Gram staining represents a foundational and widely used technique in microbiology laboratories worldwide. This differential staining method is designed to quickly classify bacteria into two large groups based on their cellular characteristics. The technique provides an immediate, visual distinction that informs initial identification and guides subsequent research or clinical decisions. Danish bacteriologist Hans Christian Gram first introduced this method in 1884. It remains a routine first step in the investigation of a sample containing microorganisms.
The Structural Basis for Classification
The success of the Gram stain relies upon fundamental differences in bacterial cell wall architecture. All bacteria possess a cell wall layer composed of a polymer called peptidoglycan, but the thickness and surrounding structures vary significantly between the two major groups.
Gram-positive bacteria are characterized by a thick, multilayered peptidoglycan structure, accounting for up to 90% of the cell envelope’s dry weight. This dense mesh acts like a solid barrier, retaining the primary stain during the procedure.
Gram-negative bacteria possess a much thinner peptidoglycan layer, often comprising only about 10% of the cell wall. This thin layer is sandwiched between the inner cytoplasmic membrane and a distinct outer lipid membrane. The presence of this outer membrane, which contains lipopolysaccharides, makes the Gram-negative cell wall more complex. This difference in cell wall composition dictates how each type of bacterium reacts to the staining reagents.
The Procedure: Four Critical Steps
The Gram staining process involves a precisely timed sequence of four reagent applications to achieve the differential result. The first step is applying the primary stain, crystal violet, a purple dye that permeates the cell walls of all bacteria. Crystal violet molecules enter and bind to components within the peptidoglycan layer, staining both Gram-positive and Gram-negative cells deep purple. This initial staining ensures all bacterial cells are visibly marked.
Next, a mordant, typically Gram’s iodine solution, is applied. The iodine complexes with the crystal violet molecules inside the cell, forming the crystal violet-iodine (CV-I) complex. This complex is physically larger than the original crystal violet molecule. This complex formation strengthens the bond between the dye and the cellular material in preparation for decolorization.
The third and most time-sensitive step is the application of a decolorizing agent, usually ethanol or acetone. When the decolorizer hits the Gram-negative cells, the outer lipid membrane is dissolved, creating pores and allowing the CV-I complex to escape through the thin peptidoglycan layer. Conversely, the solvent acts on the thick peptidoglycan wall of Gram-positive cells by dehydrating it, causing the mesh structure to constrict and tighten. This constriction effectively traps the large CV-I complexes within the Gram-positive cell, preventing the purple color from washing out.
Since Gram-negative cells are now colorless, a counterstain is needed to make them visible. The final step involves flooding the smear with safranin, a contrasting red or pink dye. Safranin easily penetrates the decolorized Gram-negative cells, staining them pink or red. Although safranin is absorbed by Gram-positive cells, its lighter pink color is masked by the intense, trapped purple primary stain.
Distinguishing Gram-Positive from Gram-Negative
The final examination of the stained sample under a microscope yields a clear, dichotomous result for immediate classification. Bacteria that successfully retain the primary crystal violet-iodine complex appear purple or dark blue and are identified as Gram-positive. These cells have thick, dehydrated peptidoglycan walls that successfully resisted the washing action of the decolorizer.
Conversely, bacteria that appear pink or red have been stained by the safranin counterstain and are classified as Gram-negative. These cells failed to retain the purple stain due to their thin peptidoglycan layer and the dissolution of their outer lipid membrane during the alcohol wash. This rapid, color-based classification provides clinicians with initial information long before full bacterial culture results are available.
The distinction between purple and pink is important in a clinical context because the difference in cell wall structure directly influences the susceptibility of the bacteria to certain antibiotics. For example, some antibiotics primarily target the thick peptidoglycan layer, making them more effective against Gram-positive organisms. Using the Gram stain result, a healthcare provider can select a targeted, initial antibiotic therapy while awaiting definitive culture and sensitivity results.