Summary
The purity of six European non-certified samples of Pyronin Y was compared with that of two American samples certified by the Biological Stain Commission. The methods used were spectrophotometry and a Methyl Green-Pyronin staining test (both as applied by the Biological Stain Commission), thin layer chromatography, mass spectrometry, determination of pH, and content of some electrolytes. It was found that none of the European batches of Pyronin Y passed the complete test as prescribed by the Biological Stain Commission. Their dye content was uniformly low (between 5 and 19%). Furthermore, thin layer chromatography and mass spectrometry revealed that two of the dye samples contained no Pyronin Y or only traces.
It is concluded that assessment of an unknown sample of a dye labelled Pyronin Y should be initiated with thin layer chromatography. The pH and content of electrolytes in an aqueous solution of the dye should also be determined in order to obtain reproducible staining results. Finally, the value of the work performed by the Biological Stain Commission is underlined, although more sophisticated methods are necessary for testing the purity of dyestuffs.
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References
Clemens, H. J. &Toepfer, K. (1968a) Physikalisch-chemische Eigenschaften von kommerziellen Thiazinfarbstoffen. 1. Bestimmung des Farbstoffgehaltes und der Löslichkeit in Wasser und Äthylalkohol.Acta histochem. 31, 117–25.
Clemens, H. J. &Toepfer, K. (1968b) Physikalisch-chemische Eigenschaften von kommerziellen Thiazinfarbstoffen. 2. Qualitative und quantitative Untersuchungen über die Verunreinungen der Farbstoffe und das ‘Umlösen’ zum Erhalt eines höheren Reinheitsgrades.Acta histochem. 31, 126–34.
Darrow, M. A., Knapp, F., Stotz, E. &Conn, H. J. (1949) A comparison of American stains with recent German products.Stain Technol. 24, 1–8.
Dobres, H. L. &Moats, W. A. (1968) Qualitative analysis by thin-layer chromatography of some common dyes used in biological staining.Stain Technol. 43, 27–30.
Editorial (1926) Constitution of Commission on Standardization of Biological Stains.Stain Technol. 1, 4–10.
Egerton, G. S., Gleadle, J. M. &Uffindell, N. D. (1967) Purification of organic dyes, particularly of the anthraquinone series.J. Chromatogr. 26, 62–71.
Emery, A. J., Hazen, F. K. &Stotz, E. (1950) Spectrophotometric characteristics and assay of biological stains. III. The xanthenes.Stain Technol. 25, 201–8.
Holmes, W. C. &Peterson, A. R. (1930) The analysis of Neutral Red and of the Pyronins.Stain Technol. 5, 91–6.
Horobin, R. W. (1969) The impurities of biological dyes: their detection, removal, occurrence and histological significance — a review.Histochem. J. 1, 231–65.
Horobin, R. W. (1970) Dextrin and salt as impurities of histological dyes. Estimation and removal.Histochemie 22, 39–44.
Horobin, R. (1973) Effects on staining of the presence of various kinds of dye impurities.Acta histochem. Suppl.13, 269–80.
Horobin, R. W. &Goldstein, D. J. (1974) The influence of salt on the staining of tissue sections with basic dyes: an investigation into the general applicability of the critical electrolyte concentration theory.Histochem. J. 6, 599–609.
Horobin, R. W. &Murgatroyd, L. B. (1967) A comparison of rapid electrophoretic and chromatographic methods for the investigation of common histological dyes.Histochemie 11, 141–51.
Horobin, R. W. &Murgatroyd, L. B.(1969) The identification and purification of Pyronin and Rhodamine dyes.Stain Technol. 44, 297–302.
Kasten, F. H. (1962) Comparison of Pyronin dyes obtained from various commercial sources. 1. History.Stain Technol. 37, 265–75.
Kasten, F. H., Burton, V. &Lofland, S. (1962) Schiff-type reagents in cytochemistry. 2. Detection of primary amine dye impurities in Pyronon B and Pyronin Y (G).Stain Technol. 37, 277–91.
Kurnick, N. B. (1952) Histological staining with Methyl-Green-Pyronin.Stain Technol. 27, 233–42.
Lillie, R. D. (1977) Conn's Biological Stains, 9th edn. pp. 566–567, 590. Baltimore: Williams & Wilkins.
Marshall, P. N. (1976) Thin-layer chromatography of some cationic dyes commonly used in histology.J. Chromatogr. 129, 277–85.
Mowry, R. W. (1980) Report from the president. The Biological Stain Commission: its goals, its past and its present status.Stain Technol. 55, 1–7.
Mowry, R. W. &Kasten, F. H. (1975) Conference Report: The importance of dye purification and standardization in biomedicine.Stain Technol. 50, 65–81.
Rottier, P. B. (1953) Spectrophotometry of dyes: 1. Methyl Green. 2. Pyronin.Stain Technol. 28, 265–73.
Scott, J. E.(1967) On the mechanism of the Methyl Green-Pyronin stain for nucleic acids.Histochemie 9, 30–47.
Scott, J. E. (1972) Lies, damned lies- and biological stains.Histochem. J. 4, 387–9.
Singer, M. (1952) Factors which control the staining of tissue sections with acid and basic dyes. InInternational Review of Cytology (edited byBourne, G. H. andDanielli, J. F.), Vol. 1, pp. 211–255. New York: Academic Press.
Stahl, E. (1967)Dünnschicht-Chromatographie. Ein Laboratoriumshandbuch. 2nd edn, pp. 588–590. Berlin, Heidelberg, New York: Springer-Verlag.
Stotz, E., Conn, H. J., Knapp, F. &Emery, A. J. (1950) Spectrophotometric characteristics and assay of biological stains.Stain Technol. 25, 57–68.
Telford, C. B. (1974) Nucleic acids and Methyl Green-Pyronin: Fixation, Pyronin purification and theory of staining.Can. J. Med. Technol. 36, 1–24.
Toepfer, K. H. (1973) Reinheitsgrad und Verunreinigung von Handelsfarbstoffen. Störender Einfluss auf histologische und histochemische Färbeverfahren.Acta histochem. Suppl.13, 261–8.
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Lyon, H., Andersen, A.P., Andersen, I. et al. Purity of commercial non-certified European samples of Pyronin Y. Histochem J 14, 621–630 (1982). https://doi.org/10.1007/BF01011894
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DOI: https://doi.org/10.1007/BF01011894