Ziehl–Neelsen stain

The Ziehl–Neelsen stain, also known as the acid-fast stain, is a bacteriological staining technique used in cytopathology and microbiology to identify acid-fast bacteria under microscopy, particularly members of the Mycobacterium genus. This staining method was initially introduced by Paul Ehrlich (1854–1915) and subsequently modified by the German bacteriologists Franz Ziehl (1859–1926) and Friedrich Neelsen (1854–1898) during the late 19th century. The acid-fast staining method, in conjunction with auramine phenol staining, serves as the standard diagnostic tool and is widely accessible for rapidly diagnosing tuberculosis (caused by Mycobacterium tuberculosis) and other diseases caused by atypical mycobacteria, such as leprosy (caused by Mycobacterium leprae) and Mycobacterium avium-intracellulare infection (caused by Mycobacterium avium complex) in samples like sputum, gastric washing fluid, and bronchoalveolar lavage fluid. These acid-fast bacteria possess a waxy lipid-rich outer layer that contains high concentrations of mycolic acid, rendering them resistant to conventional staining techniques like the Gram stain. After the Ziehl–Neelsen staining procedure using carbol fuchsin, acid-fast bacteria are observable as vivid red or pink rods set against a blue or green background, depending on the specific counterstain used, such as methylene blue or malachite green, respectively. Non-acid-fast bacteria and other cellular structures will be colored by the counterstain, allowing for clear differentiation.

Procedure

A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells.

"Acid alcohol" refers to an alcohol (reagent/solvent-grade; often denatured for tax reasons) solution of hydrochloric acid. Water is present in some versions. The percentage refers to the acid content by volume.

Mechanism explanation

The mechanism of action of the Ziehl–Neelsen stain has long been discussed and is not completely understood. Historically, it was believed to involve a chemical reaction between the acidic dyes and the cell walls of the bacteria. The acidity of the dyes was thought to cause them to bind strongly to the lipid-rich cell walls—particularly to mycolic acids—resulting in the selective staining of only those cells that possess a thick, waxy envelope. This traditional view held that these cell-wall lipids were responsible for the retention of the primary dye after acid–alcohol decolourisation, explaining the “acid-fast” property of mycobacteria and related organisms. The Ziehl–Neelsen stain is a two-step staining process. In the first step, the tissue is stained with a basic fuchsin solution, which stains all cells pink. In the second step, the tissue is incubated in an acid-alcohol solution, which decolourises all cells except for acid-fast cells, which retain the colour and appear red. The mechanisms by which this colour is produced were historically thought to involve an interaction of basic fuchsin with cell-wall components, creating a stable dye complex responsible for the observed red coloration. Recent studies have provided a more detailed and experimentally supported interpretation of this process, however alternative hypotheses to the structures stained, including nucleic acids were proposed. Fluorescence and confocal microscopy show that the primary dyes (carbol fuchsin and auramine O) actually localise to intracellular nucleic acids (DNA and RNA) rather than to the cell wall itself. In this updated model, the cell envelope’s role is mainly to prevent the loss of these dyes during acid–alcohol washing, rather than to act as the dye-binding target. This intracellular localisation explains the characteristic “beading” pattern observed in Ziehl–Neelsen and fluorescent acid-fast stains, corresponding to the bacterial nucleoids. Similar nucleic-acid staining patterns are seen in other acid-fast organisms such as Cryptosporidium, Cystoisospora, and Schistosoma spp. These findings indicate that acid-fast stains function primarily as nucleic-acid stains whose retention depends on cell-wall integrity rather than direct binding to mycolic acids.

Organisms that stain as acid-fast

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