A New Principle in Polychrome Staining: A System of Automated Staining, Complementary to Hematoxylin and Eosin, and Usable as a Research Tool

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 mμ acrylic sections.     

An improved polychrome staining method for thick epoxy sections.

Improved polychrome staining of 1-1.5 mum epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial cells are stained deep blue, connective tissue pink, and fat cells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

A simplified method for staining mast cells with astra blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

An Improved Polychrome Staining Method for Thick Epoxy Sections

Improved polychrome staining of 1-1.5 μm epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial ells are stained deep blue, connective tissue pink, and fat ells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

Romanowsky Staining with Buffered Solutions, III. Extension of the Method to Romanowsky Stains in General

Solutions at 0.3 g. per 100 cc. of equal parts of glycerin and methyl alcohol of various Wright, Giemsa, Leishman and Balch stains and similar eosinates of thiazene dyes give satisfactory wholesale staining of sections without differentiation when buffered with citric-acid and sodium-phosphate. Prestaining with alum hematoxylin adds to depth, density and permanence of nuclear staining, but decreases clarity. A satisfactory modification of Mayer's acid hemalum is described. The reaction should be pH 4.2 for neutral formalin or Orth fixation, pH 4.6 for acid formalin, pH 5.0 for Zenker formalin and pH 6.5 for ethyl or methyl alcohol or Carney fixation. Toluidine blue phloxhiate is found to be a quite desirable stain and its preparation is described. Clarite and clarite are definitely superior to neutral Canada balsam, and somewhat inferior in regard to fading compared with liquid petrolatum as mounting media for these Romanowsky stains.     

The staining of Brunner's gland and other neutral mucins by carmine, hematoxylin and orcein in alkaline solutions.

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orcein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

Improved nuclear staining with mordant blue 3 as a hematoxylin substitute.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely results very resemble a good hematoxylin and eosin.     

Lacto-Phenol Preparations

More or less permanent mounts of fungi, algae, root tips, epidermis, germinating spores, and other small objects may be made readily by transferring the material to Amann's lacto-phenol containing anilin blue, W. S. or acid fuchsin, used singly or mixed. The addition of 20 to 25% of glacial acetic acid to these mixtures is frequently advantageous; or material may be stained with various dyes—acid fuchsin, anilin blue, W. S. (cotton blue), rose bengal, phloxine, hematoxylin—in aqueous solutions containing 5% of phenol, and then mounted in lacto-phenol, 50% glycerin or phenolglycerin, depending on the dye used. The phenol solutions of acid fuchsin and anilin blue are acidified with acetic acid and those of rose bengal and phloxine are made slightly alkaline with ammonium hydroxide. The addition of ferric chloride to acid fuchsin or acidified hematoxylin may improve staining. Fixation may be preferable but may be omitted, especially with fungi. Formulae for the mounting media and ten staining mixtures are given.     

Mordant Blue 3: a Readily Availablx Substitute for Hematoxylin in the Routine Hematoxylin and Eosin Stain

Mordant blue 3 may be used as a suhstitute for hematoxylin in hematoxylin and eosin stains. The staining solution consists of 0.25 g dye, 40 ml of 10% iron dam, 5 ml of cone H₂SO₄, and 955 ml of dirtilled H₂O. Staining the is 5 minutes, followed by differentiation in acid water or acid alcohol. After blueing, the seaions are counterstained with emin. Results closely resemble the hematoxylin and eosin stain.     

Van Gieson's picrofuchsin. The staining mechanisms for collagen and cytoplasm,and an examination of the dye diffusion rate model of differential staining

The staining mechanism of van Gieson's picrofuchsin was studied by use of simple protein model systems and tissue sections, and by spectrophotometry and dialysis experiments. At the endpoint of the staining reaction (equilibrium) cytoplasm is yellow. Dye dilution experiments demonstrated that the highest affinity in the tissue section — picrofuchsin system is between binding sites in cytoplasmic protein and acid fuchsin. Nevertheless sections that were first stained in acid fuchsin (AcF) and then in picrofuchsin ended up with cytoplasm stained yellow. It was concluded that differences in the dye diffusion rates and differences in the permeability of tissue components cannot be invoked to explain the differential staining result. Model experiments with dissolved proteins demonstrated a positive relationship between protein concentration and uptake of picric acid (PA) from picrofuchsin. From this and experiments with additives (sodium dodecylsulphate, urea etc.) and organic solvents, it is proposed that coagulant interchain cross-linking at the high protein concentration of the cytoplasm masks potential dye-binding sites. This affects high affinity dyes with multiple binding sites more than small dyes, and so puts AcF at a disadvantage compared to PA. Staining of non-collagen proteins is mainly by hydrophobic bonding, involving ionic attractions, apolar bonds, and release of water. This mode of binding is relatively strong, decreases swelling and leads to slow dye exchange. Dye binding to collagen is mostly by hydrogen bonds, but in aqueous dye solvent nonpolar residues and charged residues may also participate. This structure remains relatively open during and after dye-binding, and the bound dye ions are therefore easily exchanged for other dye ions. Address at which the main part of the investigation was carried out     

