Fact-checked by Grok 2 weeks ago

Auramine O

Auramine O is a synthetic diarylmethane and fluorescent , chemically known as 4,4'-(imidocarbonyl)bis(N,N-dimethylaniline) monohydrochloride, with the molecular C₁₇H₂₂ClN₃ and a of 303.83 g/. It appears as yellow needle-like crystals that are sparingly soluble in water but soluble in and phenol, and it exhibits at approximately 432 nm and at approximately 500 nm under fluorescence microscopy. Primarily utilized in , Auramine O binds to mycolic acids in the walls of acid-fast , such as , enabling their visualization as bright yellow-green fluorescent rods against a dark background when excited by UV or . This fluorochrome method offers higher sensitivity and faster detection compared to traditional carbol fuchsin-based Ziehl-Neelsen , allowing examination at lower magnifications (×400–500) and identifying more per field. The fluorescent staining technique using Auramine O was developed in the late and first reported as an effective fluorochrome for staining tubercle in 1938 by Hagemann, who combined it with acid-alcohol decolorization to enhance specificity. The technique gained prominence through the work of Richards in , who demonstrated its binding to mycolic acids and improved its protocol for routine use in sputum smears for . Often paired with as a in the Truant method, Auramine O has become a standard in low-resource settings for rapid screening of acid-fast , though it may also stain non-tuberculous mycobacteria and certain parasites like . Despite its efficacy, the dye is classified as a possible based on animal studies, necessitating careful handling in laboratory environments.

Chemical Properties

Molecular Structure and Formula

Auramine O, also known as C.I. Basic Yellow 2, is a diarylmethane characterized by its ion core. Its IUPAC name is 4-[4-(dimethylamino)benzenecarboximidoyl]-N,N-dimethylaniline . The molecular formula of Auramine O is C<sub>17</sub>H<sub>22</sub>ClN<sub>3</sub>, reflecting the composition of two para-dimethylaminophenyl groups linked to a central carbon-nitrogen moiety and a . The is 303.83 g/mol. Structurally, Auramine O features a central carbon atom double-bonded to an NH<sub>2</sub><sup>+</sup> group, forming an iminium ion [(4-(CH<sub>3</sub>)<sub>2</sub>N-C<sub>6</sub>H<sub>4</sub>)<sub>2</sub>C=NH<sub>2</sub><sup>+</sup>] that is stabilized by the electron-donating dimethylamino substituents on the flanking phenyl rings. This configuration exists in the hydrochloride salt form, with Cl<sup>-</sup> as the anion. The iminium structure can be textually represented as: 
   (CH₃)₂N-C₆H₄
          |
(CH₃)₂N-C₆H₄-C=NH₂⁺ Cl⁻
where the central C=NH<sub>2</sub><sup>+</sup> connects the two 4-(dimethylamino)phenyl groups. Auramine O originates from , which is 4,4'-bis(dimethylamino) [(4-(CH<sub>3</sub>)<sub>2</sub>N-C<sub>6</sub>H<sub>4</sub>)<sub>2</sub>C=O], through an formation process that replaces the carbonyl oxygen with the NH<sub>2</sub><sup>+</sup> group. This diarylmethane framework contributes to its classification among cationic fluorescent dyes used in various applications.

Physical Properties

Auramine O appears as yellow needle-like crystals in its pure form. It has a of 267 °C, at which point it decomposes. The of the compound is approximately 1.07 g/cm³. Auramine O is odorless. Auramine O is sparingly soluble in water (approximately 10 g/L at 20 °C), but soluble in ethanol (20 g/L at 20 °C) and phenol. Under normal conditions, Auramine O is stable, though it is incompatible with strong oxidizing agents and sensitive to light exposure. As a fluorescent dye, Auramine O exhibits an absorption maximum at approximately 432 nm and emits yellow-green fluorescence with a peak at 499 nm.

