High throughput virus plaque quantitation using a flatbed scanner

The plaque assay is a standard technique for measuring influenza virus infectivity and inhibition of virus replication. Counting plaque numbers and quantifying virus infection of cells in multiwell plates quickly, accurately and automatically remain a challenge. Visual inspection relies upon experience, is subjective, often time consuming, and has less reproducibility than automated methods. In this paper, a simple, high throughput imaging-based alternative is proposed which uses a flatbed scanner and image processing software to quantify the infected cell population and plaque formation. Quantitation results were evaluated with reference to visual counting and achieved better than 80% agreement. The method was shown to be particularly advantageous in titration of the number of plaques and infected cells when influenza viruses produce a heterogeneous population of small plaques. It was also shown to be insensitive to the densities of plaques in determination of neutralization titres and IC50s of drug susceptibility. In comparison to other available techniques, this approach is cost-effective, relatively accurate, and readily available.

► A high-throughput imaging method was proposed to quantify virus infected cells and plaques. ► A flatbed scanner was used to scan multi-well plates. Homemade software was used for quantitation. ► Good agreement was demonstrated with visual estimation of plaque counts and plaque size. ► It was used to quantify virus titration and titres in virus neutralization and drug susceptibility. ► The method is flexible, simple and cost-effective, and can be easily setup in many laboratories.