Gram staining is a foundational method in microbiology, developed by Hans Christian Gram in 1884. The technique remains widely used today as a rapid and cost-effective way to classify bacteria into two broad groups based on their cell wall characteristics. Performing a Gram stain is typically the first step in the laboratory identification process, providing a quick initial division of the bacteria present. This classification system immediately aids in directing further analysis and treatment decisions.
Gram Positive Versus Gram Negative
The most immediate result of a Gram stain is the color of the bacteria when viewed under a microscope, which allows for their classification as either Gram-positive or Gram-negative. Gram-positive bacteria retain the initial stain and appear deep purple or blue. Gram-negative bacteria, however, do not retain the primary stain and instead take on the color of a second stain, appearing pink or red.
This color distinction provides a scientist or clinician with immediate information about the organism. Knowing whether an infection is caused by a Gram-positive or Gram-negative bacterium instantly narrows the list of potential pathogens, guiding initial clinical decision-making.
Cell Wall Structure and Staining
The difference in staining results stems entirely from the distinct architecture of the bacterial cell walls. Gram-positive bacteria possess a thick, mesh-like layer of peptidoglycan, which is a polymer of sugar and amino acids, forming up to 90% of the cell envelope. This substantial layer acts like a sponge, trapping the stain complex within the cell.
Gram-negative bacteria, by contrast, have a much thinner layer of peptidoglycan, making up only about 10% of their cell wall. This thin layer is sandwiched between the inner cell membrane and an outer membrane that contains lipids and lipopolysaccharides. When a decolorizing agent is applied, the high lipid content of the outer membrane is dissolved, allowing the stain complex to easily wash away from the thin peptidoglycan layer. The thick peptidoglycan wall of Gram-positive cells, being dehydrated and shrinking, physically constricts to prevent the stain from escaping.
The Four Step Staining Process
The Gram staining procedure involves a precise sequence of four steps, utilizing chemical reagents to achieve differential coloring. The first step is applying the primary stain, crystal violet, which enters both Gram-positive and Gram-negative cells, coloring all bacteria purple. Next, Gram’s iodine, which functions as a mordant, is added.
The iodine interacts with the crystal violet to form a large, insoluble crystal violet-iodine complex (CV-I) within the cell walls of the bacteria. The third and most delicate step is decolorization, typically using an alcohol or acetone wash. This wash removes the stain from Gram-negative cells but leaves the CV-I complex trapped within the Gram-positive cell wall.
Finally, a counterstain, safranin, is applied. Since the Gram-positive cells are already stained purple, they are unaffected by this red dye. The now-colorless Gram-negative cells absorb the safranin, causing them to appear pink or red. This final step ensures the Gram-negative bacteria are visualized during microscopic examination.
Clinical Significance
The ability of the Gram stain to classify bacteria quickly makes it a rapid diagnostic tool in clinical settings. Results can be available in minutes, long before more detailed culture results are ready, which can take days. This speed is important in diagnosing serious infections like sepsis, pneumonia, or urinary tract infections.
The primary role of the Gram stain is guiding the immediate selection of initial, broad-spectrum antibiotics, often referred to as empiric therapy. Since certain antibiotics are more effective against Gram-positive bacteria while others target Gram-negative organisms, the stain result allows clinicians to tailor the initial treatment choice. For instance, if Gram-positive cocci are identified in a blood sample, the clinician can immediately select an effective antibiotic regimen. This rapid, targeted approach ensures the patient receives appropriate treatment without delay and helps reduce the unnecessary use of powerful, broad-spectrum antibiotics, which is a core strategy in managing antibiotic resistance.