Synthesis and Production

Chemical Synthesis

Auramine O is primarily synthesized on a laboratory scale through the condensation of , chemically known as 4,4'-bis(dimethylamino), with in the presence of as a dehydrating agent. The reaction proceeds by heating the mixture to 150-160 °C for 2-4 hours, often in a sealed tube to contain volatile gas and prevent side reactions. This process involves the formation of the salt structure of Auramine O . The simplified reaction equation for this key step is: (CH_3)_2NC_6H_4 - CO - C_6H_4N(CH_3)_2 + NH_4Cl \rightarrow [(CH_3)_2NC_6H_4]_2C=NH^+ \, Cl^- + H_2O Typical yields for this condensation range from 80-90%, depending on the purity of the starting materials and precise control of heating conditions. An alternative synthetic route starts with the condensation of N,N-dimethylaniline and to produce Michler's base, or 4,4'-methylenebis(N,N-dimethylaniline). This intermediate is then oxidized, typically using an agent like or cupric chloride, to yield Michler's ketone, which undergoes the aforementioned imine formation with or . This multi-step approach allows for preparation from more readily available precursors but introduces additional purification needs between stages. Following , the crude Auramine O is purified by recrystallization from or water- mixtures, which effectively removes impurities such as unreacted Michler's ketone and inorganic salts, resulting in a product with greater than 80% content. This step ensures the compound's suitability for analytical or applications by enhancing and .

Industrial Manufacturing

The industrial manufacturing of Auramine O began in the late , with the dye first introduced in by European chemical firms, including major German producers like , which pioneered large-scale synthesis amid the rapid growth of the synthetic sector. Early production was centered in (Switzerland, , the , and ), later expanding to the , before shifting predominantly to in modern times. Commercial production typically employs a multi-step batch process in specialized reactors, starting with the condensation of N,N- and to form Michler's (4,4'-bis(N,N-dimethylamino)), followed by reaction of this with additional to yield the final Auramine O . The process often proceeds in heated vessels under controlled conditions, such as 170–180°C, incorporating , , and to facilitate the transformation, with variations allowing for continuous flow in larger facilities to enhance throughput. While laboratory-scale synthesis provides a foundational method, industrial adaptations emphasize safety enclosures and effluent treatment to manage volatile intermediates like and . Key innovations in the improved process efficiency, notably through patented methods incorporating or related compounds as catalysts to boost yields by 12–20% over prior techniques, reducing waste and raw material costs in the step. For instance, replacing a portion of conventional salts with at least 0.7 parts per part of Michler's per the 1961 American Cyanamid patent enabled higher conversion rates, up to 85%, in ammonia-saturated environments. As of , global of Auramine O was estimated at approximately 1,000 tonnes annually, with the majority manufactured in —primarily and —to supply , , and microbiological markets. in industrial output adheres to standards set by the Biological Stain Commission, which certifies commercial batches for content typically ranging from 80% to 85% purity, ensuring consistency for applications requiring high intensity. Analytical methods like HPLC and , refined since the 1970s, verify purity and absence of impurities during .

Applications

Diagnostic Staining in Microbiology

Auramine O serves as a key fluorescent in the of acid-fast , particularly mycobacteria, by binding selectively to mycolic acids in their cell walls, which imparts a yellow-green when excited by light at approximately 430-460 nm, with emission peaking around 500 nm. This binding mechanism enhances visibility under fluorescence microscopy, allowing acid-fast organisms to stand out against a dark background. The technique is especially valuable for detecting , the causative agent of , as well as in and various (NTM) in clinical samples such as and tissue biopsies. The standard procedure, known as the Truant auramine-rhodamine , involves preparing a smear from the specimen, heat-fixing it, and applying a primary stain solution containing 0.1% Auramine O dissolved in a phenol-glycerol vehicle for 15 minutes at 37°C to facilitate penetration and binding. The slide is then decolorized with 0.5% in 70% for 2-3 minutes to remove unbound dye, followed by counterstaining with 0.5% for 2-4 minutes to quench background . Examination occurs under a using a blue-light source, where acid-fast appear as bright yellow-green rods. This method was originally described by Truant et al. in 1962, building on earlier work by Hagemann in 1938, and gained popularity in the 1950s-1960s due to improved microscopic equipment and its efficacy in clinical settings. The (WHO) has recommended auramine-based microscopy, particularly with (LED) systems, for tuberculosis diagnosis in resource-limited settings since 2011, as it aligns with global efforts to enhance case detection. Compared to the traditional Ziehl-Neelsen (ZN) stain, auramine O offers several advantages, including a shorter overall processing time of 2-5 minutes for reading slides versus 10-20 minutes for ZN, higher that enables detection of as few as 10-100 per slide, and the ability to scan large areas at low magnification (e.g., 25x ) without , reducing observer fatigue. These features make it particularly suitable for high-volume screening in endemic areas, where it has demonstrated up to 10% greater than ZN, especially in paucibacillary cases. However, limitations include potential non-specific from non-acid-fast organisms, cellular debris, or structures like , which can lead to false positives and necessitate confirmatory testing. The counterstain with mitigates this to some extent, but specificity remains lower than ZN in low-prevalence settings.

