Current issues of interest to vendors and users of dyes and biological stains.

Jan 10, 2015

Certified dyes [Updated October 2022]

The following additions and changes have been made since 2002, the year of publication of the 10th edition of Conn’s Biological Stains and of a detailed account of tests then used in the Commission’s assay laboratory (Penney et al. 2002). The following notes (alphabetically by names of dyes) refer to new tests, revised standards, and substances added to the list of stains for which certification is available. Companies that sell dyes for use as biological or microscopical stains should keep in touch with the Biological Stain Commission.


Horobin RW, Kiernan JA (2002) Conn’s Biological Stains, 10th ed. Oxford, UK: BIOS Scientific Publishers.

Penney DP, Powers JM, Frank M, Willis C, Churukian C (2002) Analysis and testing of biological stains —The Biological Stain Commission Procedures. Biotechnic & Histochemistry 77(5-6): 237-275.


Alcian blue 8G (CI 74240) and other alcian blue dyes. The production of alcian blue 8G (as formulated for CI 74240, Ingrain blue 1) was discontinued many years ago (Scott 1973, 1996). Dyes currently sold as alcian blue (with or without the 8G suffix and the Colour Index number or name) are copper phthalocyanines with a variety of solubilizing cationic side-chains. Some dyes sold as “alcian blue 8GX” are certainly not the dye once sold with that name. An ongoing collaboration of the BSC with the Department of Colour and Polymer Science, University of Leeds, UK, is investigating the chemistry of several phthalocyanine dyes sold as alcian blue. Preliminary results (Ali et al. 2022) indicate that at least four different solubilizing side-chains occur in the various products.

An additional criterion for certification (formation of an acid-insoluble pigment on addition of alkali) was introduced in 2008, and the acceptable range of the absorption maximum was extended because samples with absorption maxima outside the previously specified range (Penney et al 2002) passed other tests and performed well in the required staining methods (Lyon & Kiernan 2008). The currently acceptable range of the visible absorption maximum for alcian blue is 605-634 nm. If an alkali-induced precipitate redissolves on acidification, the sample is identified as an “alcian blue variant” rather than “alcian blue 8G or equivalent”. Both types of alcian blue must stain glycosaminoglycans and acid glycoproteins, but not nucleic acids, from a solution at pH 2.5. The BSC also tests for stability (non-precipitation) of an aqueous solution of the dye that also contains 0.5 M magnesium chloride at pH 5.7. Such stability is necessary for a batch of the dye to be used in Scott’s critical electrolyte concentration (CEC) techniques for staining glycosaminoglycans.

A complete description of the BSC’s current tests and requirements for certification of alcian blue dyes was published in 2021 (Fagan et al. 2020; Click here for the paper). The document includes explanations of the types and names of alcian blue dyes, and of the recent changes in the BSC’s tests and standards.

Each certificate issued by the BSC for an alcian blue batch will state the assayed dye content of the powder; this is a useful guide to companies and individuals making staining solutions, because the alcian blues  used in published techniques usually contained less than half as much colorant as some currently marketed batches. The BSC’s certificate also explains the staining methods for which each certified batch of alcian blue has been found to be suitable.


Ali, S., Dapson, R.W., Horobin, R.W., Kiernan, J.A. & Kazlauciunas, A. 2022. At least four distinct blue cationic phthalocyanine dyes sold as alcian blue raises the question: what is alcian blue? Biotechnic & Histochemistry 97(1): 11-20.

Fagan, C., Dapson, R. W., Horobin, R. W. & Kiernan, J. A. 2020. Revised tests and standards for certification of alcian blue dyes by the Biological Stain Commission. Biotechnic & Histochemistry 95(5): 333-340.

Lyon HO, Kiernan JA (2008) News from the Biological Stain Commission. Biotechnic & Histochemistry 83(3-4):201-203.

Penney DP, Powers JM, Frank M, Willis C, Churukian C (2002) Analysis and testing of biological stains—The Biological Stain Commission Procedures. Biotechnic & Histochemistry 77(5-6): 237-275.

Scott JE (1973) Alcian dyes: I.C.I. cease manufacture and release details of composition. Histochemie 37: 379 380.

