Commercial Varieties of Nuclear Fast Red; Their Behaviour in Staining After Autoradiography

Dye marketed as nuclear fast red by various British firms (mixtures of anthraquinones by B. D. H. and G. T. Gurr, a quinone-imine dye by E. Gurr and Kodak) were investigated as nuclear stains particularly in autoradiography where the uptake of haematoxylin hindered visualization of the grains; in this case the uptake of S³⁵ by the epiphyseal plate of the tibiae of mice. A 0.1% solution of the anthraquinone dye CI 60760 (Nuclear fast red; G. T. Gurr) in saturated potassium aluminium sulphate gave a good nuclear stain and was used in the staining sequence: Alcian blue—nuclear fast red—tartrazine. These dyes barely coloured the emulsions used (both stripping film and gel-form emulsion) so that visualization of the silver grains and histological detail were good. The strains were applied after development and other processing of the emulsion was complete.     

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.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

New fluorescence reactions in DNA cytochemistry. 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes

Metal-dye complexes are widely applied in light microscopic techniques for chromatin staining (e.g., hematoxylin and carmine), but fluorescent complexes between phosphate-binding cations and suitable ligands have been little used. Preformed and postformed Al complexes with different anionic dyes induced strong and selective fluorescence reactions in nuclei from chicken blood smears, frozen sections, paraffin-embedded sections and Epon-embedded sections of mouse and rat tissues, mitotic chromosomes, meiotic chromosomes and kinetoplasts of Trypanosoma cruzi epimastigotes. The DNA-dependent fluorescence of these structures showed a very low fading rate. The emission colors were related to the ligand. The most suitable compounds for forming fluorescent Al chelates were 8-hydroxyquinoline, morin, nuclear fast red and purpurin. Staining with diluted carmine solutions and InCl3 mordanting, followed by 8-hydroxyquinoline, also induced chromatin fluorescence. After treating isolated mouse chromosomes with the preformed complex Al-nuclear fast red, x-ray microanalysis indicated a P:Al:dye binding ratio of about 40:15:1. The selectivity, stability and easy formation of these fluorescent Al complexes are obvious advantages for their use as new cytochemical probes in cytologic studies.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

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.     

News from the Biological Stain Commission

Abstract

The origins of repeated hematoxylin shortages are outlined. Lack of integration in the hematoxylin trade exacerbates the problems inherent in using a natural product. Separate corporations are engaged in tree growth and harvesting, dye extraction, processing of extracts to yield hematoxylin, and formulation and sale of hematoxylin staining solutions to the end users in biomedical laboratories. Hematoxylin has many uses in biological staining and no single dye can replace it for all applications. Probably, the most satisfactory substitutes for aluminum-hematoxylin (hemalum) are the ferric complexes of celestine blue (CI 51050; mordant blue 14) and eriochrome cyanine R (CI 43820; mordant blue 3, also known as chromoxane cyanine R and solochrome cyanine R). The iron-celestine blue complex is a cationic dye that binds to nucleic acids and other polyanions, such as those of cartilage matrix and mast cell granules. Complexes of iron with eriochrome cyanine R are anionic and give selective nuclear staining similar to that obtained with acidic hemalum solutions. Iron complexes of gallein (CI 45445; mordant violet 25), a hydroxyxanthene dye, can replace iron-hematoxylin in formulations for staining nuclei, myelin, and protozoa.     

Schiff-Type Reagents in Cytochemistry. 2. Detection of Primary Amine Dye Impurities In Pyronin B and Pyronin Y(G)

Many batches of pyronin B (C.I. 45010), pyronin Y or G (C.I. 45005), and acridine red (C.I. 45000) produce positive Feulgen or PAS reactions when their 0.25% solutions are saturated with SO₂ and used on acid-hydrolyzed or periodate-oxidized tissue sections. These dyes behave as Schiff-type reagents and stain aldehyde-containing structures orange, brown, pink, red, or violet, depending on the particular batch used. The most frequent contaminants are violet and are nonfluorescent. Aldehyde groups are stained by these dyeSO₂ solutions as is shown by using unhydrolyzed controls in the Feulgen reaction and unoxidized controls in the PAS reaction, and by dye solutions lacking SO₂. Other procedures included reactions with aldehyde-blocking reagents, treatment with deoxyribonuclease and diastase, and extraction of nudeic acids with trichloroaeetic acid. The standard Schiff reagent was used in the Same procedures as a basis for comparing results. Since the Schiff-aldehyde reaction requires a dye with a primary amine group and since true pronins contain only secondary or tertiary amines, the positive histochemical results are evidently caused by dye contaminants possessing primary amine groups. The PAS reaction is more sensitive than the Feulgen reaction in detecting dye contaminants. Tissues used were chiefly formalin-fixed mouse intestine and ascites cells. Seventy-five commercial pyronins were studied from 21 different firms. Among 19 batches of pyronin B, 14 were found to contain primary amine dye contaminants. Among 39 batches of pyronin Y(G), 19 contained similar primary amine dye contaminants. Of the 8 batches of acridine red tested, 7 were found to contain primary amine dye contaminants. Nine commercial mixtures of methyl green-pyronin were studied and 4 were found to be likewise contaminated, but these reactive dye contaminants in them are apparently not associated with methyl green. A tabulated summary of the pyronin batches containing primary amine contaminants, and a list of sources and distributors of pyronin dyes are included.     

