The Gram staining procedure is a foundational technique in microbiology, enabling the classification of bacterial species based on the composition of their cell walls. This differentiation is achieved through a precise sequence of chemical treatments, where each reagent performs a specialized function to color or decolorize the cells. Among these steps, the application of a mordant is a decisive action that chemically prepares the primary stain for retention within the bacterial structure. The mordant does not stain the cell itself but rather facilitates the interaction necessary for the subsequent steps to distinguish between different types of bacteria.
Defining the Mordant in Staining
A mordant is a substance that acts as a fixative, significantly enhancing the binding of a dye or stain to a biological substrate. The term itself is derived from the Latin word mordere, which means “to bite,” historically referring to how the substance helped the dye “bite” onto the fiber. In the context of Gram staining, the mordant is Gram’s Iodine, an aqueous solution containing iodine and potassium iodide.
The application of the primary stain, Crystal Violet, is a relatively simple process where the positively charged dye penetrates the cell wall of all bacteria. However, without the use of a fixative, the initial stain would be easily washed away during the later rinsing and decolorization stages. The role of the mordant is to address this solubility issue by acting as a molecular anchor.
By introducing Gram’s Iodine, the procedure ensures that the initial purple coloration is secured within the cellular components. The mordant chemically prepares the cell for the ultimate differential wash that defines the Gram stain’s utility.
The Chemical Mechanism of Dye Fixation
The chemical action of the mordant begins immediately after the primary stain, Crystal Violet (CV), has saturated the cell. Crystal Violet is a basic dye that dissociates in solution into a positively charged chromogen (CV+), which is drawn to the negatively charged components within the bacterial cell. This small, cationic dye molecule readily enters both types of bacterial cells.
When Gram’s Iodine is applied, the iodine ions (I- or I3-) penetrate the cell and interact directly with the CV+ ions. This interaction initiates a chemical reaction known as metathetical anion exchange, where the small chloride anion from the Crystal Violet is replaced by the bulkier iodide. The result is the formation of a much larger, insoluble molecular complex known as the Crystal Violet-Iodine (CV-I) complex.
The formation of the CV-I complex is the specific mechanism of dye fixation. The newly created complex is significantly larger than the original Crystal Violet molecule and is no longer water-soluble. At this stage, both Gram-positive and Gram-negative bacteria contain this large, trapped purple complex and appear identically stained. This fixation step is a prerequisite for the final differentiation between the two bacterial types.
How the Mordant Creates Differential Staining
The creation of the large, insoluble CV-I complex by the mordant sets the stage for the most distinguishing step in the procedure: decolorization. The ability of the two main bacterial groups to retain this large complex when exposed to an organic solvent, typically an alcohol or acetone mixture, is what allows for differential staining. This retention is directly linked to the structural differences in their cell walls.
Gram-positive bacteria possess a thick, multi-layered cell wall composed primarily of peptidoglycan. When the alcohol-based decolorizer is introduced, it acts as a dehydrating agent, causing the thick peptidoglycan layer to shrink and tighten. This dehydration effectively constricts the pores within the mesh-like structure, trapping the large, insoluble CV-I complex inside the cell. The result is that Gram-positive bacteria resist decolorization and retain the initial purple stain.
In stark contrast, Gram-negative bacteria have a much thinner layer of peptidoglycan, which is covered by an outer lipid membrane. The decolorizer, acting as a lipid solvent, dissolves this outer membrane, creating large gaps in the cell envelope. Because the underlying peptidoglycan layer is so thin, it cannot effectively shrink or tighten enough to retain the large CV-I complexes. This allows the large complex to wash out of the cell easily, leaving the Gram-negative bacteria colorless and ready to accept the counterstain.