Copyright © 2022 JoVE Journal of Visualized Experiments jove.com February 2022 • 180 • e63063 • Page 1 of 8 Use of Crystal Violet to Improve Visual Cytopathic Effect- based Reading for Viral Titration using TCID50 Assays Alba Frias-De-Diego 1 , Elisa Crisci 1 1 College of Veterinary Medicine, Department of Population Health and Pathobiology, North Carolina State University Corresponding Author Elisa Crisci ecrisci@ncsu.edu Citation Frias-De-Diego, A., Crisci, E. Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays. J. Vis. Exp. (180), e63063, doi:10.3791/63063 (2022). Date Published February 12, 2022 DOI 10.3791/63063 URL jove.com/video/63063 Abstract Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines. Introduction Viral titration via TCID50 assay is a commonly used technique in infectious disease research 1 . Although variations on the math behind this method have been proposed over time 1,2,3,4 , the currently applied methods of infection detection rely on visual confirmation through the presence of cytopathic effect (CPE) using microscopy 5 . To confirm CPE visualization more objectively on TCID50 assays, immunocytochemical (ICC) intracellular staining targeting the proteins of the virus is one of the most-commonly used methods 6 as different viruses can produce varying forms of CPE. In our case, the cell morphological changes are similar when infected with both Influenza A virus (IAV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), where infected cells round up and detach from the plate. In the case of PRRSV, it causes a CPE known as "total destruction", where all cells end up detaching from the well. IAV on the other hand, can present both total destruction and an additional CPE known as "subtotal destruction" where a small number of cells do not detach after infection 7 . However, this technique is time consuming to perform and requires