Virus Plaque Assay: A Technique to Measure Infectious Herpes Simplex Virus via Quantification of Plaques Formed in Monolayer of Virus-Infected Cells

Abstract

Source: Sadowski, L. A. et al. Plaquing of Herpes Simplex Viruses. J. Vis. Exp. (2021)

This video describes the virus plaque assay of the infectious herpes simplex virus or HSV. The diluted viral particles are inoculated to the monolayer of epithelial cells. Upon incubation, the virus forms plaques and is counted visually to determine the infectious virus titer.

Protocol

1. Seeding of the Vero cells

1. The day before initiating the plaque assay, trypsinize Vero cells and resuspend them in regular Dulbecco's Modified Eagles Medium (DMEM) and supplement as per standard cell culture methodology. Resuspend the trypsinized cells in 10 mL of DMEM per T-75 flask of the confluent Vero cells (~10⁷ cells).
   NOTE: DMEM is used for plaque assays instead of alpha-MEM because it slightly slows the cell growth rate and favors better plaque assay results.
2. Count the cells by one's preferred method (e.g., a standard hemocytometer with Trypan Blue exclusion).
3. Seed the cells at 4 x 10⁶

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12-well plates	Corning	3512
6-well plates	Corning	3516
Alpha-MEM	Lonza	12169F
Antibiotic/antimycotic	Gibco	15240096
Crystal violet	Alfa Aesar	B2193214
DMEM	Gibco	11965092
Dulbecco's PBS (no Mg++ or Ca++)	Gibco	14190144
Fetal calf serum	Millipore-Sigma	TMS-013-B
L-alanyl-L-glutamine (Glutamax)	Gibco	GS07F161BA
Hemacytometer	Thermo Fisher	02-671-54
Methylcellulose	Millipore-Sigma	27-441-0
Quaternary agent (Lysol I.C.)	Thermo Fisher	NC9645698
Trypan Blue	Corning	25900CI
Trypsin	Cytiva	SH30042.01
Vero cells	ATCC	CCL-81