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A plaque assay is a cell-culture method that counts infectious virus. A layer of susceptible cells is infected with diluted virus and covered with a gel or semisolid overlay. Each clear spot of dead cells (a plaque) is counted as one plaque-forming unit (PFU), and the result is reported as PFU per mL. It measures infectious particles, not total particles, and works only for viruses that kill their host cells.
Definition and scope
A recent Viruses paper describes plaque assays as “one of the most accurate methods for directly quantifying infectious replicating viruses,” with the result expressed as plaque-forming units per mL (PFU/mL) (Murray et al., 2025). A 2026 Journal of General Virology paper on honey bee viruses describes the same logic: an infected monolayer of cells is overlaid with a polymer matrix that limits viral particle diffusion, each plaque effectively denotes an infectious viral particle, and it is referred to as a p.f.u. (McMenamin and Goblirsch, 2026).
The method sits within virology as a way to quantify infectivity, and it is the usual reference point when a lab needs a titer for a stock of virus, such as the titering step described in the lentiviral transduction protocol guide.
How a plaque assay works in practice
The published methods share a common logic rather than a single recipe.
- Permissive cells. The assay needs a cell type that is permissive to infection by the virus of interest (McMenamin and Goblirsch, 2026).
- Dilution of the sample. The inoculum is diluted so that individual plaques can be counted; the honey bee study counted wells where the dilution of inoculum yielded 5-100 plaques per well. That window is one study’s choice, not a universal rule. See the serial dilution guide for the arithmetic behind dilution series.
- Overlay. The overlay restricts particle movement, so that newly produced viruses can only infect neighboring cells (Murray et al., 2025). That is what turns each infection into a discrete, countable spot. The honey bee study tested a standard solid agarose overlay and a semisolid carboxymethyl cellulose (CMC) overlay.
- Fixing, staining and counting. In the influenza study, cells were fixed with 60:40 acetone:methanol for 20 minutes at room temperature, counterstained with crystal violet, and the plaques were counted. This describes that study’s method only.
When conditions are suboptimal, the honey bee authors note that a large variation in plaque size or diffuse plaque borders may result, which makes counting less reliable.
Plaque assay vs fluorescent focus assay vs qPCR
Each readout answers a slightly different question. The table sets out what each one reports and the strength and limit each paper states.
| Method | What it reports | Stated strength | Stated limit |
|---|---|---|---|
| Plaque assay | Infectious virus as PFU/mL | Direct quantification of infectious replicating virus (Murray et al., 2025) | Only works with viruses that form visible plaques by killing host cells (Murray et al., 2025) |
| Fluorescent focus assay (FFA) | Infected foci | Can quantify non-lytic viruses (Murray et al., 2025) | Counts infected foci rather than plaques, so it is not a PFU measurement (Murray et al., 2025) |
| qRT-PCR | Viral load | Described by the honey bee authors as more rapid, accessible and sensitive for viral load (McMenamin and Goblirsch, 2026) | Reports viral genome, which is a different question from infectious titer |
For a practical look at the nucleic-acid route, see the qPCR and RT-qPCR guide.
What a plaque count does and does not tell you
Use this checklist when reading or reporting a PFU/mL figure.
- It counts infectious virus. A plaque forms only when a virus infects cells and spreads to neighbors, so PFU reflects infectivity.
- It requires visible plaques. Viruses that do not kill their host cells to form visible plaques cannot be measured this way (Murray et al., 2025).
- PFU is not a particle count. The influenza authors state that one cannot know whether there is a one-to-one ratio of plaques to infectious viruses.
- It depends on the cells. A permissive cell type is required, so a titer is specific to the cell line and virus pairing used.
- Conditions affect readability. Variable plaque size or diffuse borders can follow from suboptimal conditions.
Where it matters for labs and supply decisions
Because a plaque assay is built from cells, an overlay and a staining step, the items a lab typically has to line up are those named in the methods above:
- cells permissive to the virus, maintained under standard cell culture practice;
- an overlay material: the honey bee study compared solid agarose with semisolid CMC;
- fixative and a counterstain: the influenza study used acetone:methanol and crystal violet;
- containment suited to the virus being handled; see the biosafety levels compared.
These are examples from published methods, not a required list. A lab should follow the protocol validated for its own virus and cell line.
Common misunderstandings
“One plaque equals one virus particle.”
The honey bee paper treats each plaque as effectively denoting an infectious particle, but the influenza paper cautions that a one-to-one ratio cannot be assumed. Treat PFU as a measure of infectious units.
“Any virus can be titered by plaque assay.”
No. The method only suits viruses that form visible plaques by killing host cells. Non-lytic viruses need another readout such as a fluorescent focus assay.
“qPCR replaces the plaque assay.”
qRT-PCR is faster and more sensitive for viral load according to the honey bee authors, but it answers a different question from infectious titer.
FAQ
What does PFU/mL mean?
Plaque-forming units per milliliter: the number of plaques counted, adjusted for the dilution and volume of inoculum, expressed per mL of the original sample.
Why is an overlay used?
The overlay restricts particle movement so newly produced viruses can only infect neighboring cells, producing discrete plaques (Murray et al., 2025).
Can a plaque assay measure non-lytic viruses?
No. A fluorescent focus assay offers the ability to quantify non-lytic viruses (Murray et al., 2025).
Sources
- Murray J, Nogales A, Martinez-Sobrido L, Martin DE, Sancilio FD, Tripp RA. Evaluating Probenecid or Oseltamivir Inhibition of Influenza A Virus Replication Through Plaque Assay or Fluorescent Focus Assay Using Non-Structural Protein 1-H1N1 Venus Reporter Virus. Viruses 17(3):335, 2025. doi:10.3390/v17030335. https://europepmc.org/article/PMC/PMC11945664 (CC BY 4.0). Date checked: 2026-10-29.
- McMenamin A, Goblirsch M. Quantitation of cytopathic honey bee viruses using the viral plaque assay method. J Gen Virol 107(7), 2026. doi:10.1099/jgv.0.002304. https://europepmc.org/article/PMC/PMC13412448 (CC BY 4.0). Date checked: 2026-10-29.
