Assaying Actual Hematein Content of Commercial Hematoxylins and Hemateins

Hematein-free hematoxylin (HFH) was prepared by a modification of the procedure of Palmer and Lillie (Histochemie, 5: 44-54, 1965). Fifty mg of HFH were dissolved in 5 mg of ethylene glycol and then 45 nil of an aqueous solution of 2.25 gm KAl(SO₄)₂. 12H₂O and 5.445 mg KIO₃ were added. Since this amount of KIO₃ would be sufficient to oxidize 25 mg of HFH to hematein we have termed this half-oxidized hematoxylin (HOH). The peak absorbance (560 nm) of this purple solution remained constant for at least a week. With omission of the KIO₃ the solution was colorless. A curve was constructed by plotting absorbance against concentration of hematein in HOH at various dilutions. For analyses of hematein content of commercial hematoxylins 50 mg of sample and 100 mg of hydroquinone were dissolved in 5 ml of ethylene glycol and then 45 ml of a 5% solution of KAl(SO₄)₂. 12H₂O were added. The addition of the hydroquinone stabilized the absorbance for about 5 min. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. Eleven samples of hematoxylin certified by the Biological Stain Commission had hematein concentrations varying from 0.01 to 0.43%. For analyses of the available hematein content of commercial hemateins, 50 mg of sample were dissolved in 10 ml of ethylene glycol, then 45 ml of water and 45 ml of 5% KAI(SO₄)₂. 12H₂O added. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. In 9 samples of hematein from 4 different sources the active hematein content varied from 19 to 97%.     

Application of current chemical concepts to metal-hematein and-brazilein stains

Summary Current chemical concepts were applied to Weigert's, M. Heidenhain's and Verhoeff's iron hemateins, Mayer's acid hemalum stain and the corresponding brazilein compounds. Fe⁺⁺⁺ bonds tightly to oxygen in preference to nitrogen and is unlikely to react with lysyl and arginyl groups of proteins. Binding of unoxidized hematoxylin by various substrates has long been known to professional dyers and was ascribed to hydrogen bonding. Chemical data on the uptake of phenols support this theory. Molecular models indicate a nonplanar configuration of hematoxylin and brazilin. The traditional quinonoid formula of hematein and brazilein was revised. During chelate formation each of the two groups of the dye shares an electron pair with the metal and contributes a negative charge to the chelate. Consequently, the blue or black 2:1 (dye:metal) complexes are anionic. Olation of such chelates affects the staining properties of iron hematein solutions. The color changes upon oxidation of hematoxylin, reaction of hematein with metals, and during exposure of chelates to acids can be explained by molecular orbital theory.Without differentiation or acid in dye chelate solutions, staining patterns are a function of the metal. Reactions of acidified solutions are determined by the affinities of the dye ligands. Brazilein is much more acid-sensitive than hematein. This difference can be ascribed to the lack of a second free phenolic –OH group in brazilein, i.e. one hydrogen bond is insufficient to anchor the dye to tissues. Since hematein and brazilein are identical in all other respects, their differences in affinity cannot be explained by van der Waals, electrostatic, hydrophobic or other forces.     

Application of current chemical concepts to metal-hematein and -brazilein stains

Current chemical concepts were applied to Weigert's, M. Heidenhain's and Verhoeff's iron hemateins, Mayer's acid hemalum stain and the corresponding brazilein compounds. Fe bonds tightly to oxygen in preference to nitrogen and is unlikely to react with lysyl and arginyl groups of proteins. Binding of unoxidized hematoxylin by various substrates has long been known to professional dyers and was ascribed to hydrogen bonding. Chemical data on the uptake of phenols support this theory. Molecular models indicate a nonplanar configuration of hematoxylin and brazilin. The traditional quinonoid formula of hematein and brazilein was revised. During chelate formation each of the two oxy- groups of the dye shares an electron pair with the metal and contributes a negative charge to the chelate. Consequently, the blue or black 2:1 (dye:metal) complexes are anionic. Olation of such chelates affects the staining properties of iron hematein solutions. The color changes upon oxidation of hematoxylin, reaction of hematein with metals, and during exposure of chelates to acids can be explained by molecular orbital theory. Without differentiation or acid in dye chelate solutions, staining patterns are a function of the metal. Reactions of acidified solutions are determined by the affinities of the dye ligands. Brazilein is much more acid-sensitive than hematein. This difference can be ascribed to the lack of a second free phenolic -OH group in brazilein, i.e. one hydrogen bond is insufficient to anchor the dye to tissues. Since hematein and brazilein are identical in all other respects, their differences in affinity cannot be explained by van der Waals, electrostatic, hydrophobic or other forces.     

