The Gram stain is a foundational diagnostic technique in microbiology that provides a rapid method for classifying bacteria. It sorts the microbial world into two large groups based on differences in cellular architecture. This initial classification serves as a starting point for identifying the cause of an infection, making it a routine procedure in clinical laboratories.
The Staining Result: What Color is Gram Negative?
Gram-negative bacteria appear pink or red under the microscope after the full staining procedure is complete. This distinct coloration results from the bacteria taking up the final dye, known as the counterstain (typically safranin). In contrast, Gram-positive bacteria retain the initial primary stain, causing them to appear purple or blue.
Overview of the Gram Staining Procedure
The Gram stain is a four-step differential staining process that exploits structural differences in bacterial cell walls. First, the primary stain, crystal violet, colors all bacterial cells purple. Next, Gram’s iodine solution is added, acting as a mordant to form a large, insoluble crystal violet-iodine complex within the cell.
The third step is decolorization, where a solvent like alcohol or acetone is rapidly applied to the slide. This solvent washes the purple complex out of Gram-negative cells, leaving them colorless. Finally, the counterstain, safranin (pink or red), is applied and taken up by the colorless Gram-negative cells, while the purple Gram-positive cells remain unchanged.
The Cell Wall Difference: Why Gram Negative Bacteria Stain Pink
Gram-negative bacteria appear pink due to the unique structure of their cell wall, which differs substantially from Gram-positive organisms. They possess a relatively thin layer of peptidoglycan, the rigid mesh-like polymer that forms the cell wall, which is sandwiched between two lipid membranes. Crucially, Gram-negative bacteria have an outer membrane composed largely of lipopolysaccharides (LPS) that covers the thin peptidoglycan layer.
When the decolorizing agent, such as alcohol, is applied, it dissolves the lipids in this outer membrane. The removal of this lipid-rich barrier and the thinness of the underlying peptidoglycan layer allow the large, purple crystal violet-iodine complex to easily wash out of the cell. Once colorless, the cell absorbs the pink counterstain, safranin, making it visible as a pink or red bacterium.
Importance of Gram Classification in Medicine
Determining a bacterium’s Gram classification is a fundamental step in the clinical management of infections. The Gram stain is often the first test performed on a sample because it provides a rapid result, typically within minutes. This speed is valuable for guiding initial, or empirical, antibiotic therapy before full culture results are available.
The cell wall structure that dictates the pink versus purple staining directly impacts antibiotic effectiveness. For instance, many antibiotics, such as penicillin, interfere with peptidoglycan synthesis. Gram-negative bacteria are often more resistant to these drugs because the protective outer membrane acts as a barrier. Knowing the organism is Gram-negative (pink) directs the medical provider to choose an antibiotic that can successfully penetrate this outer membrane.