The Gram stain is a foundational technique in microbiology, developed by Hans Christian Gram in 1884. This differential staining procedure is one of the first and fastest tests performed for initial bacterial identification, dividing nearly all bacteria into two main categories: Gram-positive and Gram-negative. Its significance lies in quickly narrowing down potential infection causes in clinical settings, guiding the physician toward appropriate preliminary treatment options before slower culture results are available.
The Scientific Basis of Differential Staining
The ability of the Gram stain to separate bacteria into two groups is rooted in the structural differences of their cell walls. Gram-positive bacteria possess a thick, multilayered cell wall composed primarily of peptidoglycan (60% to 90% of the structure). Gram-negative bacteria, by contrast, have a much thinner layer of peptidoglycan, which is surrounded by an outer membrane made of lipids and lipopolysaccharide.
The staining process begins with the primary stain, crystal violet, which is taken up by the cell walls of both types of bacteria. Next, a mordant, Gram’s iodine solution, is added, forming a crystal violet-iodine complex (CV-I) inside the cell walls. The subsequent step is the application of a decolorizer, typically an alcohol or acetone mixture, which attempts to wash the CV-I complex out of the cells.
In Gram-positive cells, the alcohol dehydrates the thick peptidoglycan layer, causing it to shrink and effectively trapping the CV-I complex inside. Conversely, in Gram-negative cells, the solvent dissolves the outer lipid membrane and passes through the thin peptidoglycan layer, allowing the CV-I complex to easily wash out. This leaves the Gram-positive cells stained purple, while the Gram-negative cells become colorless before the final step.
Detailed Steps for Performing the Gram Stain
The Gram stain procedure begins with preparing a bacterial smear on a clean glass slide, which must be thin enough for clear observation. A small amount of culture is mixed with water, spread thinly, and allowed to air dry completely. The smear is then “heat-fixed” by quickly passing the slide through a gentle flame; this kills the bacteria and adheres them firmly to the glass so they do not wash off during subsequent steps.
The first reagent, crystal violet, is flooded over the fixed smear for approximately 30 to 60 seconds to stain all cells purple. The excess stain is then gently rinsed off with water, taking care not to dislodge the fixed smear. Following this, the mordant, Gram’s iodine solution, is applied for another 30 to 60 seconds, which facilitates the formation of the crystal violet-iodine complexes within the cell walls.
The most time-sensitive step involves applying the decolorizer, usually an ethanol-acetone mixture. The slide is held at an angle and the decolorizer runs over the smear for only 5 to 10 seconds, or until the runoff appears clear. Immediately rinsing the slide with water stops the decolorization process, preventing the stain from being washed out of the Gram-positive cells.
Finally, a counterstain, typically safranin (a light pink or red dye), is applied for 30 to 60 seconds. Gram-positive cells remain purple and do not take up this new color. However, the decolorized Gram-negative cells absorb the safranin, turning them pink. After a final gentle rinse with water and air-drying, the slide is ready for microscopic examination.
Analyzing and Reporting the Results
To analyze the finished slide, the specimen is observed under a microscope using the oil immersion objective. The differential staining yields two distinct colors for immediate classification: Gram-positive bacteria appear deep violet or blue, while Gram-negative bacteria appear pink or red. This color distinction is paired with an observation of cellular morphology, which refers to the bacteria’s shape and arrangement.
The two most common shapes are cocci (spherical or round) and bacilli (rod-shaped). The arrangement of the cells is also noted, such as cocci appearing in clusters or bacilli arranged in chains. A complete report combines these observations, describing the organisms as, for example, “Gram-positive cocci in clusters” or “Gram-negative bacilli.”
Accurate interpretation requires avoiding common technical artifacts that can lead to misidentification, often related to the decolorization step. Over-decolorization (solvent left on too long) strips the crystal violet from Gram-positive cells, causing them to incorrectly appear pink. Conversely, under-decolorization prevents the dye from washing out of Gram-negative cells, causing them to incorrectly appear purple. Another common error occurs when the bacterial culture is too old, leading to an unreliable, “Gram-variable” result.