Scott JE (1996) Alcian blue now you see it, now you don’t. Eur. J. Oral Sci. 104: 2 9.


Basic fuchsine and pararosaniline.  High quality pararosaniline (CI 42500, Basic red 9) has been available for many years. Until now, the BSC’s requirement for certification of pararosaniline or basic fuchsine has been a dye content of at least 88%, determined by titration with titanous chloride as prescribed by Peterson et al (1934).

Basic fuchsine traditionally is a mixture containing 2, 3 or 4 of the related aminotriarylmethane dyes pararosaniline, rosaniline (CI 42510), new fuchsine (CI 42520, Basic violet 2) and magenta II
(which has no CI number). Most samples of basic fuchsine submitted to the BSC in recent years contain only pararosaniline. They have been Certified for the required biological applications of basic fuchsine even though they probably would have met the more stringent testing required for samples submitted as pararosaniline.

Samples recently submitted to the BSC as basic fuchsine and found to be mixtures of triphenylmethane dyes have assayed below the previously required 88% but have performed in a satisfactory manner as ingredients of Schiff’s reagent and the endo culture medium for coliform bacilli, and in stains for acid-fast bacteria. Basic fuchsine composed of mixed dyes is a less expensive product than pararosaniline and is suitable for the same biological and medical applications, with the sole exception of preparing Gomori’s or similar aldehyde-fuchsine stains (Mowry and Emmel 1977). To acomodate these batches of basic fuchsine mixtures, the BSC has reduced the minimum dye content for dyes submitted as basic fuchsine from 88% to 80%. These batches will also be evaluated for component percentages, so that batches more heavily composed of pararosaniline and rosaniline are acceptably identified versus unacceptable ones consisting of mostly new fuchsine. For dyes submitted as pararosaniline, the standard remains at 88%. Biological tests for these dyes remain as described by Penney et al. (2002).

Inexpensive basic fuchsine containing mixed triphenylmethane dyes must be sufficiently free of brown and yellow (acridine) impurities to make a clean Schiff reagent (colorless after shaking with no more than 0.5 grams of activated charcoal per gram of the original dye). This test has been a requirement for certification of basic fuchsine (including pararosaniline) for many years (Lillie 1977; Penney et al., 2002).

The reduced assay requirement for basic fuchsine may make BSC-certified batches of the dye mixture available for clinical diagnostic purposes in places where pararosaniline cannot be afforded.

A complete description of the BSC’s tests and requirements for certification of basic fuchsine and pararosaniline is in preparation and will be submitted to Biotechnic & Histochemistry for independent peer review and eventual publication.

Pararosaniline and new fuchsine are the only members of the basic fuchsine group commercially available as single dyes. New fuchsine is the preferred amine for making a diazonium salt widely used in immunohistochemistry to localize secondary antibodies labelled with alkaline phosphatase. The BSC does not currently offer testing of new fuchsine. Is there a demand for
certified new fuchsine? Please let us know.


Lillie RD (1977) Conn’s Biological Stains, 9th ed. Baltimore: Williams & Wilkins.

Mowry RW, Emmel VM (1977) The production of aldehyde fuchsin depends on the pararosaniline (C.I. No. 42500) content of basic fuchsins which is sometimes negligible and is sometimes mislabeled. Journal of Histochemistry and Cytochemistry 25: 239.

Penney DP, Powers JM, Frank M, Willis C, Churukian C (2002) Analysis and testing of biological stains – the Biological Stain Commission procedures. Biotechnic & Histochemistry 77: 237-275.

Peterson AR, Conn HJ, Melin CG (1934) Methods for the standardization of biological stains. Part IV. The triphenylmethane derivatives. Stain Technology 9: 41-48.


Carmine (CI 75470).  This is used as a food dye, with the name “carmine” applied to carminic acid, carmine or sometimes aminocarminic acid. Only true carmine is certified by the Biological Stain Commission. A new spectrophotometric test (Dapson 2005) distinguishes the three dyes and also provides an assay. For a review of carmine and related dyes, see Dapson (2007).


Dapson RD (2005) A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid. Biotechnic & Histochemistry 80(5-6): 201-205).

Dapson RD (2007) The history, chemistry and modes of action of carmine and related dyes. Biotechnic & Histochemistry 82(4-5): 173-187.


