Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

Summary

This protocol shows an accurate and objective approach to visualize viral titrations using crystal violet, by comparing it with optical microscopy and immunocytochemical staining.

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 research¹. 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 microscopy⁵. To confirm CPE visualization more objectively on TCID50 assays, immunocytochemical (ICC) intracellular staining targeting the proteins of the virus is one of t....

Protocol

1. Titration protocol

NOTE: Use a cytopathic virus infecting adherent cells. For this demonstration, Influenza A Virus (IAV) of swine origin (A/California/07/2009/(H1N1)) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 2, strain NC 1-7-4 were used.

1. Titrate these viruses in 96 well plates for 7 days in a biosafety cabinet located in a Biosafety Level 2 (BSL-2) laboratory.
   1. To perform these titrations, seed 96 well plates with the requ.......

Discussion

Viral titrations are routinely used in virology research, with PFU detection and TCID50 assays most commonly used^1,2,3,4. Both methods rely on the detection of CPE in infected cells, and even though they can be visually assessed via microscopy, a staining is usually applied to obtain a more objective result or to even reduce incubation times. In the case of TCID50, one of the most comm.......

Materials

Name	Company	Catalog Number	Comments
96-well cell culture plates	Genesee	25-221	Clear, flat bottom
AEC solution	Thermo Fisher	1122
Crystal violet	Thermo Fisher	C581-25; C581-100
DMEM	Corning	10-017-CV
Fetal bovine serum	BioWest	S1480
Paraformaldehide	Thermo Fisher	J19943
Primary Influenza Antibody	Bioss	BS-0344R
Primary PRRSV Antibody	Bioss	BS-10043R
Saponin	Thermo Scientific	AAA1882014
Secondaty antibody	Invitrogen	31460
Tris Hydrochloride	Thermo Scientific	AM9856