Other Uses

In histology and cytology, Auramine O functions as a fluorescent analog of the Schiff reagent, facilitating the detection of aldehydes in tissue sections through variants of the periodic acid-Schiff (PAS) staining method, where it produces yellow-green fluorescence in nuclei and other structures. This application leverages its binding affinity to produce enhanced visualization under fluorescence microscopy, particularly for carbohydrate-rich components. Auramine O is utilized as a basic yellow dye in industrial applications, including the coloration of , textiles, , and inks, where it provides bright yellow hues suitable for , products, and printing. Its adoption in these sectors has declined due to recognized and environmental concerns, limiting it to non-food contact materials. Historically, Auramine O has served as a mild antiseptic agent in low concentrations, exhibiting inhibitory effects against certain bacteria due to its chemical structure as a diarylmethane dye, with early 20th-century studies recommending it for disinfection purposes. In research settings, Auramine O acts as a fluorescent probe in flow cytometry for analyzing cellular components, such as reticulocytes and urinary sediments, where it stains RNA and DNA to enable quantitative detection. It also supports environmental monitoring by detecting acid-fast bacteria and microorganisms in water and soil samples, aiding assessments of microbial contamination. Although explored in photodynamic therapy studies for its photosensitizing potential, its primary research utility remains in fluorescence-based bacterial identification rather than clinical PDT applications. Auramine O is prohibited as a food colorant in the European Union and the United States owing to its carcinogenic properties, as classified by international health authorities. Despite these bans, instances of illegal adulteration in spices and foodstuffs persist, prompting regulatory surveillance to mitigate health risks from unauthorized use.

Safety, Toxicity, and Regulation

Health and Environmental Hazards

Auramine O exhibits primarily through oral and dermal routes, with an oral LD50 of 1490 mg/kg in rats and 480 mg/kg in mice, indicating it is . Dermal exposure is also concerning, as the LD50 is 300 mg/kg in mice, suggesting possible absorption and potential for or burns upon contact. Additionally, direct contact causes serious eye irritation, manifesting as redness, pain, and potential corneal damage. Chronic exposure to Auramine O is associated with carcinogenic risks, classified by the International Agency for Research on Cancer (IARC) as Group 2B, possibly carcinogenic to humans, based on limited evidence in humans and sufficient evidence in experimental animals. Epidemiological studies from manufacturing workers in the 1970s through the 2000s have linked occupational exposure to increased incidence of , with relative risks elevated up to 20-fold in some cohorts exposed to Auramine O during production processes. Auramine O demonstrates mutagenic potential, testing positive in several Ames bacterial mutagenicity assays, particularly with metabolic activation, indicating its ability to induce reverse mutations in Salmonella typhimurium strains. This genotoxicity is attributed to its planar aromatic structure, which facilitates DNA intercalation and strand breaks in vitro and in vivo. In the environment, Auramine O persists in aqueous systems due to its moderate , with a log Kow value of approximately 3.5, promoting adsorption to sediments and potential in benthic organisms. It is toxic to aquatic life, exhibiting an of 0.093 mg/L for such as Selenastrum capricornutum over 72 hours, and broader LC50 values ranging from 0.3 to 4.8 mg/L across , daphnids, and , leading to long-term adverse effects in ecosystems. Primary exposure routes for Auramine O include inhalation of dust or aerosols in laboratory and industrial settings, which can irritate respiratory tracts, and dermal contact during handling or staining procedures, facilitating absorption through intact skin.

