JoVE Encyclopedia of Experiments
Microbiology
0 views • 2:54 min • August 31st, 2026
Take a multi-well plate containing a culture of adherent mammalian cells.
Inoculate the wells with serial dilutions of a cytopathic virus and incubate.
The virus enters the host cells, replicates, and produces progeny virions.
A high viral load induces visible cytopathic effects, or CPE, including morphological changes and cell detachment.
Remove the medium and wash the wells twice with a buffer to remove detached cells.
Incubate with a fixative to preserve cellular morphology.
Remove the fixative and wash the wells twice with a buffer to eliminate residual fixative.
Incubate with crystal violet, a membrane-permeable, cationic dye that binds to anionic proteins and DNA in the fixed cells.
Remove excess dye.
Unstained wells at a high virus concentration show virus-induced CPE, while stained wells at lower virus concentrations show viable cells.
Count the stained and unstained wells across dilutions, then use these counts in a mathematical method to estimate the viral infectious titer.
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.