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Short answer: Auramine-rhodamine staining is a fluorescent acid-fast stain. It uses two dyes, auramine O and rhodamine B, to make mycobacteria glow against a dark background under a fluorescence microscope. One manufacturer’s instructions describe it as recommended for qualitative procedures in the fluorescent microscopic detection of mycobacteria. A positive smear is presumptive, not a final result.
Definition and scope
Mycobacterial cell walls contain mycolic acid, which complexes with certain dyes and resists decolorization by acid-alcohol. That property is called acid-fastness. In auramine-rhodamine staining, the dyes bind the mycolic acids, an acid-alcohol decolorizer removes stain from everything else, and a potassium permanganate counterstain makes tissue and debris non-fluorescent. Acid-fast bacilli then appear bright yellow-green or reddish-orange under the microscope.
The manufacturer’s instructions for use (IFU) call acid-fast microscopy the most rapid initial step in diagnosis and in learning how many acid-fast bacilli are present. The stain is sold for in vitro diagnostic use by properly trained individuals. This page explains the concept and the supply and quality questions around it. It does not give a bench procedure; follow your laboratory’s validated method and the kit’s own instructions.
Auramine staining
“Auramine staining” is the shorter name for the fluorescent acid-fast approach built on the dye auramine O. Truant and colleagues, working in 1962, used auramine and rhodamine separately and in combination, and found the combination most satisfactory.
Auramine-rhodamine staining
“Auramine-rhodamine” names the two-dye combination. The classical formulation listed in the IFU contains auramine O, rhodamine B, glycerol, phenol and demineralized water, with proportions adjusted as required to meet performance standards.
How it is used in practice
In practice the stain is one step in a smear workflow. The IFU lists what the user must supply beyond the stain itself: a quality control slide or control organisms, an acid-alcohol decolorizer, potassium permanganate counterstain, glass slides, a heat source, a microscope and immersion oil. Interpretation is by fluorescence: acid-fast organisms fluoresce reddish-orange against a dark background, while non-acid-fast material does not fluoresce or may appear pale yellow.
The evidence trail runs back several decades. The IFU credits Hagemann (1937) with describing fluorescent dyes for acid-fast bacilli, and notes that Bennedsen and Larsen (1966) reported higher yield and substantially reduced smear examination time compared with carbolfuchsin.
A more recent study from an African population with high HIV prevalence (Shea et al., Journal of Clinical Microbiology, 2009) evaluated auramine-rhodamine fluorochrome microscopy against culture. Sputum was liquefied with 10% dithiothreitol and concentrated by centrifugation at 3,000 x g. Sensitivity was 93.1% (67 of 72 culture-positive specimens) and specificity was 100% (32 of 32 culture-negative specimens). Those figures describe concentrated sputum in one study with culture as the reference; they are not a guarantee for other specimen types or preparations.
Types and related stains
The stains in this family share one principle: the mycolic acid wall holds the dye through acid-alcohol. They differ in how the signal is read and in what the laboratory needs to read it.
| Question | Auramine-rhodamine (fluorochrome) | Carbolfuchsin stains (Ziehl-Neelsen, Kinyoun) |
|---|---|---|
| Sensitivity and specificity | A review of 45 studies cited by Shea et al. concluded that fluorescent microscopy is more sensitive than and as specific as carbolfuchsin stains | Shea et al. report the Kinyoun stain performed worse than Ziehl-Neelsen; see Ziehl-Neelsen staining |
| Equipment cost | Shea et al. name the high cost of purchasing and maintaining fluorescence equipment and the short life of mercury vapor lamps, and suggest LEDs as an inexpensive alternative | Shea et al. describe the Kinyoun stain as technically the easier to use, though reported to be inferior to Ziehl-Neelsen |
| Confirmation of a positive | The IFU says a positive fluorescent smear may be restained by Ziehl-Neelsen or Kinyoun | Named in the IFU as restain methods |
The same fluorescence principle underlies other lab methods; for the physics, see fluorescence spectroscopy. For a different differential stain, see the Gram stain.
Where it matters for labs and supply decisions
The IFU gives a concrete checklist that a laboratory can compare against the product it holds. These items come from one manufacturer’s instructions; other products may differ.
- Controls: lots are tested with an acid-fast stain control slide, and the user is expected to run a QC slide or control organisms. The IFU states that if aberrant QC results are noted, patient results should not be reported. See CLIA quality control requirements for the regulatory side.
- Condition of the reagent: do not use it if the red clear liquid has changed color, if it is expired, or if it has deteriorated.
- Storage: 20-25 °C in the original container.
- Hazard labelling: the IFU lists a possible cancer hazard and describes the reagent as poison, combustible and irritant. Review it with your hazard communication program.
- Not supplied with the stain: decolorizer, permanganate counterstain, slides, a heat source, a microscope and immersion oil.
- Equipment upkeep: the study authors flag fluorescence equipment cost and lamp life as limits of the method.
Common misunderstandings
A positive smear is a final diagnosis
No. The IFU calls a positive result presumptive evidence of the presence of mycobacteria.
A negative smear rules out infection
No. The IFU states that a negative smear does not indicate that the specimen will be culturally negative, so culture methods must be employed.
The counterstain is cosmetic
It is functional. Potassium permanganate renders tissue and debris non-fluorescent, which reduces the chance of artifacts.
FAQ
What is auramine staining?
It is the fluorescent acid-fast method in which auramine O, often combined with rhodamine B, binds mycolic acids in mycobacterial cell walls so the organisms fluoresce under a fluorescence microscope.
What is auramine-rhodamine staining used for?
Per the manufacturer’s IFU, it is recommended for qualitative procedures in the fluorescent microscopic detection of mycobacteria. It is not a stand-alone diagnosis.
Does a negative smear rule out tuberculosis?
No. The IFU states that a negative staining reaction does not indicate that the specimen will be culturally negative, so culture methods must be employed.
How is a positive fluorescent smear followed up?
The IFU says a positive fluorescent smear may be restained by the conventional Ziehl-Neelsen or Kinyoun procedure, and that a positive reaction is presumptive evidence of mycobacteria. This page does not give bench steps; follow your laboratory’s validated method.
What does a laboratory need besides the stain?
The IFU lists a quality control slide or control organisms, decolorizer, potassium permanganate counterstain, demineralized water, glass slides, a heat source, a microscope and immersion oil.
Sources
- Thermo Fisher Scientific (Remel), TB Auramine-Rhodamine instructions for use, IFU40090 (revised November 2004): https://documents.thermofisher.com/TFS-Assets/MBD/Instructions/IFU40090.pdf. Date checked: 2026-10-29.
- Shea YR et al. High sensitivity and specificity of acid-fast microscopy for diagnosis of pulmonary tuberculosis in an African population with a high prevalence of HIV. J Clin Microbiol 2009;47(5):1553-1555: https://pmc.ncbi.nlm.nih.gov/articles/PMC2681839. Date checked: 2026-10-29.