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.     

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.     

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Abstract

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.     

Aluminon: its limited application as a reagent for the detection of aluminum species

The triammonium salt of aurin tricarboxylic acid, commonly referred to as aluminon, forms a dye that has been used for the colorimetric determination of Al(III) species. We have reviewed the pertinent literature on the reaction of aluminon with respect to the metallic species that form colored aluminon complexes. The effects of experimental variables, such as time, temperature, and pH, upon the color development of the aluminon complex are also presented. Organic and inorganic species, particularly Be(II) and Fe(III), which can affect color formation, are described. The use of aluminon as a histochemical staining agent for the detection of aluminum requires verification by atomic absorption spectrophotometric analysis or other quantitative techniques.     

Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin

The 3,3'-diethylthiacarbocyanine (DiSC(2)(5)) dye is able to aggregate on full matched PNA-DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-beta-cyclodextrin (Succ-beta-CyD) to the solutions containing PNA-DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC(2)(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.     

Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique

Using Allium cepa chromosomes after 5-bromo, 2'-deoxyuridine (BrdU) incorporation, we studied several acid and basic dyes and fluorochromes for their potential as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) technique. All of the dyes and fluorochromes investigated showed a photosensitizing capacity which was slightly lower than 33258 Hoechst in the cases of daunomycin, phloxin, fluorescein, thioflavine T and nuclear fast red, and somewhat higher in the case of eosin Y. Observation and cytophotometric analysis of differentially Giemsa-stained sister chromatids when eosin Y was used as the photosensitizing agent revealed the unsubstituted chromatid to be reddish violet in colour (absorption maximum, 550 nm), while the BrdU-substituted chromatid was blue or pale violet blue (absorption maximum, 580 nm). These results indicate that eosin Y is a useful photosensitizing dye which could be used as a substitute for 33258 Hoechst in the FPG staining technique.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

The flocculation performance of Tamarindus mucilage in relation to removal of vat and direct dyes

A food grade natural mucilage, extracted from the seeds of Tamarindus indica pods, is used as a flocculant for removal of solubilised vat (golden yellow) and direct dye (direct fast scarlet) in aqueous solutions. The maximum removal obtained was 60% for golden yellow after 2 h and was 25% for direct fast scarlet after 1 h. The optimum mucilage dose was 10 mg/l and 15 mg/l for golden yellow and direct fast scarlet, respectively. The pH values also seem to affect the percent removal of both the dyes significantly. In case of vat dye, the pH value of the test samples affected the percent removal significantly. The change was highly significant between neutral and alkaline pH. In case of direct dye, there was no significant change in percent removal at pH 7 and pH 4 whereas a significant change in percent removal was observed between pH 7 and pH 9.2. The plausible mucilage-dye interaction and flocculation mechanism has been discussed. This new flocculant works better in the case of vat dye removal compared with the direct dye.     

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.     

Analysis of the mortality experience amongst U.S. nuclear power industry workers after chronic low-dose exposure to ionizing radiation

Workers employed in 15 utilities that generate nuclear power in the United States have been followed for up to 18 years between 1979 and 1997. Their cumulative dose from whole body ionizing radiation has been determined from the dose records maintained by the facilities themselves and the REIRS and REMS systems maintained by the Nuclear Regulatory Commission and the Department of Energy, respectively. Mortality in the cohort from a number of causes has been analyzed with respect to individual radiation doses. The cohort displays a very substantial healthy worker effect, i.e. considerably lower cancer and noncancer mortality than the general population. Based on 26 and 368 deaths, respectively, positive though statistically nonsignificant associations were seen for mortality from leukemia (excluding chronic lymphocytic leukemia) and all solid cancers combined, with excess relative risks per sievert of 5.67 [95% confidence interval (CI) -2.56, 30.4] and 0.506 (95% CI -2.01, 4.64), respectively. These estimates are very similar to those from the atomic bomb survivors study, though the wide confidence intervals are also consistent with lower or higher risk estimates. A strong positive and statistically significant association between radiation dose and deaths from arteriosclerotic heart disease including coronary heart disease was also observed in the cohort, with an ERR of 8.78 (95% CI 2.10, 20.0). While associations with heart disease have been reported in some other occupational studies, the magnitude of the present association is not consistent with them and therefore needs cautious interpretation and merits further attention. At present, the relatively small number of deaths and the young age of the cohort (mean age at end of follow-up is 45 years) limit the power of the study, but further follow-up and the inclusion of the present data in an ongoing IARC combined analysis of nuclear workers from 15 countries will have greater power for testing the main hypotheses of interest.