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

On the mechanism of sequence iron-hematein stains

Summary As a prerequisite for the histochemical study of sequence iron-hematoxylin stains the iron alum-acidified hematein procedure was developed which does not require differentiation.Histochemical blocking and extraction procedures demonstrated that carboxyl and hydroxyl groups are essential for the binding of cationic iron.The iron alum-Prussian blue reaction colored collagen, reticulum fibers and basement membranes more intensely than muscle fibers. Treatment of tissue sections mordanted in iron alum with the acidified hematein solvent resulted in practically complete removal of iron from all tissue structures. It must therefore be concluded that the selective staining of muscle fibers, terminal bars and related structures with sequence iron-hematein stains is not due to high affinity of iron for these tissue components.Observations by R. and M. Heidenhain on sequence hematoxylin-potassium dichromate and hematoxylin-alum stains and data from modern textile chemistry indicate that the staining patterns obtained with metal-hematein sequence stains are determined by the affinity of the hematein moiety for certain tissue structures.     

A Simple Assay Procedure for Mixtures of Hematoxylin and Hematein

A simple and rapid method for the simultaneous quantitative analysis of mixtures of hematoxylin and hematein uses the molar extinction coefficients of the pure substances calculated by Lalor and Martin (1959). Absorbance measurements of the samples dissolved in methanol are made at wavelengths of 292 nm and 445 nm, the wavelengths of maximum absorption of hematoxylin and hematein respectively. The hematoxylin absorbance at 292 nm is corrected for the presence of hematein.

Using this method it was found that of 12 commercial samples labelled “hematoxylin” all contained at least 90% of the compound. Hematein contents of these samples fell in the range 0.1% to 6.8%. In 9 commercial samples labelled “hematein” the hematein contents fell in the range 1.2% to 90.7%. The hematoxylin contents of these samples fell in the range of 1.0% to 82.7%.

This paper describes also a chromatographic method for the identification of hematein and its oxidation products.     

Progress in the Standardization of Stains Certain Less Commonly Used Stains

The Commission must acknowledge the assistance recently given by two commercial concerns in developing two new products, namely, a pure type of hematein and a special grade of acid fuchsin.     

Progress in the Standardization of Stains

The Commission must acknowledge the assistance recently given by two commercial concerns in developing two new products, namely, a pure type of hematein and a special grade of acid fuchsin.     

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

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.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining.

The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed.     

Concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB)

Summary A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color -D-glucosyl and -D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.This investigation was supported in part by a Grant-in-Aid from the Japanese Education Ministry (1975)     

Spectroscopic and staining studies of the ripening and overripening of aluminum hematoxylins