Handling Precautions and Regulations

Auramine O is classified under the Globally Harmonized System (GHS) as Dangerous, with key hazard statements including H302 (harmful if swallowed), H351 (suspected of causing cancer), and H411 (toxic to aquatic life with long lasting effects). When handling Auramine O, appropriate (PPE) must be worn, including gloves, safety glasses or goggles, protective clothing, and a lab coat; respiratory protection such as a P3 is recommended when dusts or powders are generated, and operations involving powders should be conducted in a to minimize risks. For storage, Auramine O should be kept in a cool, dry, well-ventilated area away from light and incompatible materials, in tightly sealed containers to prevent degradation and accidental release; access should be restricted and locked where possible. Disposal of Auramine O and contaminated materials must follow local, national, and international regulations for hazardous waste, typically involving incineration or specialized chemical treatment at approved facilities, without mixing with other wastes. Regulatory restrictions on Auramine O include its prohibition as a food additive or colorant in the European Union under Regulation (EC) No 1333/2008, which authorizes only listed substances for food use, and similarly by the U.S. Food and Drug Administration (FDA), where it is considered an illegal color additive subject to import detention under Import Alert 45-02. In the EU, it is registered under REACH (EC) No 1907/2006 for industrial applications but restricted under Annex XVII entry 75 from use in tattoo inks and permanent make-up; no specific OSHA permissible exposure limit (PEL) exists, but general guidelines for handling hazardous dyes apply to occupational settings. In case of exposure, emergency procedures include immediately washing affected skin or eyes with plenty of water for at least 15 minutes while removing contaminated clothing, rinsing the mouth if swallowed, and seeking medical attention; for inhalation, move to fresh air and provide oxygen if breathing is difficult, with professional medical advice required for any suspected or prolonged .

