4.1: Introduction to Phages

Learning Objectives

• Define "bacteriophage" and their role in microbiology.
• Differentiate between and describe lytic and lysogenic bacteriophage lifecycles.
• Describe how, as obligate intracellular pathogens, bacteriophages are able to be grown in culture.
• Explain the purpose and demonstrate proficiency in performing a plaque assay.
  • Define a "plaque".
  • Learn how to perform serial dilutions
  • Acquire the skills necessary to cultivate bacterial lawns for the purpose of plaque formation.
• Interpret plaque assay results accurately, proficient in calculating the total virus titer. 

 

1. What are Bacteriophages?

Bacteriophages, commonly referred to as phages, are viruses which specifically infect bacteria. As obligate intracellular pathogens, phages lack their own cellular machinery necessary for replication. Therefore, they rely heavily on bacteria, and must hijack their host’s cellular machinery to reproduce, often killing them in the process.

Bacteriophages are nearly ubiquitous in nature and exhibit a wide variety of shapes and sizes. Generally, they are much smaller and more structurally simple than bacteria, since they do not carry many of the components necessary for replication. Most consist of a protein coat surrounding a viral genome, with a tail used for attachment and injection of its genome (Figure 1). This might not seem like much, but these are all the components required for bacteriophages to infect their hosts.

 

Figure 1: (a) An electron microscope image of a bacteriophage attaching to a bacterium. (b) Diagram of a general bacteriophage structure. (Credit: Microbiology an OpenStax textbook.)

 

2. Bacteriophage Lifecycles

Phages can undergo two primary life cycles when infecting bacteria: the lytic cycle and the lysogenic cycle.

 

2.1 The Lytic Cycle

During the lytic cycle, phages immediately begin replicating after infecting a bacterial cell, ultimately resulting in the lysis, or destruction, of the host. There are five distinct stages included in the lytic cycle (Figure 2):

1. Attachment: The phage attaches to the surface of a bacterial cell. Most phages have a narrow host range, meaning they are only capable of attaching and infecting one species or strain of bacteria. While much rarer, some phages have a broad host range and are capable of infecting several species of bacteria rather than just one.
2. Penetration: During this stage, the bacteriophage’s tail acts like a hypodermic needle and injects the viral genome into the bacterial cell. It is important to note, the only component of the phage that enters the bacterial cell is the viral genome, all other components (including the capsid, tail, etc.) remain outside the bacterium.
3. Biosynthesis: Once inside, the viral genome induces the synthesis of proteins that hijack the host cell. The hijacked cell then becomes a puppet the bacteriophage uses to produce new viral components (capsid proteins, tail fibers, etc.) and replicate its viral genome.
4. Maturation: The viral components made during the biosynthesis stage self-assemble to form new phages.
5. Lysis: Viral proteins produced during the biosynthesis stage, such as lysozyme, will break down the cell wall/membrane, causing the bacteria to rupture or burst. The newly produced phages are then released and free to spread throughout their environment and infect new bacterium.

 

Figure 2: The lytic lifecycle is depicted above. There are five stages of the bacteriophage lytic lifecycle, which results in phage production and host cell death. (Credit: Microbiology an OpenStax textbook.)

 

2.2 The Lysogenic Cycle

In contrast, during the lysogenic cycle, the phage does not immediately begin replicating after infecting a bacterium. Instead, the phage incorporates its viral genome into the host genome, creating a prophage (Figure 3). These prophages do not lyse, or destroy the host cell, rather, they remain dormant and are replicated along with the bacterium. However, specific conditions, such as stress, can trigger them to reactivate, causing the prophage to enter the lytic cycle. Since a prophage contains its own genes, its incorporation into the bacterial genome can induce phenotypic changes in the host. This is known as lysogenic conversion and is responsible for increasing the virulence of many bacterial pathogens through the introduction of toxin genes. Vibrio cholera and Clostridium botulinum are examples of pathogenic bacteria in which many of their toxin producing genes originate from prophages (Parker et al., 2016).

 

Figure 3: This figure depicts the lysogenic cycle. During this cycle, instead of outright killing their host, a bacteriophage will incorporate their genome into the host cell and remain dormant. However, this dormant prophage always has the potential to reactive and enter the lytic cycle. (Credit: Microbiology an OpenStax textbook.)

 

3. Culturing Bacteriophages

The biggest challenge when culturing bacteriophages, or any virus in general, is that they are incapable of replicating by themselves. If you put 1 trillion bacteriophages into sterile media, and placed them in an incubator, nothing would happen, not a single new bacteriophage would form. To culture bacteriophages, they must have an ample supply of hosts. Therefore, when growing bacteriophages, microbiologists must place them in the presence of the bacterium they are able to infect.

