Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins

The most distinctive property of aldehyde fuchsin is its staining of certain nonionic proteins and peptides in unoxidized cells and tissues. These substances include granules of pancreatic islet B cells, elastic fibers and hepatitis B surface antigen. Aldehyde fuchsin made from two different basic fuchsins, each certified by the Biological Stain Commission and labelled C.I. (Colour Index) No. 42500 (pararosanilin), did not stain pancreatic B cells at all. Stain Commission's records and retesting showed that each of the "faulty" basic fuchsins was not pararosanilin, but rosanilin, whose Colour Index number is 42510. These basic fuchsins were labelled with the wrong Colour Index number when packaged. Additional basic fuchsins were coded by V.M.E. and tested by R.W.M. for their capacity to make satisfactory aldehyde fuchsins. Only certain of these aldehyde fuchsins stained unoxidized pancreatic islet B cells. The same aldehyde fuchsins stained elastic fibers strongly. Each basic fuchsin whose aldehyde fuchsin was judged satisfactory proved to be pararosanilin. Aldehyde fuchsin solutions made from other basic fuchsins stained elastic fibers only weakly and did not stain pancreatic B cells at all in unoxidized sections. Each basic fuchsin whose aldehyde fuchsin was unsatisfactory proved to be rosanilin. It appears that only aldehyde fuchsin made from pararosanilin stains unoxidized pancreatic B cell granules dependably. We found that basic fuchsins from additional lots of Commission-certified pararosanilin and rosanilin were also labelled with incorrect Colour Index numbers when packaged. Steps were taken to prevent recurrences of such mislabelling which has made it difficult until now to correlate differences in the properties of pararosanilin and rosanilin. A table is provided of all basic fuchsins that have been certified by the Biological Stain Commission since 1963 when they began the practice of subdesignating basic fuchsins according to whether they are pararosanilins or nonpararosanilins. The consumer can readily determine from the certification number on the label the correct subdesignation of any Commission-certified basic fuchsin listed here. Until now, mislabelling of some lots of pararosanilin as rosanilin and vice-versa has confused and frustrated the users of basic fuchsins in other applications such as the carbol fuchsin staining of tubercle bacilli and certain cytochemical tests, e.g. esterase and acid phosphatase, that utilize hexazotized pararosanilin as a coupling reagent. Consumers experiencing trouble with any Commission-certified dye should look to the Biological Stain Commission for help. This is an important reason for purchasing, whenever possible, only Biological Stain Commission certified dyes.     

The Biological Stain Commission's Quality Control Laboratory operations and improved traceability of certified stains

Abstract

The Biological Stain Commission (BSC) is a quality control laboratory that certifies biological dyes for staining cells and tissues. Originally, a single lot of a certified dye was sold to histologists. Today, companies frequently change their lot numbers as part of regulatory efforts. When a certified dye undergoes a lot number change, the BSC must re-certify this dye to verify that it is identical to the one certified earlier. The BSC has improved how these lot changes are monitored using a redesigned BSC certification label. Certification labels always have been issued by the BSC and are attached to every bottle of “BSC certified dye” that is sold. The new BSC certification label has added security features and currently bears both the BSC certification number and the manufacturer batch lot number. The result is improved security and traceability of certified dyes.     

Report from the President

Although the original Commission on Standardization of Biological Stains was first organized in 1921, it was not until 1944 that this was incorporated as an independent, nonprofit organization known as the Biological Stain Commission (see Clark 1974). The certification of dyes, as indicated by special labels purchased by manufacturers or vendors for attachment to the dye containers, originated with the parent organization and has continued to this day. The objectives of the Biological Stain Commission (BSC) are 1) to identify and standardize the content and performance of dyes and dye preparations used in staining biological tissues and products, 2) to issue labels of certification to companies that buy these to inform consumers that their certified dyes meet the specifications of the BSC, 3) to carry out and to support investigations on dyes and their performance, 4) to publish scientific data concerning biological stains and their use, and 5) to maintain, through scientific meetings and correspondence, an active “dialogue” among scientific and industrial personnel concerned with biological stains. The present report summarizes Commission activity and some of the changes that have occurred during the past five years.     

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.     

Aldehyde Pararosanilin (True Aldehyde Fuchsin) Stains Purified Insulin, Oxidized or Not, Following Adequate Fixation with Either Formalin or Bouin'S Fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from “basic fuchsin” whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC.

Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content.

We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam® sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.     

Purity of commercial non-certified European samples of Pyronin Y

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.     

