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Analytical Chem JoVE (Open Access) Citable · DOI

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

DOI: 10.3791/52065-v
What you'll learn
  • ✓ Perform plaque assays to quantify viral titers on host cell monolayers
  • ✓ Compare semisolid overlay versus liquid microcrystalline cellulose overlay systems
  • ✓ Calculate viral concentrations from plaque counts and dilution factors
  • ✓ Apply plaque assay techniques across multiple virus families
Protocol

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Difficulty
intermediate
Total time
~3–5 days (includes cell preparation, viral infection, fixation, and plaque visualization)
Model organism
Mammalian cell culture monolayers (species/line not specified in abstract)
Biosafety
BSL-2

Steps

1
Prepare cells and reagents for assay

Set up host cellular monolayers and prepare overlay solutions (semisolid and microcrystalline cellulose formulations) required for the plaque assay.

▶ 01:30
2
Perform serial dilutions and infect monolayers

Create serial dilutions of virus stock and inoculate prepared cell monolayers with diluted virus to establish infection conditions for plaque formation.

▶ 03:25
3
Fix and stain infected cell monolayers

Remove overlay medium, fix cells with appropriate reagent, and apply staining protocol to visualize plaques on the monolayer.

▶ 05:34
4
Count plaques and determine viral titer

Count individual plaques on stained monolayers and apply dilution factor calculations to determine viral concentration in plaque-forming units per milliliter.

▶ 06:34
5
Analyze results and compare overlay systems

Review plaque assay data comparing traditional semisolid overlays with novel liquid microcrystalline cellulose overlay performance across virus families tested.

▶ 07:02
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