Summary Spectroscopic study of alum hematoxylins during the course of ripening, optimal status and final deterioration reveals an absorption spectrum with, maxima at about 560 m and at about 430 m. Early in the course of ripening the optical density ratio D560/D430 rises above 3.0 and this ratio is maintained at 3–4 through most of the useful life of the stain batch. In the final stage of deterioration the density at 430 m, rises and that at 560 fails, so that in late deterioration stages D560/D430 may fall as low as 0.5.As ripening progresses the density at 560 m, gradually rises so that when it reaches a value of 0.350 (1 cm cell, dilution 10 mg hematoxylin/1 liter) adequate staining is achieved. This level seems the same whether ripening be done by aeration, oxygen bubbling, sodium iodate or mercuric acetate. As ripening progresses further the D560 value may rise above 0.800 with air or iodate oxidation. Maximum values are usually lower with iodate oxidation. This finding tends to agree with the ancient impression that natural ripening provides the best alum hematoxylins.In agreement with the known variable hematein content (20–100%) of comercial hematoxylins the required doses of sodium iodate to reach initial satisfactory staining performance are variable and considerably lower than the precalculated stoichiometric amount for the complete conversion of fully reduced hematoxylin to hematein. The often quoted figure of 200 mg per gram hematoxylin agrees closely with the amount of potassium iodate which would be needed to convert 1 gm pure crystalline hematoxylin (M.W. 356.32) completely to hematein. It was found that 80 mg NaIO₃ per gram of reduced hematoxylin was adequate and for commercial samples 40–60 mg usually sufficed.The final overoxidation product of hematoxylin appears to be a complex mixture, not readily reduced back to hematein. Since 185.13 mg NaIO₃ is the theoretical amount for complete conversion to hematein of fully reduced hematoxylin, the amount of iodate required to attain maximum optical density at 560 m, is always less than that. The observed amounts range from 60 to 140 mg per gram of hematoxylin, and excesses apparently operate to convert hematein to further oxidation products.

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Zusammenfassung Alaun-Hämatoxylin zeigt bei spektroskopischen Untersuchungen während der Reifung, der besten Färbungszeit und beim endgültigen Zerfall 2 Absorptionsmaxima. Sie liegen etwa bei 560 m und bei 430 m. Das Verhältnis der optischen Dichte D560/D430 steigt im Laufe der Reifung bereits früh auf 3,0 und bleibt während der Brauchbarkeit der Farblösung beständig zwischen 3,0 und 4,0. Im letzten Stadium des Zerfalls steigt die optische Dichte der Farblösung bei dem Maximum von 430 m während die von 560 m sinkt, so daß zu dieser Zeit D560/D430 bis auf 0,5 abfallen kann.Bei der Reifung steigt die optische Dichte langsam an. Wenn ein Wert von 0,350 (1 cm Zelle, 10 mg Hämatoxylin/Liter) erreicht ist, werden die Färbungen zufriedenstellend. Es scheint gleichgültig zu sein, ob die Reifung durch Luft, durch Hindurchblasen von Sauerstoff oder chemisch mittels Natriumjodat oder Quecksilberacetat erfolgt ist. Bei fortschreitender Reifung kann der D560-Wert bei Anwendung von Luft oder Jodat auf über 0,800 steigen. Die Maximalwerte sind bei Jodatoxydation jedoch stets etwas niedriger als bei Luftoxidation. Diese Befunde scheinen mit dem alten Eindruck übereinzustimmen, daß natürliche Reifung das beste Alaun-Hämatoxylin erzeugt.Da bekanntlich der Hämateingehalt der komerziellen Hämatoxiline stark schwankt (zwischen 20 und 100%), ist auch die zur Herstellung einer zufriedenstellenden Farblösung erforderliche Menge an jodsaurem Natrium sehr unterschiedlich. Sie ist beträchtlich niedriger als die im voraus berechnete stöchiometrische Menge für die vollständige Oxydation des voll reduzierten Hämatoxylins zu Hämatein. Die oft genannte Zahl von 200 mg NaJO₃ pro Gramm Hämatoxylin stimmt genau mit der Menge von KJO₃ überein, die benötigt wird, um 1 g kristallines Hämatoxylin (M.W. 356,52) in Hämatein zu verwandeln. Es wurde festgestellt, daß pro Gramm voll reduzierten Hämatoxylins 80 mg NaJ0₃ angemessen sind und daß gewöhnlich für Handelsware 40–60 mg genügen.Ein durch endgültige Überoxydation von Hämatoxylin entstandenes Produkt scheint eine komplexe Mischung zu sein, die nicht leicht wieder zu Hämatein reduziert werden kann. Da 185,13 mg NaJO₃ die theoretische Menge für die vollständige Oxydation von voll reduziertem Hämatoxylin zu Hämatein ist, ist die benötigte Menge von Jodat, um die höchste optische Dichte (D560) zu erreichen, immer etwas geringer als 185,13 mg. Die beobachteten Mengen liegen zwischen 60 und 140 mg pro Gramm Hämatoxylin. Überschüsse scheinen Hämatein in weitere Oxydierungsprodukte zu verwandeln.

