Gram Staining & Major Bacterial Pathogens
How the Gram stain works, how to read it, and a structured map of the most clinically important Gram-positive and Gram-negative pathogens.
Interactive Diagram
Tap a teal label to learn about each structure.
Mechanism of the Gram Stain
Crystal violet enters both cell types. Iodine forms a large crystal violet–iodine complex. The alcohol decolorizer extracts lipids from the thin Gram-negative wall and the complex leaks out, while the thick, dehydrated Gram-positive peptidoglycan traps it. Safranin counterstains the colorless Gram-negative cells pink, so Gram-positive appear purple and Gram-negative pink. Over-decolorization falsely makes Gram-positives look negative; under-decolorization does the reverse, so technique matters.
Gram-Positive Cocci
Staphylococcus (catalase-positive clusters) includes S. aureus (coagulase-positive, causes abscesses, osteomyelitis, toxic shock) and S. epidermidis (coagulase-negative, device infections). Streptococcus (catalase-negative chains) is classified by hemolysis: beta-hemolytic group A (S. pyogenes — pharyngitis, cellulitis, rheumatic fever), group B (S. agalactiae — neonatal sepsis), and alpha-hemolytic viridans group (endocarditis) and S. pneumoniae (pneumonia, meningitis, otitis). Enterococcus causes UTIs and nosocomial infections and is notable for intrinsic vancomycin resistance (VRE).
Gram-Positive Rods
Bacillus anthracis (anthrax, capsule of poly-D-glutamate), Clostridium species (C. perfringens gas gangrene, C. tetani tetanospasmin, C. botulinum botulinum toxin, C. difficile pseudomembranous colitis), Corynebacterium diphtheriae (diphtheria toxin inhibiting elongation factor 2), and Listeria monocytogenes (intracellular, cold-grown in deli meats, neonatal meningitis). Listeria is the exception: it is a Gram-positive rod that is catalase-positive and motile at room temperature with tumbling motility.
Gram-Negative Cocci and Coccobacilli
Neisseria (gonorrhoeae and meningitidis) are diplococci requiring chocolate/Thayer–Martin media; they oxidize glucose. Haemophilus influenzae needs factor X and V (chocolate agar) and causes meningitis, epiglottitis, and otitis in unvaccinated children. Bordetella pertussis causes whooping cough and is cultured on Bordet–Gengou agar. Brucella and Francisella are small intracellular coccobacilli that are laboratory hazards.
Gram-Negative Rods (Enterics and Beyond)
Enterobacteriaceae (E. coli, Klebsiella, Salmonella, Shigella, Proteus, Yersinia) ferment glucose, are oxidase-negative, and grow on MacConkey agar. Pseudomonas aeruginosa is a non-fermenter, oxidase-positive, and produces pyocyanin and a sweet grape-like odor. Vibrio cholerae causes rice-water diarrhea and grows on TCBS agar. Helicobacter pylori is a spiral, urease-positive organism causing gastritis and ulcers. Campylobacter jejuni is a curved, oxidase-positive rod causing bloody diarrhea.
