Primary stain
The sole purpose of this reagent is to stain the cells with the selected color. A spot of red pigment stains the surrounding cells red, purple stains the cells purple, and similarly with other pigments depending on the type of cell staining.
Mordant
This reagent function is to increase the efficiency of staining by forming a soluble complex with the primary stain. Thus, the resulting complex makes it possible to enhance the color of the primary stain and thus increase the affinity of bacteria to the pigment.
Decolorizing agent
Typically, it is a highly concentrated aqueous ethyl alcohol solution that allows the removal of excess dye after cell staining. In particular, alcohol increases the cell wall’s porosity, allowing unbound pigment molecules to be removed from the bacterial surface, whereas thicker walls of Gram-positive microorganisms more strongly anchor the color.
Counterstain
Chronologically, this reagent is used last in order to pink stain previously discolored cells. More specifically, it allows a qualitative distinction between the two cell types since the purple Gram-positive bacteria remain unchanged, whereas the clear Gram-negative microorganisms acquire a pink color. Consequently, the comparison is now based on the color difference between the bacteria.