Modified Ziehl–Neelsen stain (Cryptosporidium)

Principle

• Coccidian oocysts have a lipid-rich wall that retains carbol fuchsin after decolorization with a weaker acid than the classic AFB stain.
• Hot (modified ZN) and cold (modified Kinyoun) versions are used.

Specimen

• Fresh or formalin-fixed stool; sediment from formalin–ethyl acetate concentration improves detection.
• Duodenal aspirates or other specimens as indicated.

Reagents and equipment

• Methanol (fixative), carbol fuchsin, weak decolorizer (commonly 1% sulfuric acid, per local procedure), counterstain (methylene blue or malachite green).

Procedure

• Prepare a thin smear, air-dry and fix with methanol.
• Stain with carbol fuchsin (heated for the hot method, or longer unheated for the cold Kinyoun method).
• Rinse, then decolorize briefly with the weak acid until no more color runs off.
• Rinse and counterstain for about 1 minute; rinse, dry and examine under oil immersion.

Results

• Cryptosporidium oocysts: round, 4–6 µm, pink-red on a blue or green background; some stain weakly or appear as ghosts, and internal sporozoites may be seen.
• Cyclospora cayetanensis: 8–10 µm, variably acid-fast (from unstained to deep red); also autofluorescent under UV.
• Cystoisospora belli: large ellipsoidal oocysts (about 20–30 µm long) that are acid-fast.

Quality control and pitfalls

• Run a positive control slide containing Cryptosporidium with each batch.
• Yeasts are of similar size but stain blue-green with the counterstain; do not confuse them.
• Over-decolorizing (acid too strong or applied too long) gives false negatives.
• Shedding is intermittent; examine multiple specimens.
• Antigen detection (EIA or IFA) and PCR are more sensitive.

Clinical use

• Diagnosis of cryptosporidiosis, cyclosporiasis and cystoisosporiasis, causes of watery diarrhea, especially in children and immunocompromised patients (e.g. advanced HIV).