The Gram stain is a foundational technique in microbiology, providing a rapid method for the preliminary identification and classification of bacteria. Developed in 1884 by the Danish physician Hans Christian Gram, this differential staining procedure remains one of the most widely used tests in clinical and research settings. Its primary purpose is to quickly categorize bacterial species into two distinct groups based on the structural properties of their cell walls. The staining process gives scientists immediate, valuable information about an unknown microorganism, setting the stage for more specific testing and diagnosis.
The Four Steps of the Gram Stain Procedure
The process relies on the sequential application of four specific reagents to a bacterial sample that has been fixed onto a glass slide. The first step involves flooding the slide with the primary stain, crystal violet, which is a purple dye that permeates and colors all bacterial cells present. This ensures both Gram-positive and Gram-negative cells absorb the purple color.
Following the application of the primary stain, Gram’s iodine solution is added, acting as a mordant. The iodine interacts chemically with the crystal violet inside the bacterial cell to form a large, insoluble crystal violet-iodine (CV-I) complex. This complex is larger than the original crystal violet molecule, and its formation is necessary for the stain to be retained.
The third and most crucial stage is the application of a decolorizer, typically a solution of ethanol or acetone. This solvent treatment attempts to wash the CV-I complex out of the cells. This step determines the final staining result by selectively removing the purple complex from one group of bacteria.
Finally, the slide is treated with a counterstain, such as safranin, which is a red or pink dye. This last step stains any cells that lost the primary crystal violet color during the decolorization stage. Cells that retained the crystal violet remain purple, while those that were decolorized absorb the safranin and appear pink or red.
The Cell Wall Difference
The differing reactions to the decolorizer are attributed to fundamental differences in the composition and architecture of the bacterial cell wall. All bacteria possess a cell wall containing peptidoglycan, a polymer that provides structural strength. The amount and placement of this layer vary significantly between the two groups.
Gram-positive bacteria have a substantially thick, multi-layered peptidoglycan sheath, up to 80 nanometers thick. This layer is exposed to the exterior and contains teichoic acids. When the alcohol-based decolorizer is applied, it dehydrates the thick peptidoglycan layer, causing it to shrink and tighten.
This dehydration effectively closes the pores within the mesh, trapping the large crystal violet-iodine complexes inside the cell. Because the complex cannot escape, Gram-positive bacteria remain stained purple throughout the procedure, resisting decolorization.
Gram-negative bacteria have a much thinner peptidoglycan layer, typically only 2 to 7 nanometers thick. This thin layer is sandwiched between an inner cytoplasmic membrane and a unique outer membrane composed of lipopolysaccharides (LPS). The presence of this outer lipid bilayer is the defining structural difference.
When the decolorizer is added, the solvent rapidly dissolves the lipid components of this outer membrane, creating holes in the cell’s protective layer. The thin peptidoglycan layer cannot shrink enough to retain the large CV-I complexes, which quickly leach out. This loss of the purple stain leaves the Gram-negative cells colorless and ready to accept the counterstain.
Interpreting Gram Results
The final color observed under the microscope dictates the classification and provides immediate clues about the organism’s identity. Bacteria that retain the primary stain and appear purple are identified as Gram-positive. These bacteria are generally susceptible to antibiotics like penicillin, which targets the synthesis and cross-linking of the thick peptidoglycan layer.
Bacteria that lose the purple stain and pick up the safranin counterstain appear pink or red, classifying them as Gram-negative. The presence of the outer membrane often confers resistance to certain antibiotics because the membrane acts as a barrier, preventing drugs from reaching the peptidoglycan target. This structural difference guides the initial selection of appropriate antimicrobial therapy.
The Gram stain provides preliminary information rapidly, which is important when treating severe infections. While a full bacterial culture can take days, the Gram stain offers a result within minutes, allowing doctors to begin an initial, targeted treatment while awaiting definitive identification. Knowing the Gram status helps narrow down the possibilities.
Understanding Cellular Shapes
Beyond the color reaction, the Gram stain allows for the observation of the bacteria’s physical form, addressing cellular morphology and arrangement. The shape of an individual bacterial cell is determined by its rigid cell wall and is a stable trait used for classification.
The three fundamental bacterial shapes are the sphere, the rod, and the spiral. Spherical or oval-shaped bacteria are referred to as cocci, while rod-shaped bacteria are called bacilli. Bacteria exhibiting a curved or twisted morphology are categorized as spirilla, ranging from gently curved forms to rigid spirals or flexible spirochetes.
The way these cells organize themselves after division, known as their arrangement, also provides descriptive information. Cocci, for example, may be observed in pairs (diplococci), in chains (streptococci), or in grape-like clusters (staphylococci). Combining the Gram reaction with the morphology and arrangement offers a detailed preliminary description, such as “Gram-positive cocci in clusters” or “Gram-negative bacilli.”