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

The goal of this protocol is to produce a virus stock titration and visualize it using crystal violet staining. To do that, a general viral titration will be demonstrated followed by the staining. This titration will be performed in a 96-well plate for a period of seven days in a biosafety cabinet within a biosafety level 2 laboratory.

To do so, a 96-well plate of the preferred cell line will be seeded using the corresponding media and let grow until confluent. The virus stock will be the diluted using tenfold dilutions, by mixing 900 microliters of media and 100 microliters of virus, or the corresponding dilution. Make sure to vortex each tube to ensure proper mixing of the media and the virus and avoid errors on the dilutions.

Write the layout of your plate in the lead as well as each dilution to help during infection process. Before infection, wash using 200 microliters of PBS. Add 50 microliters of your inoculum in the corresponding wells.

Then, incubate at 37 degrees celsius in a 5%CO2 incubator for seven days. After the seven day incubation, wash using 200 microliters of PBS. Fix the cells by adding 50 microliters per well of PFA at 4%in PBS and incubate for 15 minutes at room temperature.

After incubation, wash cells again twice with 200 microliters of PBS prior to the addition of 50 microliters per well of crystal violet in water and incubate for five minutes at room temperature. Finally, aspirate crystal violet from the wells and optionally, leave the plates to air dry for two to five minutes at room temperature or wash it with 200 microliters of water to remove excessive stain before visualization. As a result, after re-staining, negative cells will be stained.

Positive wells will be cleared. This information will be used in one of the two described formulas to calculate your final viral titer. When the titer obtained with each method was compared, it showed that in monocyto chemistry staining could in some cases detect additional positive wells in comparison with crystal violet, and that likewise, both staining methods could detect more positive wells than visual evaluation via microscopy.