Acid-fastness

File:Mycobacterium tuberculosis Ziehl-Neelsen stain 640.jpg

Mycobacterium tuberculosis (stained red) in tissue (blue).

Acid-fastness is a physical property of certain bacteria, protozoa, and eukaryotic cells, as well as some subcellular structures, referring to their resistance to decolorization by acids during laboratory staining procedures.<ref name=Madison_2001>Template:Cite journal</ref><ref name=Sherris>Template:Cite book</ref> Once stained as part of a sample, these organisms can resist the acid and/or ethanol-based decolorization procedures common in many staining protocols, hence the name acid-fast.<ref name="Sherris"/>

Historically, acid-fast stains were thought to stain lipids of the cells based on the observed charectistics of cell staining under a wide range of conditions,<ref>Template:Cite journal</ref><ref>Template:Cite journal</ref> although the results were limited by the tools available, however as early as 1959 there were observations of how nucleic acids were acid fast.<ref>Template:Cite journal</ref> Dyes such as carbol fuchsin and auramine O penetrate the cell and bind to DNA and RNA, producing characteristic red or yellow-green fluorescence, respectively. The property of “acid-fastness” therefore reflects the organism’s ability to retain these dyes after acid–alcohol decolorization, a feature determined mainly by the integrity and composition of the outer cell wall rather than by any specific lipid chemistry.<ref name="Hanscheid2025">Template:Cite journal</ref>

The mechanisms of acid-fastness vary by species. In the genus Mycobacterium, the property has been traditionally attributed to the high mycolic acid content of the cell wall, which indeed contributes to dye retention and resistance to decolorization. However, many other acid-fast organisms—such as intestinal coccidia and parasitic helminths of the genus Schistosoma—lack mycolic acids yet display comparable acid-fastness, suggesting that other cell-wall structures, such as cyst walls or egg shells, may provide similar resistance to decolorization.<ref name="Hanscheid2025" />

Further histopathologic evidence supports this broader mechanism: in tissue sections, staining intensity is markedly reduced when bacterial cell walls are damaged or when xylene-based deparaffinization is used during specimen processing. A xylene-free, heat-based method has been shown to preserve cell-wall integrity and substantially improve detection of mycobacteria and other acid-fast organisms, particularly when using fluorescent Auramine O staining.<ref name="Marinho2023">Template:Cite journal</ref>

Acid-fast organisms are difficult to characterize using standard microbiological techniques, though they can be stained using concentrated dyes, particularly when the staining process is combined with heat. Some, such as Mycobacteria, can be stained with the Gram stain, but they do not take the crystal violet well and thus appear light purple, which can still potentially result in an incorrect gram-negative identification.<ref>Template:Cite journal</ref>

The most common staining technique used to identify acid-fast bacteria is the Ziehl–Neelsen stain, in which acid-fast species appear bright red against a blue background. Another method is the Kinyoun method, in which bacteria appear red against a green background. Fluorescence microscopy using auramine O—a nucleic acid–binding fluorochrome—has largely replaced these techniques in clinical laboratories due to higher sensitivity, rapidity, and safety. Rhodamine, sometimes added as a secondary dye, contributes little to sensitivity but slightly enhances contrast.<ref name="Abe_2003">Template:Cite journal</ref><ref name="Hanscheid2025" /><ref name="Marinho2023" />

Some acid-fast staining techniques

• Ziehl–Neelsen stain (classic and modified bleach types)<ref>{{#invoke:citation/CS1|citation

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• Kinyoun stain, a development of ZN that requires no heating; variants:
  • Alternative dyes (Victoria blue instead of fuchsin, picric acid instead of methylene blue), which is useful to color-blind people and materials where the classical ZN/Kinyoun dyes provide insufficient legibility.<ref>Theory and Practice of Histological Techniques, John D Bancroft, 6th ed, p314</ref>
  • Moeller's method
  • Dorner's method<ref>Dorner, W. 1926. Un procédé simple pour la colouration des spores. Le Lait 6:8–12.</ref> (acid alcohol decolorizer) without the Schaeffer–Fulton<ref>Template:Cite journal</ref> modification (decolorize by water)<ref>{{#invoke:citation/CS1|citation

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• • Detergent method, using Tergitol 7, nonionic polyglycol ether surfactants type NP-7 for decolorizing<ref>Template:Cite journal</ref>
• Fite stain<ref>{{#invoke:citation/CS1|citation

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• • Fite-Faraco stain<ref>{{#invoke:citation/CS1|citation

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• • Wade Fite stain<ref>{{#invoke:citation/CS1|citation

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• Ellis and Zabrowarny stain<ref>Template:Cite journal</ref><ref>{{#invoke:citation/CS1|citation

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• Auramine-rhodamine stain
• Auramine phenol stain

Notable acid-fast structures

Very few structures are acid-fast; this makes staining for acid-fastness particularly useful in diagnosis. The following are notable examples of structures which are acid-fast or modified acid-fast:

• All Mycobacteria – M. tuberculosis, M. leprae, M. smegmatis and atypical mycobacteria.
• Certain Actinobacteria (especially aerobic ones in the order Mycobacteriales) with mycolic acid in their cell wall; not to be confused with Actinomyces, which is a non-acid-fast genus of actinomycete. Note that Streptomyces do not contain mycolic acid.
  • Nocardia (weakly acid-fast; resists decolorization with weaker acid concentrations)
  • Rhodococcus
  • Gordonia
  • Tsukamurella
  • Dietzia
• Head of sperm
• Bacterial spores, see Endospore
• Legionella micdadei
• Certain cellular inclusions e.g.
  • Cytoplasmic inclusion bodies seen in
    • Neurons in layer 5 of cerebral cortex neuronal ceroid lipofuscinosis (Batten disease).
  • Nuclear inclusion bodies seen in
    • Lead poisoning
    • Bismuth poisoning.
• Oocysts of some coccidian parasites in faecal matter, such as:
  • Cryptosporidium parvum,<ref>Template:Cite journal</ref>
  • Isospora belli<ref>Template:Cite journal</ref>
  • Cyclospora cayetanensis.<ref>Template:Cite journal</ref>
• A few other parasites:
  • Sarcocystis
  • Taenia saginata eggs stain well but Taenia solium eggs don't (can be used to distinguish)
  • Hydatid cysts, especially their "hooklets" stain irregularly with ZN stain but emanate bright red fluorescence under green light, and can aid detection in moderately heavy backgrounds or with scarce hooklets.<ref>Template:Cite journal</ref>
• Fungal yeast forms are inconsistently stained with Acid-fast stain which is considered a narrow spectrum stain for fungi.<ref>{{#invoke:citation/CS1|citation

|CitationClass=web }}</ref> In a study on acid-fastness of fungi,<ref>Wages ds, Wear dJ. acid-fastness of fungi in blastomycosis and histoplasmosis. Arch Pathol Lab Med 1982; 106:440-41.</ref> 60% of blastomyces and 47% of histoplasma showed positive cytoplasmic staining of the yeast-like cells, and Cryptococcus or candida did not stain, and very rare staining was seen in Coccidioides endospores.

References

Online protocol examples

• Ziehl–Neelsen protocol (PDF format).
• Alternate Ellis & Zabrowarny method Template:Webarchive for staining AFB.