Whereas the bacterial growth in culture is indicated by turbidity or colonies, bacteriophages do not have their own discernable features to indicate their presence. Instead, bacteriophage growth in culture is indicated by the death or lack of bacterial growth. This death or lack of growth can be visualized using a plaque assay. The steps to perform a plaque assay are as follows:

1. Create a bacterial lawn: A bacterial lawn is a thin, dense layer of bacterial growth in semisolid or soft agar, that is often laid upon a nutrient agar plate. The “soft” quality of the agar allows bacteria to grow and spread throughout, which gives the lawn a cloudy, or turbid quality.
2. Introduce phages to the lawn: Microbiologists will then introduce phages to the bacterial lawn. The phages will infect and lyse some of the bacterium present within the lawn.
3. Formation of plaques: Since bacterial growth in the lawn causes it to be turbid or cloudy, death of bacteria due to phages infection results in the formation of small, clear zones, or plaques, from the lack of bacterial growth (Figure 4).

 

Figure 4: This figure depicts several bacterial lawns grown in the presence of a phage. The clear zones or plaques indicate where bacterial cells have been killed or lysed. (Credit: Microbiology an OpenStax textbook.)

 

4. Calculating Original Virus Titer

Similar to colonies for bacteria, plaques often originate from a singular phage. Therefore, plaque number can be used to estimate the original viral titer, or the concentration of the phage in the original sample. The formula for calculating original virus titer is shown below:

Original viral titer = (Number of plaques / volume of phage plated) X (1 / dilution factor)

NOTE: The original viral titer is calculated in PFU or plaque forming units

For example:

A sample of T4 was diluted by a factor of 10^-4, and 0.1 mL of the dilution was plated on a bacterial lawn of E. coli. After a 24-hours incubation period, 56 plaques were observed on the plate. Calculate the original viral titer.

Step 1: Calculate the plaque number in diluted sample (convert all volume measurements to milliliters)

Step 2: Calculate the inverse of the dilution factor

Step 3: Calculate original viral titer by multiplying the solution from steps 1 and 2

Therefore, the original viral titer of the undiluted T4 phage sample is 5.6 x 10^6 PFR/mL or 5,600,000 PFU per mL

5. Serial Dilutions

When performing a plaque assay, a problem can arise if the original phage concentration is excessively high. A bacterial lawn inoculated with such a sample becomes completely inundated with plaques, so plentiful they overlap and become indiscernible from one another. This makes it impossible to count the total number of plaques. To remedy this, microbiologists perform serial dilutions (Figure 5). Serial diluting is the process of diluting a sample across a series of tubes, often by factor of 1:10, to obtain a desired concentration. In the case of a plaque assay, this is a concentration of phages that will produce a countable number of plaques, typically between 30 to 300. A lawn with less than 30 plaques is rarely used for calculations as it is susceptible to statistical errors. In a lawn with more than 300 plaques, the plaques begin to blend together, making it difficult to ascertain an accurate number.

Figure 5: This figure depicts the process of serial diluting a phage sample by a factor of 1:10. The first dilution is labeled 1:10, or 10^-1, with each subsequent dilution being a tenth of the previous (1:100 or 10^-2, 1:1000 or 10^-3, etc.).  Each dilution is plated. The 1:10,000 or 10^-5 concentration produces plaque number within the desired range of 30 to 300. (Credit: Figure adapted from Microbiology an OpenStax textbook)

 

Real-World Connections

Bacteriophages: A Potential Solution to Antibiotic Resistance?

The discovery of antibiotics remains one of the most significant medical breakthroughs of the 20th century. Often referred to as “magic bullets,” antibiotics revolutionized medicine by becoming the first treatment to effectively target and eliminate bacterial infections without causing harm to patients. As a result, once-devastating diseases such as leprosy, plague, and tuberculosis became manageable and, in many cases, curable.

However, the effectiveness of antibiotics is increasingly threatened by the emergence of antibiotic-resistant bacteria. Many bacterial pathogens have evolved mechanisms to evade the effects of conventional antibiotic therapy. This growing resistance has prompted an urgent search for alternative treatment strategies.

One promising alternative is the use of bacteriophages—viruses that specifically infect and destroy bacteria. For every bacterial species, including human bacterial pathogens, there exists at least one corresponding bacteriophage capable of infecting it. Therefore, bacteriophages could be harnessed as a weapon against bacterial infections.

Bacteriophages offer several advantages over traditional antibiotics. Their host specificity allows them to target pathogenic bacteria without disrupting the patient’s beneficial microbiota. Moreover, because bacteriophages cannot infect human cells, they present a low risk of complications in patients. In fact, the U.S. Food and Drug Administration (FDA) has already approved the use of a bacteriophage to reduce the risk of Listeria contamination in ready-to-eat meats (Clark, Douglas, and Choi, 2018).

Despite their potential, bacteriophage therapy faces notable challenges. The high specificity that makes them so effective also limits their broad applicability. Each bacterial infection would require a unique phage for treatment, demanding rapid and accurate diagnosis to match the causative pathogen to the correct bacteriophage treatment. This requirement complicates treatment and diagnosis which highlights the need for continued research on bacteriophage therapy.

 

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 6.1: Viruses" by Parker et al., Openstax, Digital: ISBN-13: 978-1-947172-23-4 is licensed under CC BY 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

Image Citations

Figure 1, "Microbiology Chapter 6.1: Viruses" by Parker et al., Openstax, Digital: ISBN-13: 978-1-947172-23-4 is licensed under CC BY 4.0

Figure 2, "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

Figure 3, "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

Figure 4, "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

Figure 5, Modified from "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