View the PDF The Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition.

In microbiology and molecular biology, it is used for staining bacteria, histological slide staining, DNA staining, etc. It also shows antibacterial and antifungal properties, hence used in sterilization and disinfection. In Gram Staining, it is used as a basic dye in the ionized form of CV+ and Cl-.

This staining technique uses two stains; crystal violet as primary stain and safranine as a counterstain. Those bacteria with Gram-positive cell walls will retain primary stain and appear violet or purple. These bacteria are termed Gram-Positive bacteria.

It is routinely used in clinical settings to help identify bacteria causing an infection. Although widely used, Gram staining has several limitations, such as the time required for manual staining and slide analysis, variability due to operator technique and safety risks from the chemicals involved.