Learn — Specimen collection, staining, culture & diagnostics

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Acid-fast stain (Ziehl-Neelsen)

The Ziehl-Neelsen stain is the differential stain that demonstrates acid-fastness, the ability of mycobacteria to resist decolourisation by acid after being stained with hot carbol fuchsin, a property they owe to the mycolic acid in their waxy cell wall. It answers in an hour a question that culture takes six weeks to answer, and it tells the nurse whether the patient in the bed is infectious to everyone around him.

  • The method in order: flood the heat-fixed smear with strong carbol fuchsin, heat until it steams for about 5 minutes without boiling or letting it dry, wash, decolourise with 20 to 25 percent sulphuric acid or 3 percent acid-alcohol, wash again and counterstain with methylene blue or malachite green for about a minute.
  • Acid-fast bacilli appear as bright red slender beaded rods against a blue or green background, because everything else in the sputum is decolourised and takes up the counterstain.
  • The strength of the decolouriser is graded to the organism: 20 to 25 percent sulphuric acid for Mycobacterium tuberculosis, 5 percent for Mycobacterium leprae, 1 percent for Nocardia, and the modified Ziehl-Neelsen with 1 percent sulphuric acid for the coccidian parasites Cryptosporidium, Cyclospora and Isospora.
  • Microscopy is insensitive: roughly 10,000 bacilli per millilitre of sputum are needed for a positive smear against 10 to 100 per millilitre for culture, so a negative smear never excludes tuberculosis. Fluorescence microscopy using auramine on a light-emitting-diode microscope is more sensitive and much faster to read and has replaced Ziehl-Neelsen for screening in many laboratories.
  • Under India's National Tuberculosis Elimination Programme the first test for a person with presumptive tuberculosis is an upfront molecular test, a nucleic acid amplification test such as CBNAAT or Truenat, which gives a result within hours and reports rifampicin resistance at the same time; smear microscopy has moved to follow-up and treatment monitoring rather than being the entry point.
  • Culture remains the reference standard: Lowenstein-Jensen egg medium grows rough, tough, buff-coloured colonies in about 6 to 8 weeks because the generation time is 15 to 20 hours, while automated liquid culture in a mycobacteria growth indicator tube is positive in about 1 to 2 weeks, and both allow drug susceptibility testing.
  • Specimen collection is a nursing procedure with rules: two good-quality sputum specimens, one on the spot and one early morning, of thick purulent sputum in a wide-mouth leak-proof container, collected in the open air or a designated sputum booth and never in a closed room, toilet or the ward, with the nurse standing to the side and out of the line of the cough.
  • A smear- or NAAT-positive pulmonary case is nursed with airborne precautions: a single room with the door closed, negatively pressured where available, a fit-checked N95 respirator for staff, a surgical mask on the patient whenever he leaves the room, and cough etiquette with open windows and sunlight as the cheapest controls there are.

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