Viral titration is essential for quantifying infectious virus in research, vaccine production, and clinical diagnostics. The plaque assay is the gold standard for measuring infectious viral titer.
The Plaque Assay Principle
When a virus infects a monolayer of susceptible cells in a culture dish, it replicates and spreads to adjacent cells. After several rounds of infection, a localized zone of cell death (a plaque) becomes visible. Each plaque is assumed to originate from a single infectious virus particle.
The process:
- Grow a confluent monolayer of susceptible cells (e.g., Vero cells for many viruses) in a multi-well plate.
- Infect the monolayer with serial dilutions of the virus sample.
- Allow virus adsorption for 1–2 hours with periodic rocking.
- Overlay the cells with a semi-solid medium (agarose, methylcellulose, or carboxymethyl cellulose) that prevents the virus from spreading through the liquid medium, limiting infection to cells adjacent to the original infection site.
- Incubate for 2–10 days (depending on the virus) until plaques are visible.
- Fix and stain the monolayer with crystal violet, neutral red, or formalin. Plaques appear as clear areas against a stained cell background.
Count plaques and calculate: plaque-forming units per mL (PFU/mL) = (number of plaques) / (dilution × volume plated in mL).
TCID₅₀ Assay
The TCID₅₀ (50% Tissue Culture Infectious Dose) assay is an endpoint dilution method that does not require plaque visualization. Serial dilutions of the virus are added to replicate wells of a 96-well plate containing susceptible cells. After incubation, wells are scored for cytopathic effect (CPE). The TCID₅₀ is the dilution at which 50% of the wells show CPE, calculated by the Reed–Muench or Spearman–Kärber method.
TCID₅₀/mL values can be converted to PFU/mL (approximately 1 PFU ≈ 0.7 TCID₅₀ units), but the relationship is not exact and depends on the virus and cell type.
Fluorescent Focus Assay
For viruses that do not produce clear plaques or CPE, viral proteins are detected by immunofluorescence. The infected monolayer is fixed and stained with fluorescent antibodies against a viral antigen. Fluorescent foci are counted under an epifluorescence microscope, giving focus-forming units per mL (FFU/mL).
Hemagglutination Assay
Some viruses (influenza, paramyxoviruses) have hemagglutinin proteins that bind and agglutinate red blood cells. Serial dilutions of the virus are mixed with RBCs in a V-bottom plate. The hemagglutination titer is the highest dilution that still causes agglutination (a diffuse lattice of RBCs that does not settle into a button). The HA assay is fast and inexpensive but measures total viral particles, not infectious ones.
