What Is a Phage Assay and How Is It Performed?

A bacteriophage, or “phage,” is a virus that targets and replicates within bacteria. To study these ubiquitous microorganisms, scientists use a laboratory method known as a phage assay. This technique is designed to detect and count the number of active phage particles in a sample, allowing researchers to understand a viral population’s concentration and viability. The assay provides a reliable way to visualize and quantify these bacteria-infecting viruses.

The Plaque Assay Method

The most established technique for a phage assay is the soft agar overlay method. The process begins by serially diluting a sample containing phages to systematically reduce its concentration. Each dilution is then mixed with a high concentration of a suitable host bacteria, which must be in its logarithmic phase of growth to ensure susceptibility to infection. This mixture is suspended in a tube of molten, soft agar kept at a temperature that keeps it liquid without killing the bacteria.

This mixture is poured over a petri dish containing a solid nutrient agar base and swirled to create a thin, even top layer. The soft agar solidifies, immobilizing the bacteria and phage particles. The plates are then placed in an incubator for 12 to 24 hours at a temperature optimal for bacterial growth. During this incubation, uninfected bacteria multiply to form a dense, cloudy layer across the plate known as a bacterial lawn.

Against this cloudy lawn, clear zones called plaques may appear, representing areas where bacteria have been destroyed by phages. The formation of a plaque is initiated by one infectious phage particle infecting a single bacterial cell. After replicating inside the host, the phage causes the bacterium to burst and release new phage progeny. These newly released phages then infect adjacent bacteria, repeating the cycle and creating a visible zone of clearing.

Quantifying Phage Concentration

A plaque assay provides a quantitative measure of the phage concentration in the original sample. Each plaque on the bacterial lawn is presumed to have originated from a single infectious viral particle. This assumption allows scientists to count the plaques and calculate the concentration, referred to as the phage titer. The final titer is expressed in Plaque Forming Units per milliliter (PFU/mL).

To determine the titer, a plate with a countable number of plaques, usually between 30 and 300, is selected. This range is considered statistically reliable and avoids plaques merging, which makes counting difficult. The number of plaques is counted and used in a calculation that accounts for the dilution factor and the volume plated. The formula effectively reverses the dilution to find the initial phage count.

Variations of Phage Assays

While the soft agar overlay assay is standard for quantification, other variations exist. A simpler alternative is the spot assay. In this method, a bacterial lawn is prepared, and then small drops of different phage dilutions are placed onto the agar surface. After incubation, zones of clearing where the drops were applied indicate the presence of phages. This technique is qualitative and used to quickly determine if a sample contains active phages.

Another variation is the enrichment assay, used when the phage concentration in a sample is expected to be extremely low. Direct plating of environmental water or soil samples would likely yield no plaques. The sample is first incubated in a liquid broth with a host bacteria culture. This step allows any phages present to multiply, amplifying the population to a detectable level before performing a plaque or spot assay.

Applications in Science and Medicine

Phage assays are used across numerous scientific and medical fields. A prominent application is in the development of phage therapy. With antibiotic resistance becoming a global health threat, phages are investigated as an alternative for treating bacterial infections. Assays are used to isolate, purify, and determine the dosage of therapeutic phages targeted against specific pathogenic bacteria.

The assays also extend to environmental monitoring. Phages that infect coliform bacteria, which are associated with fecal matter, can serve as indicators of water contamination. In the food industry, phage assays are employed to detect phages that infect foodborne pathogens like Salmonella and Listeria. This helps ensure food safety by monitoring for bacterial contamination in processing environments.

The phage assay is also used in basic research. For decades, it has been applied in molecular biology and genetics to investigate virus-host interactions, study gene function, and understand the mechanisms of viral replication. Its simplicity and effectiveness in isolating and quantifying viruses have been central to building our modern understanding of virology.