Revised procedures for the certification of carmine (C.I. 75470, Natural red 4) as a biological stain

Carmine is one of the original dyes certified by the Biological Stain Commission (BSC). Until now it has lacked both an assay procedure for dye content and a means to positively identify the dye. The methods for testing carmine in the laboratory of the BSC have been revised to include spectrophotometric examination at pH 12.5-12.6 to determine that the dye is carmine (λ_max=530-335 nm). The maximum absorbance of a solution containing 100 mg of dye per liter of water, adjusted to pH 12.5-12.6, which provides a relative measure of dye content, should lie in the range 1.2 to 1.8. If the dye is not carmine, spectrophotometry at pH 1.9-2.1 shows whether it is carminic acid (λ_max=490-500 nm) or 4-aminocarminic acid (λ_max=525-530 nm). The latter two dyes, which are also called carmine when sold as food colorants, have physical properties different from those of true carmine. The functional tests for carmine as a biological stain are Orth's lithium-carmine method for nuclei, Southgate's mucicarmine method for mucus, and Best's carmine method for glycogen.     

A Modification of the Technic of Handling Chick Embryos

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission, in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Methods for the Standardization of Biological Stains: Part IV

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission, in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Improving Biological Dyes and Stains: Quality Testing Versus Standardization

This paper discusses the impact of both standardization and quality testing of dyes and stains in biology and medicine. After a brief review of why standardized dyes and stains are not presently available commercially, two types of testing and ways of improving dye quality are described. National or international organizations could be established to define standardization of dyes and stains. Standardization would be specifically defined as a list of physico-chemical parameters such as elaborated in this paper. Commercial batches of comparable quality may be labeled by the supplier as “standard dye.” a procedure currently performed by the European Council for Clinical and Laboratory Standardization (ECCLS). Also recommended to improve dye quality is commercial dye testing by independent laboratories with subsequent certification for use. This sort of quality control is currently carried out in the United States by the Biological Stain Commission (BSC). The advantages and disadvantages of both techniques and the use of image analysis for the definition of standards are discussed. A combination of both the BSC testing protocols and the ECCLS standards should be established for extended quality control of biological dyes and stains.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid.

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

A method for determining identity and relative purity of carmine,carminic acid and aminocarminic acid

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5–12.6) followed by a qualitative scan of a low pH (1.90–2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

The Purity of Fluorescent Dyes: a Report Delivered to the Scientific Session of the Annual Meeting of the Biological Stain Commission

The Biological Stain Commission occasionally has been requested to certify fluorochromes as biological stains. Although formal certification is unlikely in the near future, the Commission is nevertheless concerned with the quality of these reagents. Commercial samples of fourteen fluorochromes were investigated for the presence of fluorescent organic impurities using reverse phase thin layer chromatography. Our findings suggest that some fluorochrome dyes are pure, but most are impure. Most fluorochromes vary in purity among vendors and among batches sold by single vendors. Impurities may be present at such high concentration that little of the presumed compound is present. Some impurities behave quite differently from the nominal dye. This may either create confusion or it might be useful. In the latter case, however, the impurity may occur only in a single batch. Impurities result from problems related to organic syntheses, separations, and economics. Solving those problems is often expensive, and what is expensive may not be performed. Fortunately, knowledge of synthetic chemistry often permits identification of fluorochromes likely to be impure. Moreover, predictions of likely staining effects of particular impurities can be made if appropriate structure-activity models are available. Possible actions by the Commission aimed at limiting the problems resulting from impurities of fluorescent dyes are noted.     

Staining Problems with Eosin Y: A Note from the Biological Stain Commission

In 1980, eosin Y was the certified dye with which technologists encountered most problems. The specific problem most frequently brought to the attention of the Biological Stain Commission was that solutions of eosin Y formed a precipitate and failed to stain cytoplasm red when used as a counterstain to hematoxylin.     

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

News from the BSC No. 8

Abstract

In the 8th and following issues of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the BSC's International Affairs Committee will present information from a meeting held in Ghent, Belgium on 15–18 June 2009 concerning the progress achieved by the International Standards Organization Committee ISO/TC 212 Clinical Laboratory Testing and in Vitro Diagnostic Test Systems since the last meeting held in Vancouver, Canada in 2008. A note on the meaning and significance of E numbers found on the labels of foodstuffs and beverages sold for human consumption concludes this edition of News from the Biological Stain Commission.     

News from the Biological Stain Commission No. 15

In the 15^th issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission’s International Affairs Committee presents information from the plenary meetings of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on August 22–24, 2012 in Berlin, Germany. An additional discussion of the use of food dyes in India also is included.     

General Considerations: Methods for the Standardization of Biological Stains: Part I

This is the introduction to a series of papers giving the assay methods employed in the testing of dyes submitted to the Commission on Standardization of Biological Stains. These methods are used in determining whether or not to allow the sale of any particular batch of dye as a certified stain. The present paper takes up general considerations, discussing the relative merits of various analytical methods designed both for qualitative and quantitative analysis. The advantages of determining light “absorption ratios” for qualitative purposes, and of titanous chloride reduction for quantitative purposes are pointed out. It is further indicated that in spite of all the refinements that have yet been made in chemical and optical methods of analysis, great weight must be placed on biological testing in determining the quality of any given sample.