentration of original sample __________ PFU/mL.
CONCLUSIONS
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Why is it important to perform plaque assays using bacteria that are in the log phase of growth rather than in the stationary or death phase?
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If no bacterial lawn appears on a plaque assay plate, the cause could be either too few bacteria or too many viruses. Since both conditions produce similar-looking results (a plate with no visible lawn), virologists use a control plate to determine the actual cause. What should be added to the control plate to distinguish between these two possibilities, and why?
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How would exposing a bacterial lawn carrying a lysogenic virus to a sub-lethal dose of ultraviolet (UV) light affect plaque formation? Would you expect the number of plaques to increase or decrease? Explain your reasoning based on the effect of UV light on the lysogenic cycle.
4. Why is it important to spread the virus and bacteria mixture evenly across the agar surface when preparing a plaque assay? How could uneven spreading affect your results?