Gram staining is a foundational microbiological technique developed by the Danish bacteriologist Hans Christian Gram in 1884. This differential staining method rapidly classifies bacteria into two primary categories. Its main purpose is to quickly distinguish between bacteria based on inherent differences in their cellular architecture. The procedure remains one of the first steps performed on a clinical sample to characterize an unknown microorganism.
How Bacterial Cell Walls Determine Staining
The outcome of the Gram stain procedure is entirely dependent on the structural composition of the bacterial cell wall. Bacteria are broadly separated into two groups based on the thickness of a layer known as peptidoglycan. This polymer provides structural integrity and defines the staining reaction that occurs when chemicals are applied.
Gram-positive bacteria possess a thick, multi-layered cell wall composed primarily of peptidoglycan, which can constitute up to 90% of the cell envelope. This thick layer sits directly outside the cell membrane, and there is no secondary outer membrane present. The density of this mesh-like structure is what enables these organisms to retain the primary stain throughout the procedure.
In contrast, Gram-negative bacteria have a thinner layer of peptidoglycan, often consisting of only a few layers sandwiched between two membranes. The outer membrane is a distinct feature of Gram-negative cells, composed of phospholipids and lipopolysaccharides (LPS). This lipid-rich membrane plays a defining role in the decolorization step, which dictates the final color of the cell.
The Four Stages of the Staining Procedure
The Gram staining procedure involves a precise sequence of four stages, each utilizing a specific reagent to achieve the final differential color. The process begins with the application of the primary stain, crystal violet, a purple dye. When applied to the heat-fixed bacterial smear, the crystal violet permeates the cell walls and membranes, staining all bacterial cells a deep purple color.
Following the primary stain, a solution of Gram’s iodine is added to the slide, acting as a mordant. The iodine forms a large, insoluble complex with the crystal violet dye inside the cell, known as the Crystal Violet-Iodine (CV-I) complex. This complex is a larger molecule than the original dye, which helps to fix the stain within the cellular structure.
The third step involves the application of a decolorizer, usually a mixture of alcohol and acetone. In Gram-positive cells, the alcohol causes the thick, dehydrated peptidoglycan layer to shrink, effectively trapping the large CV-I complexes inside the cell. Because the dye complex cannot escape the tightened mesh, the Gram-positive cells remain purple.
Conversely, the alcohol decolorizer interacts differently with the Gram-negative cell wall structure. The solvent quickly dissolves the lipid-rich outer membrane of the Gram-negative cells. Once the outer barrier is compromised, the thin underlying peptidoglycan layer is unable to retain the CV-I complex, allowing the purple dye to be washed away, leaving the Gram-negative cells colorless.
The procedure concludes with the addition of the counterstain, safranin, a contrasting red or pink dye. Since the Gram-positive cells are already intensely purple, the lighter pink safranin is not apparent against the darker primary stain. However, the previously decolorized Gram-negative cells readily absorb the safranin, causing them to take on a visible pink or red color.
Practical Applications and Interpreting Outcomes
The final interpretation of a Gram stain involves observing two characteristics under a high-power microscope. The first characteristic is the color, which classifies the organism as either Gram-positive or Gram-negative. Cells that appear deep purple or blue are Gram-positive, while those that show a pink or red hue are Gram-negative organisms.
Simultaneously, the observer determines the shape, or morphology, of the bacteria present in the sample. Bacteria are classified as cocci (spherical), bacilli (rod-shaped), or spirilla (spiral). This shape information is combined with the color result, allowing the report to specify “Gram-positive cocci” or “Gram-negative bacilli.”
The ability of this technique to provide rapid classification makes it a valuable diagnostic tool in clinical settings. When a patient presents with a suspected bacterial infection, a Gram stain can be performed on a sample within minutes, such as blood, urine, or cerebrospinal fluid. This quick result offers healthcare providers an immediate, preliminary identification of the general type of pathogen.
This information is used to guide the initial selection of empirical antibiotic therapy before culture and sensitivity results become available, which can take days. Because Gram-positive and Gram-negative bacteria differ in cell wall chemistry, they often respond differently to various classes of antibiotics. Knowing the Gram status of the organism allows for a more targeted treatment plan to be initiated quickly, improving patient outcomes.