What are the limitations of dye exclusion assays?

Dye exclusion assays are one of the most widely used methods for estimating the proportion of viable cells in a cell population. Although the procedure is simple and straightforward, it does have a few limitations as listed below: 

• Dye exclusion assays cannot differentiate between healthy cells and viable cells that have lost or are losing their function. 
• The procedure is labor-intensive and time-consuming for large samples. 
• Counting errors may occur due to several reasons including air bubbles, poor cell dispersion, contamination of reusable counting chambers, inter-user variations, or incorrect dilution. 
• Trypan blue dye is toxic to mammalian cells. Even viable cells are eventually stained with trypan blue as the toxic die permeates their cell membrane, resulting in the death of the cell about 5 to 30 minutes after exposure. 
• The procedure is suitable only for cell suspensions. Monolayers need trypsinization.