Viruses are microscopic entities that can only replicate inside the living cells of an organism. Quantifying the concentration of these particles in a laboratory sample, a process known as titration, is fundamental to virology. While many methods exist to count the total number of viral particles, a significant portion of these particles may be non-functional or incapable of causing infection. Therefore, researchers require a method that specifically measures only the infectious capacity of a viral stock.
What is a Plaque Forming Unit (PFU)?
A Plaque Forming Unit, or PFU, is a unit of measure that represents the number of infectious virus particles per unit volume of a sample. The term is derived from the plaque assay, an established technique designed to count only the viruses that successfully infect a host cell and replicate. PFU is the standard measurement for the concentration of infectious virus, often expressed as PFU/mL.
The PFU count is always less than the total number of physical viral particles in a sample because not every particle is capable of initiating a productive infection. The assay relies on the formation of a “plaque,” which is a localized area of dead or damaged host cells. This clearing forms when a single infectious virus particle infects a cell, multiplies, kills the original cell, and subsequently spreads to and kills the surrounding cells.
The core principle is that each distinct plaque visible on the cell culture dish is assumed to have originated from a single infectious viral unit. By counting these plaques, scientists can calculate the concentration of viable, infection-competent viruses. This unit is analogous to the colony-forming unit (CFU) used to quantify viable bacteria.
The Process of the Plaque Assay
The determination of PFU begins with preparing a confluent monolayer of host cells susceptible to the virus. This continuous layer of cells ensures no gaps are mistaken for viral damage later. The virus sample, whose concentration is unknown, must first be subjected to serial dilution, often by a factor of ten.
The purpose of serial dilution is to ensure that at least one dilution contains a low enough concentration of infectious particles to produce a countable number of distinct, non-overlapping plaques. A small volume of each diluted virus sample is then added to separate host cell monolayers and incubated briefly to allow the virus particles to attach to the cells. After this initial infection period, the liquid medium is removed, and a semi-solid medium, such as agar or carboxymethylcellulose, is added as an overlay.
This semi-solid overlay prevents the newly replicated virus particles from freely spreading throughout the liquid culture medium. Instead, the virus is restricted to infecting only the immediately adjacent cells, leading to the localized cell death that forms the visible plaque. The culture is incubated for several days to allow the plaques to develop sufficiently. The cells are then stained, typically with a dye like crystal violet or neutral red, which stains living cells but not dead ones.
The plaques appear as clear, unstained zones against the background of stained, healthy cells, making them easy to count. The final PFU/mL is calculated using a simple formula: the number of counted plaques is divided by the product of the volume of the virus added and the dilution factor used for that well. For instance, if 35 plaques are counted from a 0.1 mL volume of a 1:100,000 (or 10^-5) dilution, the original stock titer is 3.5 x 10^7 PFU/mL.
Why PFU is Essential for Viral Research
The PFU measurement is the standard method for quantifying infectious virus because it directly measures the biological function of the virus: its ability to infect and replicate. This makes it a necessary tool for developing and testing vaccines. Vaccine manufacturers must ensure that every batch of a live-attenuated vaccine contains a consistent, precisely measured dose of infectious virus to elicit a reliable immune response without causing disease.
In antiviral drug screening, PFU is used to accurately measure a drug’s efficacy by comparing the PFU count in treated and untreated viral cultures. A successful antiviral compound will significantly reduce the number of PFUs by inhibiting the virus’s ability to infect cells or replicate. This provides a direct, quantitative measure of the drug’s effectiveness in reducing the infectious burden.
PFU measurements are also fundamental in basic virology research, particularly for determining the Multiplicity of Infection (MOI). MOI is the ratio of infectious virus particles (PFU) to the number of host cells in a culture. Researchers use MOI to control experimental conditions, ensuring, for example, that most cells are infected by only a single virus particle. This control is necessary for studying the virus life cycle, gene expression, and virus-host interactions.
Understanding PFU Limitations
PFU only counts the infectious particles, which means the resulting titer is generally a significant underestimate of the total number of physical viral particles present in the sample. A viral stock is a mixture of infectious, non-infectious, and defective particles, all of which are physically present. Ratios of physical particles to PFU can vary widely depending on the virus and preparation, sometimes ranging from as low as 1.3:1 to over 8,000:1.
This difference between the total particle count and the PFU count provides important context for understanding the quality of a viral stock. A high particle-to-PFU ratio indicates a less efficient, or less infectious, preparation. Furthermore, the PFU count is dependent on the specific cell line used for the assay, as well as factors like the pH and incubation medium.
Changing the host cell line can alter the virus’s ability to attach and replicate, meaning a stock measured on one cell type may yield a different PFU on another. Therefore, PFU is not an absolute, universal measure of concentration but rather a measure of infectious titer under specific, defined laboratory conditions.