The Gram stain is a fundamental diagnostic procedure used in microbiology for the classification of bacteria. This technique serves as a powerful means of differentiating nearly all bacterial species into one of two major groups. By providing preliminary information about the microbe present in a sample, the stain guides healthcare professionals toward more focused diagnostic testing and appropriate early treatment choices.
The Staining Procedure
The Gram stain technique involves a sequence of four chemical applications to a heat-fixed smear of the bacterial sample. The first reagent applied is Crystal Violet, which acts as the primary stain, coloring all bacterial cells deep purple. Following this initial staining, a solution of Gram’s Iodine is added, functioning as a mordant to enhance the primary stain’s adherence to the cellular material.
Next, a decolorizing agent, typically a mixture of alcohol and acetone, is briefly washed over the slide. This is the most time-sensitive step, as it selectively removes the Crystal Violet from some cells but not others. The final step involves applying Safranin, a contrasting red dye, which serves as the counterstain. This ensures that any cells that lost the purple color during decolorization are stained a visible pink or red color.
Structural Basis for Differentiation
The ability of the Gram stain to differentiate between bacteria relies on the inherent chemical and physical properties of their cell walls. Bacteria are categorized as Gram-positive or Gram-negative based on their distinct cell envelope architecture. The primary structural component responsible for this differentiation is peptidoglycan, a lattice-like polymer layer that provides structural integrity to the cell.
Gram-positive bacteria possess a remarkably thick, multi-layered peptidoglycan sheath that can account for up to 90% of the cell wall material. This dense, highly cross-linked structure lacks an outer lipopolysaccharide membrane, featuring only the plasma membrane beneath the peptidoglycan. When the mordant (iodine) is added to the Crystal Violet, they form a large, insoluble complex within this peptidoglycan matrix.
The subsequent application of the alcohol-acetone decolorizer causes the thick peptidoglycan layer of Gram-positive cells to dehydrate and constrict. This shrinking effectively seals the pore structure of the cell wall, physically trapping the large Crystal Violet-iodine complex inside the cell. Consequently, Gram-positive cells retain the initial dark purple stain despite the decolorization step.
In contrast, Gram-negative bacteria have a complex, two-membrane structure with a significantly thinner peptidoglycan layer. This layer is only a small fraction of the cell wall’s total composition, often less than 10%, and is situated between an inner plasma membrane and an outer lipopolysaccharide (LPS) membrane. The decolorizer rapidly dissolves the high-lipid content of the Gram-negative outer membrane.
With the outer barrier compromised, the thin, leaky peptidoglycan layer cannot retain the large Crystal Violet-iodine complex. The complex is quickly washed out of the cell, leaving the Gram-negative bacterium colorless.
Interpreting Results: Color and Grouping
Results are interpreted under a light microscope based on both the color and the arrangement of the cells. Gram-positive organisms appear deep purple or blue-violet, a result of retaining the Crystal Violet-iodine complex within their thick cell walls. Gram-negative organisms, having lost the primary stain, are visualized as pink or red because they absorb the Safranin counterstain.
Beyond the color classification, the way bacteria organize themselves provides important information. Cocci (spherical cells) often exhibit distinct groupings that reflect their division pattern. Bacilli (rod-shaped bacteria) typically arrange themselves as single cells, pairs, or chains. Observing these arrangements in conjunction with the Gram reaction provides a more complete preliminary identification.
Common Arrangements
- Streptococci (chains formed by division in a single plane)
- Staphylococci (grape-like clusters formed by division in multiple planes)
- Diplococci (cells remaining attached in pairs after division)
- Tetrads (square clusters of four cells)
- Diplobacilli (rod-shaped cells in pairs)
- Streptobacilli (rod-shaped cells in chains)
Identifying Bacterial Morphology
The specific morphology, or shape, of the bacterial cells is the second major piece of information gained from a Gram stain. The overall shape, combined with the Gram reaction, significantly narrows the possibilities for identification. The three primary morphologies are cocci, bacilli, and spirilla.
Cocci are spherical or round cells, although they can sometimes appear slightly oval or bean-shaped. A common clinical example of a Gram-positive coccus is Staphylococcus aureus, which is often seen in clusters. Bacilli are cylindrical or rod-shaped organisms, which can vary in length and diameter; an example of a Gram-negative bacillus is Escherichia coli.
The third morphology, spirilla, encompasses all curved or spiral-shaped bacteria. These can range from a gently curved rod, sometimes called a vibrio, to a rigid, corkscrew-like form. A clinically relevant example of a spiral-shaped bacterium is Helicobacter pylori, a Gram-negative organism associated with stomach ulcers.