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Supported by a Student Fellowship of the Biological Stain Commission

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Supported in part by Research Grant No. C-4816 National Cancer Institute, National Institutes of Health.     

The reaction of unsaturated fats with the acid hematein test

Summary The reaction of saturated and unsaturated oils and fatty acids was examined in vitro with the Baker's acid hematein test. It has been found that oils whose molecules contain fatty acid components of two or more double bonds give a positive reaction with the acid hematein technique. The intensity of the reaction runs parallel with the number of double bonds.     

Notes on Technic: Amyl Acetate as a Clearing agent for Refractory Plant Materials

Thin-layer chromatographic systems are described for the analysis of various preformed metal complex dyes (aluminon-chromium(III), carminic acid-aluminum, carminic acid-chromium(III), carminic acid-iron(III), oelestine blue-chromium(III), gallamine blue-chromium(III), gallocyanin-chromium(III), hematein-aluminum, hematein-chromium(III), purpurin-aluminum) and their parent dyes. Certain of there dyes have also been analysed by agar-gel electrophoresis or gel-filtration chromatography. The merits of the three analytical methods are discussed.     

Thin layer chromatography of certain preformed metal complex dyes used in biological staining.

Thin-layer chromatographic systems are described for the analysis of various performed metal complex dyes (aluminon-chromium (III), carminic acid-aluminum, carminic acid-chromium (III), carminic acid-iron (III), celestine blue-chromium (III), gallamine blue-chromium (III), gallocyanin-chromium (III), hematein-aluminum, hematein-chromium (III), purpurin-aluminum) and their parent dyes. Certain of these dyes have also been analysed by agar-gel electrophoresis or gel-filtration chromatography. The merits of the three analytical methods are discussed.     

Thin Layer Chromatography of Certain Preformed Metal Complex Dyes Used in Biological Staining

Thin-layer chromatographic systems are described for the analysis of various preformed metal complex dyes (aluminon-chromium(III), carminic acid-aluminum, carminic acid-chromium(III), carminic acid-iron(III), oelestine blue-chromium(III), gallamine blue-chromium(III), gallocyanin-chromium(III), hematein-aluminum, hematein-chromium(III), purpurin-aluminum) and their parent dyes. Certain of there dyes have also been analysed by agar-gel electrophoresis or gel-filtration chromatography. The merits of the three analytical methods are discussed.     

Effects of additives on alum hematoxylin staining solutions.

All additives tested (ethyl alcohol, glycerine, chloral hydrate, ethylene and propylene glycol, and citric, malonic and maleic acids) in varying degrees limited the conversion of hematein to insoluble compounds. Peak absorbances increased slightly in hematoxylin solutions containing citric, malonic and maleic acids, but decreased with other additives, and in controls. After four months storage the absorbance in all solutions increased about 50%, acidity increased and staining effectiveness increased.     

A Simplified Acid Hematein Test for Phospholipids

The acid hematein test of Baker was simplified by the following method: Tissues were fixed in either formal-calcium or formal-calcium-cadmium, frozen-sectioned in a cryostat, cbromatized with potassium dichromate, washed in water, stained in acid hematein solution, washed in water again, differentiated in borax-ferricyanide, dehydrated in alcohols, cleared in xylene, and finally mounted in balsam. The process requires only 2 days instead of the 6 days necessary for the original test of Baker. The technique stains phospholipid in mitochondria and in other cellular elements, and is recommended especially for mitochondria