References

  1. [1]
    C.I. Basic Yellow 2
    **Summary of CID 17170 from PubChem:**
  2. [2]
    Acid Fast Bacteria - StatPearls - NCBI Bookshelf
    Aug 7, 2023 · The fluorochrome procedure primarily utilizes one of two dyes, the auramine-O dye, or the auramine-rhodamine dye. Auramine-O is a hydrochloride ...Introduction · Procedures · Normal and Critical Findings · Complications
  3. [3]
    Auramine O - an overview | ScienceDirect Topics
    Auramine O produced brilliant yellow fluorescence from stained organisms and the technique became popular in the United States through the work of Richards ( ...<|control11|><|separator|>
  4. [4]
    [PDF] Auramine-Rhodamine Fluorescence - Acid Fast Bacteria - WebPath
    Work in a well ventilated area, preferable under a hood. Avoid contact and inhalation of chemicals. Auramine O: possible carcinogen to humans. Animal studies; ...
  5. [5]
    C.I. Basic Yellow 2 | C17H22ClN3 | CID 17170 - PubChem
    Auramine O is a hydrochloride obtained by combining 4,4'-carbonimidoylbis(N,N-dimethylaniline) with one molar equivalent of hydrogen chloride.
  6. [6]
    Auramine O | C17H22ClN3 - ChemSpider
    Molecular formula: C17H22ClN3. Average mass: 303.834. Monoisotopic mass: 303.150225 ... [IUPAC name – generated by ACD/Name]. Hydrogenchlorid --4,4 ...
  7. [7]
    [PDF] RoC Profile: Michler's Ketone
    Michler's ketone is a chemical intermediate used in the synthesis of at least 13 dyes and pigments, particularly auramine derivatives (NCI. 1979, HSDB 2009) ...
  8. [8]
    Health and Environmental Risks of Incense Smoke - PubMed Central
    Apr 26, 2022 · AuO is a diarylmethane hydrophobic dye yellow needle-like crystals ... DNA binding receptivity of auramine O, a sensitive fluorescent stain added ...
  9. [9]
    [PDF] AURAMINE AND AURAMINE PRODUCTION 1. Exposure Data
    This process can lead to auramine that contains Michler's ketone, probably from the hydrolysis of the target dye. Auramine is used as a colourant for paper ...
  10. [10]
    AURAMINE O | 2465-27-2 - ChemicalBook
    Sep 25, 2025 · It is effective in detecting positive cases of tuberculosis. Auramine O binds to the mycolic acid in the bacterial cell wall.
  11. [11]
    [PDF] Auramine-O - SAFETY DATA SHEET
    established by the region specific regulatory bodies. 9. Physical and chemical properties. Physical State. Powder Solid. Appearance. Light green. Odor. Odorless.
  12. [12]
    Auramine O: A Fluorescent Dye for Microscopy, Research, and ...
    Nov 4, 2025 · Auramine O: A Fluorescent Dye for Microscopy, Research, and Industrial Applications ... Protect from light, moisture, and strong oxidizing agents.
  13. [13]
    Spectrum [Auramine O] - AAT Bioquest
    Auramine O is a fluorescent compound with an excitation peak at 432 nm and an emission peak at 499 nm. Other spectra of interest include: Calcofluor white 2MR, ...Missing: physical melting point density odor stability
  14. [14]
    CN103146219A - Process for preparing high-purity auramine O
    Auramine O is generally made 150-160 ℃ of heating by Michler's keton and ammonium chloride and zinc chloride, or carries out condensation by DMA and ...Missing: ketone | Show results with:ketone<|control11|><|separator|>
  15. [15]
    AURAMINE AND AURAMINE PRODUCTION - NCBI - NIH
    It was reported that a 98% pure auramine contains salts, water and Michler's ketone, an hydrolysis product (Kirsch et al., 1978). Production of auramine took ...
  16. [16]
    Statistics of the early synthetic dye industry | npj Heritage Science
    Mar 20, 2021 · More than 1200 synthetic organic colourants were introduced. Some achieved commercial success, while others were rarely used for reasons such as high cost, low ...
  17. [17]
    US3132178A - Process for preparing auramine using urea
    Auramine also is known to result from reacting Michlers ketone with urea, and zinc chloride used in place of ammonium chloride. ... auramine O free base title, ...
  18. [18]
    Glossary | Biological Stain Commission
    Jun 20, 2023 · Auramine O ... The Biological Stain Commission tests and certifies batches that meet its criteria for identification, dye content and staining ...
  19. [19]
    Auramine - an overview | ScienceDirect Topics
    Mycobacteria can be stained with auramine O, which is very useful for detecting mycobacteria directly in specimens.
  20. [20]
    Use of Light-Emitting Diode Fluorescence Microscopy to Detect Acid ...
    Auramine O is excited by blue light (wavelength, 450–480 nm) and emits in the green-yellow range (wavelength, 500–600 nm). The royal blue LED light source used ...
  21. [21]
    https://www.thelancet.com/journals/lanmic/article/PIIS2666-5247(25)00161-2/fulltext
