Chemical staining is a foundational technique in biology, used to make microscopic structures visible that would otherwise be transparent under a light microscope. Dyes are applied to cells or tissues to selectively highlight components for observation. Simply applying a stain is often not enough to ensure the color remains fixed during subsequent washing steps. In the specific context of the Gram stain, a procedure used to classify bacteria, iodine acts as a chemical assistant, universally referred to as a mordant, to secure the primary stain within the bacterial structure.
Defining the Mordant
A mordant is a chemical agent used to set or stabilize a dye onto a substrate, such as a microbial cell or a tissue sample. This chemical fixation is necessary because many biological dyes do not form a lasting bond with cell structures and would easily wash away. The term is derived from the Latin word mordere, meaning “to bite,” reflecting how the chemical helps the dye adhere tightly.
The mechanism involves the mordant binding to the dye molecule, forming a larger, more stable compound known as a coordination complex or a dye lake. This complex has a greater affinity for the biological structure than the original dye alone. The increased molecular size and altered chemical properties of this complex make it less soluble and more difficult to remove during later steps, particularly during washing or decolorization.
Many classic mordants are metal ions, such as those derived from aluminum, iron, or chromium salts. While iodine in the Gram stain is sometimes classified as a trapping agent, its practical function is to create a large, insoluble complex that anchors the dye. This action fulfills the precise role of a mordant in this microbiological context.
Iodine’s Function in Staining
In the Gram staining procedure, iodine is applied immediately after the primary dye, crystal violet. Crystal violet is a positively charged dye that initially penetrates the cell walls of all bacteria, staining them purple. Gram’s iodine solution, which contains potassium iodide and iodine, then enters the cell.
The iodine molecules, often as triiodide ions, interact chemically with the crystal violet cation. This reaction forms a much larger, insoluble macromolecular structure known as the Crystal Violet-Iodine complex (CV-I complex). This purple CV-I complex is significantly bulkier than the original crystal violet molecule.
This increase in size is the purpose of the mordant step; the large CV-I complex becomes physically trapped within the mesh-like structure of the bacterial cell wall. This interaction effectively fixes the purple color inside the cells. If the iodine step were skipped, the smaller crystal violet molecules would be easily washed out, compromising the differential nature of the stain.
Context: The Gram Staining Procedure
The addition of iodine is a non-negotiable step in the four-step Gram staining procedure, a widely used diagnostic tool in microbiology. The process begins with the primary stain, crystal violet, which colors all bacterial cells purple. Next, Gram’s iodine is added, forming the large, insoluble CV-I complex inside every stained cell.
The third step is decolorization, typically using an alcohol or acetone solution. This reagent acts differently on the two main bacterial groups, which possess distinct cell wall structures. Gram-negative bacteria have a thin layer of peptidoglycan surrounded by an outer membrane that the alcohol dissolves. This dissolution allows the large CV-I complex to be rapidly washed out, leaving the Gram-negative cells colorless.
Gram-positive bacteria have a thick, multi-layered peptidoglycan cell wall but no outer membrane. The alcohol causes this thick layer to dehydrate and shrink, which effectively closes the pores in the cell wall structure. This dehydration seals the CV-I complex inside the cell, preventing its escape. Finally, a counterstain, like safranin, is applied to color the decolorized Gram-negative cells pink, while Gram-positive cells retain the purple color.