Morphology and Staining
Domain 3 covers bacterial shape, arrangement and the staining methods used to identify them. The exam expects the sequence and purpose of each Gram-stain reagent, the acid-fast method, and the use of special stains for capsules, spores and flagella.
Core rules and concepts
- Cocci are spherical; bacilli are rod-shaped; vibrios are comma-shaped; spirilla are rigid spirals; spirochetes are flexible helices. Arrangements include diplococci, streptococci, staphylococci, tetrads and sarcinae.
- Gram stain: crystal violet (primary stain), iodine (mordant), alcohol or acetone-alcohol (decolorizer), safranin (counterstain). Gram-positive cells retain violet; Gram-negative cells lose it and take safranin.
- Ziehl-Neelsen acid-fast stain uses heat with carbolfuchsin; acid-fast bacilli resist decolorization with acid-alcohol and appear red, while non-acid-fast cells take methylene blue.
- Capsule staining often uses negative staining with India ink; endospore staining uses malachite green with heat and safranin counterstain; flagella staining uses mordants to thicken flagella.
Worked example
A sputum smear from a patient with chronic cough is stained by Ziehl-Neelsen. Red, beaded rods are seen against a blue background. Reasoning: the red rods retained carbolfuchsin after acid-alcohol, identifying acid-fast bacilli consistent with Mycobacterium tuberculosis. A Gram stain would not reliably demonstrate this organism because mycobacteria are not well stained by the Gram method.
Mark-losing traps
- Over-decolorizing Gram-positive cells makes them appear Gram-negative; old cultures can also give variable results.
- Do not call safranin the mordant; iodine is the mordant.
- Capsules are not stained by simple stains; negative staining reveals them as clear halos.
- Acid-fast staining is not the same as Gram staining, and mycobacteria are not classified as Gram-negative in routine reporting.