Why does null hypothesis testing matter for plaque quantification?

Null hypothesis testing in plaque quantification enables objective assessment of differences in viral infectivity, supporting rigorous target validation and reducing false positives in early discovery. This statistical approach ensures that observed plaque count changes are significant and actionable for R&D decisions.

How does independent variable isolation fit the trypsin activation workflow?

Isolating the effect of trypsin activation on rotavirus entry allows teams to attribute changes in plaque formation specifically to protease treatment, clarifying mechanistic pathways and supporting confident advancement of entry-targeted interventions.

What do quantitative plaque counts enable in virology assays?

Quantitative plaque counts provide a direct measure of infectious viral particles, enabling comparison across experimental conditions and supporting data-driven optimization of assay parameters and antiviral screening campaigns.

Why are replication requirements critical for cross-functional plaque assay use?

Replication ensures that plaque assay results are reproducible and reliable across teams, facilitating cross-functional collaboration and enabling consistent data interpretation for portfolio-level decision-making.

Which statistical analysis capabilities are required before implementing plaque-based readouts?

Robust statistical analysis, including calculation of mean plaque counts, standard deviations, and significance testing, is essential to validate assay performance and ensure that observed differences reflect true biological effects rather than experimental variability.

Visualizing Trypsin-Activated Rotavirus Infection Using a Plaque Assay

JoVE Encyclopedia of Experiments

Microbiology

0 views • 4:12 min • July 31st, 2026

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Begin with a diluted recombinant rotavirus suspension pre-treated with trypsin.
Trypsin cleaves outer capsid proteins, activating the viruses for efficient attachment.
Add the diluted virus suspension onto a monolayer of permissive epithelial cells and incubate.
Gently shake the plate at regular intervals to evenly distribute the viruses.
The virus enters, replicates, and forms new viruses. Aspirate the suspension to remove extracellular viruses.
Overlay the cells with a semi-solid agarose medium containing trypsin. Allow the agarose to solidify, which restricts virus spread, and incubate.
Infected cells lyse, releasing viruses and forming clear zones called plaques. Trypsin activates viruses and supports reinfection, increasing plaque formation.
Add another overlay containing a cell-permeable dye and incubate in the dark.
The dye stains viable cells red, while the plaques remain unstained.
Using a light box, visualize the plaques.
Extract the recombinant viruses from the plaques and transfer them into fresh media for downstream analysis.
Add trypsin to 100 microliters of clarified cell lysate to a final concentration of 10 micrograms per milliliter and incubate the mixture at 37 degrees Celsius for 1 hour. Then prepare a 10-fold serial dilution series ranging from ten to the negative one to ten to the negative seven in DMEM incomplete medium.
Rinse the MA104 monolayers twice with 2 milliliters of PBS and once with DMEM incomplete medium. Add 400 microliters of lysate dilutions in duplicate to the plates and incubate them at 37 degrees Celsius and 5% carbon dioxide, rocking every 10 to 15 minutes to redistribute the dilutions across the monolayer. Prepare an agarose MEM overlay solution by combining equal volumes of pre-warmed 2X EMEM with 1.5% melted and cooled agarose.
Maintain this solution at 42 degrees Celsius in a water bath and adjust the trypsin concentration to 0.5 micrograms per milliliter immediately before placing it on cells. Aspirate lysate dilutions from the six-well plate. Then rinse the cells once with 2 milliliters of incomplete medium.
Gently add 3 milliliters of the agarose MEM overlay solution onto the cell monolayer in each well. Allow the agarose to harden at room temperature. Then return the plates to the incubator.
Three days later, prepare an agarose MEM overlay solution as previously described and add neutral red to a final concentration of 50 micrograms per milliliter immediately before use. Add 2 milliliters of the overlay solution on top of the existing agarose in the six-well plates. Allow the agarose to harden and return the plate to the incubator, making sure to protect the plates with neutral red from light.
Over the next 6 hours, identify rotavirus plaques with the aid of a light box. Use disposable transfer pipettes to pick clearly defined plaques, recovering agarose plugs that extend fully to the cell layer.