Gram stain

The Gram stain procedure involves four steps: (1) crystal violet primary stain, (2) Gram's iodine as a mordant to fix the dye, (3) alcohol/acetone decolorization, and (4) safranin counterstain. Gram-positive bacteria have a thick peptidoglycan layer that traps the crystal violet-iodine complex, resisting decolorization and retaining a purple color. Gram-negative bacteria have a thin peptidoglycan layer and an outer lipopolysaccharide (LPS)-containing membrane that is disrupted by alcohol, allowing the crystal violet to wash out; they then take up the safranin counterstain, appearing pink/red. Bacterial morphology (cocci, bacilli, spirochetes) and arrangement (chains, clusters, pairs) are also assessed. Clinical significance: Gram stain results guide empiric antibiotic selection before culture results are available (e.g., vancomycin for Gram-positive cocci in clusters suggesting Staphylococcus, vs. broader coverage for Gram-negative rods). Some organisms are not well visualized by Gram stain, including Mycobacteria (acid-fast, requires Ziehl-Neelsen stain), Mycoplasma (lacks cell wall), Legionella, Chlamydia, Treponema (too thin, requires darkfield microscopy), and fungi (require KOH prep or other stains). Common exam associations: Gram-positive cocci in clusters = Staphylococcus; in chains = Streptococcus; Gram-negative diplococci = Neisseria; Gram-negative rods = Enterobacteriaceae, Pseudomonas. Gram stain is often the first test performed on CSF, sputum, blood, or wound specimens in suspected bacterial infection.