  22. [22]
    Evaluation of Auramine O staining and conventional PCR for leprosy ...
    Sep 4, 2018 · Auramine O (AO) staining is a fluorescence-based method widely used to detect mycobacterial species such as M. tuberculosis and M. leprae [18,19] ...
  23. [23]
    https://scholarlycommons.henryford.com/hfhmedjournal/vol10/iss2/2
  24. [24]
    https://dawnscientific.com/product/auramine-o/
  25. [25]
    https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/239/477/856533pis.pdf
  26. [26]
    Basic yellow 2,Auramine O,Basic yellow O ,for paper,ink ... - MIT-IVY
    Mainly used in the dyeing of hemp, paper, leather, straw woven products, rayon, etc., also used in the printing and dyeing of cotton fabrics. Its color ...
  27. [27]
    How Toxic is Auramine O in Food ? - SEN Pharma
    Feb 24, 2025 · This compound is strictly limited to industrial applications such as dyeing textiles, paper, wood, and paints. In medical laboratories, it is ...
  28. [28]
    Auramine dyes induce toxic effects to aquatic organisms from ...
    Aug 28, 2020 · The dyes Auramine and Auramine O are used in several industrial products, despite the scarce information regarding their ecotoxicity.
  29. [29]
    Auramine - an overview | ScienceDirect Topics
    The modified Ziehl-Nielsen or auramine phenol stain is used in the diagnostics of cryptosporidium cysts. The former stains acid-resistant cysts red and the ...<|control11|><|separator|>
  30. [30]
    The RELATION BETWEEN CHEMICAL CONSTITUTION AND ... - jstor
    brought about a considerable increase in antiseptic properties and Auramine О. (CH3)2N^. /-C=' ^=N(CH3)2CT nh2 was extremely active both in the case of the.
  31. [31]
    Clinical flow cytometric reticulocyte analysis - PubMed
    The flow cytometric (FCM) analysis of reticulocytes in a clinical laboratory can be accomplished using acridine orange (AO), thiazole orange (TO), auramine O ...
  32. [32]
    Urinary sediment analyzed by flow cytometry - PubMed
    Auramine O, a dye for DNA and RNA, was used for the staining of the sediment. Urine (5 ml or more) was processed by the instrument for sediment analysis.
  33. [33]
    https://www.chemimpex.com/products/00478
  34. [34]
    Food Additives & Ingredients Banned in Europe - RegASK
    Jun 20, 2022 · Banned ingredients include potassium bromate, azodicarbonamide, brominated vegetable oil, olestra, titanium dioxide, Sudan dyes, Auramine O, ...Missing: carcinogenicity spices<|control11|><|separator|>
  35. [35]
    Auramine O in foods and spices determined by an UPLC-MS/MS ...
    Auramine O (AO) is a banned food additive. This study found 27% of food and spice samples were contaminated with AO.Missing: carcinogenicity EU US
  36. [36]
    Illegal Dyes Testing | Sudan Dyes - Eurofins India
    Feb 5, 2025 · Health Risks: Linked to carcinogenic and mutagenic effects. Foods Affected: Sweets, beverages, and colourful snacks. Auramine O (Yellow Dye).
  37. [37]
    EU survey on herbs and spices finds fraud, dyes, extraneous ...
    Dec 1, 2021 · A survey on herb and spice authenticity in Europe has found potential adulteration, illegal dyes and allergens.
  38. [38]
    [PDF] Safety Data Sheet AURAMINE O, CERTIFIED C.I. 41000
    Appearance: Yellow-green powder. Odor: odorless. Vapor Pressure: not available. Odor threshold: not available. Vapor Density: not available. pH: 6.5 at 10g/l aq ...
  39. [39]
    https://www.sigmaaldrich.com/IN/en/sds/aldrich/861030
  40. [40]
    DNA-damaging activity in vivo and bacterial mutagenicity of sixteen ...
    The compounds were examined for their capability to induce alkaline DNA fragmentation in rat liver after treatment in vivo, for their mutagenicity in the ...
  41. [41]
    Solvent Yellow 34 | C17H21N3 | CID 10298 - PubChem - NIH
    Auramine O free base is a member of the class of imines that is benzophenone imine carrying two dimethylamino substituents at positions 4 and 4'.
  42. [42]
    [PDF] SAFETY DATA SHEET - TCI Chemicals
    Nov 13, 2024 · Toxicity to algae/aquatic plants. : EC50 (Selenastrum capricornutum (green algae)): 0.093 mg/l. Exposure time: 72 h. M-Factor (Acute aquatic ...
  43. [43]
    None
    ### Summary of Auramine O SDS (Sections 2, 7, 8, 13, 15)
  44. [44]
    Import Alert 45-02 - accessdata.fda.gov
    This revision to Import Alert #45-02 incorporates import alerts previously issued for illegal or undeclared food color additives.Multiple Countries · Armenia · Australia
  45. [45]
    Substances restricted under REACH - ECHA - European Union
    The list of substances restricted under REACH will be available in our new chemicals database, ECHA CHEM, since 16 September 2025.