Trypan blue is a diazo dye that has been widely used to color dead tissues or cells

selectively. The mechanism of trypan blue staining is based on it being negatively charged
and not interacting with cells unless the membrane is damaged. Indeed, undamaged cells are
very selective concerning the compounds that pass through their membrane, and thus should
not take up trypan blue. Therefore, all the cells that exclude the dye are considered viable. By
contrast, cells with damaged membranes are stained in a distinctive blue color readily
observed under a microscope. Thus, trypan blue dye is described as being a vital stain
allowing discrimination between viable cells and cells with damaged membranes that are
usually considered to be dead cells.
Several bacterial pore-forming toxins can cause host cell damage by perforating the host cell
membranes. It is conceivable that the pores formed by the toxin may allow trypan blue to
enter the host cell, which would consequently be considered to be dead. Indeed, the trypan
blue is a relatively small molecule (960 Da) and may therefore be able to penetrate into cells
through pores formed by toxins. However, the issue of whether pore-forming toxins may
cause membrane leakage without inducing immediate cell death has not been extensively
considered. In this case, trypan blue staining would reveal pore formation in the host cell
membrane but not necessarily host cell death.

MTS assay is a novel method for MTT assay as it uses a new type of tetrazolium dye, which
ultimately results in an aqueous, soluble formazan product. Here, the tetrazolium dye used in
the MTS assay is 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium. The formation of the soluble formazan dye occurs in the
presence of phenazine methosulfate (PMS), which serves as an intermediate electron
acceptor, transferring electrons from NADH to reduce the tetrazolium dye, forming the
soluble formazan product.
The direct formation of a soluble formazan product reduces the formazan product
solubilization step of the MTT assay. Therefore, MTS assay is more efficient and less time-
consuming. Also, it removes potential errors such as cell loss, which can happen during
removal of the medium and solubilizing the formazan product. Additionally, the resultant
formazan product is darker in color; this increases the sensitivity and the accuracy of the
assay.
The main difference between MTT and MTS assay is that MTT assay has an additional step
associated with the solubilization of formazan crystals whereas MTS assay is not associated
with the solubilization of formazan crystals.
MTT assay is a classical assay for measuring the cell viability to assess the effect of test
molecules on cell proliferation and cytotoxicity. However, the resultant formazan product in
the MTT assay is insoluble; hence, this assay requires an additional step to solubilize the
formazan product. In contrast, MTS assay is a novel method for measuring cell viability and
it uses a new kind of tetrazolium salt, which produces a soluble formazan, reducing one step
in the MTT assay procedure. Therefore, MTS assay is more efficient when compared to the
MTT assay. Hence, the main difference between MTT and MTS assay is the characteristics
of the formazan product.
MTT and MTS assay are two types of assays used to measure cell viability in vitro. They
help to reveal the effect of the test molecules on cell proliferation and cytotoxicity, affecting
the cell viability. Furthermore, MTT assay is more time consuming than MTS assay.
MTT assay is a classical assay for measuring the cell viability to assess the effect of test
molecules on cell proliferation and cytotoxicity. However, the resultant formazan product in
the MTT assay is insoluble; hence, this assay requires an additional step to solubilize the
formazan product. In contrast, MTS assay is a novel method for measuring cell viability and
it uses a new kind of tetrazolium salt, which produces a soluble formazan, reducing one step
in the MTT assay procedure. Therefore, MTS assay is more efficient when compared to the
MTT assay. Hence, the main difference between MTT and MTS assay is the characteristics
of the formazan product.