Eriochrome cyanine R (CI 43820, Mordant blue 3) is also called chromoxane cyanine R and solochrome cyanine R, in older books and papers. It forms red and blue complexes with ferric salts, which have been used as biological stains for more than 60 years. Solutions (iron-ECR) are stable for years, and they can be used in methods for blue staining of nuclear chromatin. Techniques and results are closely similar to alum-hematein methods, even though iron-ECR stains  basic nucleoprotein rather than DNA of chromatin.

The aqueous iron-eriochrome cyanine R complex is also a used as a regressive stain for myelin, providing (in 30 minutes at room temperature) the same result as Kluver & Barrera’s (1953) method, which takes 18 hours at 55-60C in an alcoholic solution of luxol fast blue MBS (CI 74180, Solvent blue 38).

The BSC now offers certification of eriochrome cyanine R. The tests and standards have been published (Fagan & Kiernan 2020). This dye is much less expensive than hematoxylin and has been suggested as a preferred alternative (Stefanovic et al., 2015).


Fagan C, Kiernan JA (2020) Certification procedures used by the Biological Stain Commission for eriochrome cyanine R (C.I. 43820, Mordant blue 3). Biotechnic & Histochemistry 95(5): 396-402.

Kluver H, Barrera E (1953) A method for the combined staining of cells and fibers in the central nervous system. Journal of Neuropathology and Experimental Neurology 12: 400-403.

Stefanovic D, Stefanovic M, Lalosovic D (2015) Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin? Biotechnic & Histochemistry 90: 461-469.


Nuclear fast red (CI 60760)  has been added to the list of dyes for which the Biological Stain Commission offers testing and certification (Frank et al., 2007). The criteria for certification were published in 2007. This dye is a component of a red nuclear counterstain for histochemical methods that provide blue end-products in cytoplasm (iron, mucus, heparin etc) and in extracellular materials such as cartilage matrix. Some batches of another (and very different) red dye may be sold as nuclear fast red. Caveat emptor, especially if emptor (buyer) is a company that also is a vendor (seller) of staining solutions. A useful alternative red nuclear stain is Mayer’s brazalum, which is made in the same way as Mayer’s hemalum but with brazilin instead of hematoxylin (Lillie, 1977; Kiernan, 2015).


Frank M, Dapson RD, Wickersham TW, Kiernan JA. (2007) Certification procedures for nuclear fast red (Kernechtrot), CI 60760. Biotechnic & Histochemistry 82(1): 35-39.

Lillie RD (1977) H. J. Conn’s Biological Stains. 9th ed. Williams & Wilkins, Baltimore, p.468.

Kiernan JA (2015) Histological and Histochemical Methods: Theory and Practice. 5th ed. Scion, Banbury, UK, pp.145-146,148


Orcein (CI Natural red 28) is a mixture of more than 10 dyes used for staining elastin and hepatitis B inclusions. In 2017 the BSC’s Laboratory and Certification Committee agreed to extend the acceptable range of λmax to 573-584 nm, because some batches were received that performed well in the staining tests but had absorption maxima outside the previously required range of 575-582 nm (Penney et al., 2002).

Orcein is named in the Colour Index as a “natural” dye because raw materials for its synthesis were formerly made from lichens. It has, however, been an entirely synthetic product since the 1890s (see Horobin 2002, Beecken et al. 2003).


Beecken H and 8 others (2003) Orcein and litmus. Biotechnic & Histochemistry 78(6): 289-302. [English Translation of their 1961 German paper in Angewandte Chemie 73: 665-675.]

Horobin RW (2002) Oxazines and related dyes. Ch. 12 in Horobin RW & Kiernan JA (eds), Conn’s Biological Stains, 10th ed.  pp. 277-291. Oxford, UK: BIOS Scientific Publishers.

Penney DP, Powers JM, Frank M, Willis C, Churukian C (2002) Analysis and testing of biological stains – the Biological Stain Commission procedures. Biotechnic & Histochemistry 77: 237-275.


