Microbiology
Stain and culture basics
The Gram stain sequence is crystal violet, iodine, decolorizer, safranin; Gram-negative organisms lose the crystal violet-iodine complex. Specimen quality matters as much as the culture: reject sputum with few neutrophils, and interpret urine colony counts with the collection method (catheterized versus clean catch).
Key biochemical reactions
- Catalase-positive, coagulase-positive = Staphylococcus aureus.
- Catalase-negative, bacitracin-sensitive = group A Streptococcus; optochin-sensitive = S. pneumoniae.
- Oxidase-positive = Neisseria, Pseudomonas, Aeromonas.
- Urease-positive = Helicobacter pylori, Proteus.
- Sorbitol-negative E. coli O157:H7 on MacConkey.
- TSI with acid/acid plus gas indicates lactose fermentation; alkaline/acid indicates a nonfermenter.
Susceptibility
MIC is the lowest concentration inhibiting visible growth; Kirby-Bauer zones are compared to CLIA/CAP-recognized breakpoints. MRSA is confirmed by cefoxitin or mecA/PBP2a testing; ESBL and carbapenemase producers require specific screens because routine panels can mislead.
Traps
- Normal flora in a normally sterile site (blood, CSF) is a pathogen until proven otherwise; the reverse is true for wounds.
- CSF Gram stain is only about 60-80% sensitive, so a negative stain does not exclude meningitis.
- Blood cultures require two sets of 20-30 mL total in adults before antibiotics.
- Campylobacter requires microaerophilic, 42 C conditions; Salmonella and Shigella do not.
- Acid-fast bacilli need Ziehl-Neelsen or auramine stain, not Gram stain.
Rule of thumb
If a colony is oxidase-positive, Gram-negative, and lactose-negative, think nonfermenter (Pseudomonas) before enteric pathogen.