Core concept1 exam ›
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Crystal violet -> iodine (mordant) -> alcohol/acetone decolourise -> safranin counterstain
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- Gram-POSITIVE = thick peptidoglycan retains the crystal violet-iodine complex -> PURPLE
- Gram-NEGATIVE = thin peptidoglycan + outer lipopolysaccharide membrane, dissolved by alcohol -> takes up safranin -> PINK/RED
- The outer membrane is why Gram-negatives are intrinsically resistant to vancomycin (too large to cross) and to many beta-lactams, and why they carry LPS/endotoxin
Key detail
Gram-positive
| Shape/arrangement | Organisms |
|---|---|
| Cocci in clusters | Staph aureus (coagulase +); coagulase-negative staph (epidermidis, saprophyticus - novobiocin resistant) |
| Cocci in chains/pairs | Strep pyogenes (group A, beta-haemolytic, bacitracin sensitive), Strep agalactiae (group B), Strep pneumoniae (alpha-haemolytic, optochin sensitive, bile soluble, lancet-shaped diplococci), viridans streptococci (optochin resistant), Enterococcus faecalis/faecium |
| Bacilli | Listeria monocytogenes (tumbling motility, grows at 4 degrees, intracellular), Corynebacterium diphtheriae, Bacillus anthracis/cereus, Clostridioides difficile, Clostridium perfringens/tetani/botulinum, Nocardia (partially acid-fast, branching), Actinomyces |
Gram-negative
| Shape | Organisms |
|---|---|
| Diplococci | Neisseria meningitidis, N. gonorrhoeae, Moraxella catarrhalis |
| Coccobacilli | Haemophilus influenzae, Bordetella pertussis, Brucella, Acinetobacter |
| Bacilli - lactose fermenters | E. coli, Klebsiella, Enterobacter, Citrobacter, Serratia |
| Bacilli - non-fermenters / non-lactose | Pseudomonas aeruginosa (oxidase +), Salmonella, Shigella, Proteus (swarming, urease +), Stenotrophomonas |
| Curved / spiral | Vibrio cholerae, Campylobacter jejuni (gull-wing), Helicobacter pylori |
| Anaerobes | Bacteroides fragilis, Fusobacterium, Prevotella |
Not seen or not classified on Gram stain
- Mycobacteria - waxy mycolic acid wall; Ziehl-Neelsen / auramine acid-fast stain
- Mycoplasma and Ureaplasma - no cell wall at all -> intrinsically resistant to all beta-lactams; use a macrolide, tetracycline or quinolone
- Treponema, Leptospira, Borrelia - too thin; dark-field or serology
- Chlamydia, Rickettsia, Coxiella - obligate intracellular
- Legionella - stains poorly; silver stain, urinary antigen (serogroup 1 only), PCR
- Fungi - Candida shows as Gram-positive budding yeasts; use KOH, calcofluor, silver
Clinical relevance
- The Gram stain is the first actionable result and directs empirical therapy hours before culture
- Gram-positive cocci in clusters in blood -> cover Staph aureus (flucloxacillin, or vancomycin if MRSA is possible)
- Gram-negative bacilli in blood -> Gram-negative sepsis; gentamicin plus a beta-lactam
- Gram-negative diplococci in CSF -> meningococcus; Gram-positive lancet-shaped diplococci -> pneumococcus; Gram-positive bacilli -> Listeria, so add benzylpenicillin/amoxicillin
- Vancomycin, teicoplanin and daptomycin cover Gram-positives only; aztreonam covers Gram-negatives only
- Sterile site, no organisms seen does not exclude infection - sensitivity is only ~60-80% even in culture-positive CSF, and lower after antibiotics
- Always ask whether the isolate could be a contaminant - a single bottle of coagulase-negative staph usually is; from a prosthetic device it usually is not
Correlations
- Urinary tract pathogens - nitrite reduction and biochemical identification
- Neisseria meningitidis - presentation and mortality
- Foodborne bacterial gastroenteritis
- Peritoneal dialysis peritonitis - causative organisms
- Multidrug-resistant tuberculosis (acid-fast, not Gram-stained)
4 of 4 sections written · drafted 2026-09-04