Protargol-S.  Protargol was originally a trade-name of the Winthrop Chemical Company for its brand of strong silver protein, an antiseptic made from a silver salt and peptone, and containing about 8% Ag. It is a buff to cocoa-brown powder and must not be confused with mild silver protein, a black crystalline solid made from silver oxide and gelatin or albumin, containing 20% Ag. (The term “mild” indicates a lower concentration of free, as distinct from complexed Ag+ ions.) Black products sold as protargol or silver proteinate are not suitable either for staining axons in nervous tissue by the method of Bodian (1933) or for staining ciliated protozoa. The latter application is considered essential for identification, description and classification of these organisms (Foissner 1991, 2014).

The BSC tests protargol in Bodian’s method and gives the designation protargol-S to batches that pass the test. For several years, protargol-S has not been commercially available, and protozoologists have had to rely on old stocks of the brown powder, which fortunately does not deteriorate with storage. Methods for making strong silver protein in the laboratory, suitable for staining ciliated protozoa, have been described by Pan et al. (2013), Kim & Jung (2017) and Kurilov (2017).

It is possible that brown protargol powder may again become an article of commerce. Vendors of any such products are advised to submit samples for testing and certification by the BSC.


Bodian D (1936) A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat. Rec. 65: 89-97.

Foissner W (1991) Basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa. Eur. J. Protistol. 27: 313-330.

Kim JH, Jung JH (2017) Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol. Animal Cells Syst. 21: 422-418.

Pan X, Bourland WA, Song W (2013) Protargol synthesis: an in-house protocol. Journal of Eukaryotic Microbiology 60: 609-614.

Kurilov AV (2017) Improvement of silver impregnation technique using in situ synthesized protargol. Acta Protozool. 56: 109-118.


Sirius red F3B (CI 35780)  was added in 2011 to the list of dyes for which the Biological Stain Commission offers testing and certification. The criteria for certification are published (Dapson et al.  2011). This dye has been used (as a solution in saturated aqueous picric acid), for more than 40 years, in the best and easiest staining method for collagen fibres (even very thin ones, such as reticulin) in both ordinary light microscopy and with polarization optics (Sweat et al. 1964, Puchtler et al, 1973, Junqueira et al. 1979). Similarly named “sirius red” dyes that are not CI 35780 are unsuitable for this application.


Dapson RW, Fagan C, Kiernan JA, Wickersham TW (2011) Certification procedures for sirius red F3B (CI 35780, Direct red 80). Biotechnic & Histochemistry 86(3): 133-139.

Junqueira LCU, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem. J. 11: 447-455.

Puchtler H, Waldrop FS, Valentine LS (1973) Polarization microscopic studies of connective tissue stained with picro-sirius red FBA. Beitr. Path. 150: 174-187.

Sweat F, Puchtler H, Rosenthal SI (1964) Sirius red F3BA as a stain for connective tissue. Arch. Pathol. 78: 69-72.


Thionine (CI 52000).  The spectrophotometric assay developed for this dye in 1950 was for the chloride (MW 263.7 ), which was the usual form of the dye at that time. Since about 1960, however, thionine has been manufactured as its acetate (MW 287.3). For assaying thionine (Stotz 1950), the absorbance (at maximum) of a 5 mg/l solution in water (in a 1.0 cm cell) was multiplied by 97 to calculate the percentage (w/w) content of thionine chloride in the dye powder. For thionine acetate the correct factor is 106, which is now being used. The dye contents of previously assayed batches of thionine acetate were underestimated by about 7%. Fortunately this error did not result in rejection of any samples, because the minimum dye content for certification is 85% whereas thionine acetate is usually manufactured with dye content close to 99% (Lyon & Kiernan 2008).


Stotz E, Conn HJ, Knapp F, Emery AJ (1950) Spectrophotometric characteristics and assay of biological stains. Stain Technology 25: 57-68.

Lyon HO, Kiernan JA (2008) News from the Biological Stain Commission. Biotechnic & Histochemistry 83(6): 285-288.

Dyes are becoming more expensive!

January 2018. BASF, a major dyestuff manufacturing company, recently announced that it has increased its prices for many pigments and dyes by up to 15% worldwide. The products affected are predominantly azo pigments … phthalocyanines and dyes. The company said price increases were necessary due to significantly higher raw materials costs and the rising cost of environmental regulation, health and safety, and maintainance.” See for more information. [Link updated, April 2021]