The TP-levanol fast cyanine 5RN staining procedure for proteins of the myosin-fibrin group. Fibrin staining and other remarks

The tannic acid-phosphomolybdic acid-Levanol (Supranol) Fast Cyanine 5RN (TP-L) procedure for staining muscle cells and blood platelets was used because, with this method, proteins of the myosin-fibrin group should be selectively stained. However, in human blood and blood plasma clots and in vivo thrombi, fibrin was not stained. Blood platelets probably due to their content of contractile proteins were very well stained. Apparent fibrin staining in human autopsy thrombi may be due to the staining of disintegrated platelets and the absorbance of fibrin by stained hemoglobin. Problems encountered using Nuclear Fast Red as the nuclear stain were solved by changing the dye concentration or by using a differentiating agent. Myosin staining by the TP-L method depended on the pH of the tannic-acid solution used. Raising the pH to 7.4-8.0 changed the staining result, and collagen fibers were then stained.     

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.     

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.     

Modified Whipf's Polychrome: A Connective Tissue Stain with Special Application for Demonstrating Leishmania

A polychrome stain procedure was developed to demonstrate amastigotes of the protozoan parasite Leishmania braziliensis as well as cytoplasmic and other tissue components in cutaneous lesions of infected animals. The procedure is as follows: stain nuclei for 10 minutes with an iron hematoxylin containing 0.5% hematoxylin and 0.75% ferric ammonium sulfate dissolved in 1:1 0.6 N H₂SO₄:95% ethanol; rinse 4 minutes in distilled water. Cytoplasmic staining is achieved by exposing tissues for 10 minutes to a solution containing 0.25% Biebrich scarlet, 0.45% orange G, 0.5% phosphomolybdic acid and 0.5% phosphotungstic acid in 1% aqueous acetic acid. These first two solutions are modified from Whipf's polychrome stain. Sections are differentiated for 10 seconds in 50% ethanol, rinsed in water, stained 3 minutes in 0.1% aniline blue WS in saturated aqueous picric acid, rinsed in water and differentiated for 1 minute in absolute ethanol containing 0.05% acetic acid. Mordanting overnight in 6% picric acid in 95% ethanol produced optimal results.

This procedure was applied to sectioned material from experimental animals with various protozoa. Trypanosoma cruzi, Besnoitia Jellisoni, Toxoplasma gondii and especially Leishmania braziliensis were well demonstrated. Combining cytoplasmic dyes and phosphomolybdic-phosphotungstic acids into one solution afforded differential staining of tissues by Biebrich scarlet and orange G; connective tissues were stained by this solution. Substantially improved definition of connective tissues resulted after counterstaining. This procedure differs from the Massou sequence in which connective tissues are first stained by cytoplasmic dyes along with other tissues and then destained prior to specific counter-staining. in comparing dyes structurally related to Biebrich scarlet, it was found that Crocein scarlet MOO, but not Poncenu S, was an acceptable substitute. Sirius supra blue GL and Sirius red FSBA were not useful as replacements for aniline blue WS in this procedure.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

Novel application of aniline blue. Fluorescent staining of glycogen and some protein structures

The present study shows that aniline blue can be used as a fluorescent stain for glycogen. The dye is also helpful in tracing pathological and autolytic changes in lysosomes, mitochondria, erythrocytes and nuclei, and it can also be used for demonstrating bacteria in tissue sections and smears. The techniques used are simple, rapid and inexpensive. Spectrophotometric studies on aniline blue solutions have shown that aniline blue fluorescence was enhanced by the addition of certain proteins, or of glycogen to the dye solution. In case of albumen which has the maximum effect, enhancement is dependent upon the albumen-dye ratio. The mechanism of staining is mainly due to self quenching, but there is also an evidence of the presence of hydrophobic reaction.     

Composition of J.S.B. stain and Factors Affecting its Quality

Several samples of J.S.B. stain (Jaswant Singh and Bhattacharjee, 1944) solution 1 (polychrome methylene blue) were prepared with 3-8 hr for dichromate-acid oxidation and addition of varying quantities of Na₂HPO₄ buffer for pH adjustment. Storage under severe tropical conditions and periodical checks by staining Plasmodium cynomolgi smears revealed that staining solutions oxidized 6-7 hr with a final pH of 7.8 gave optimum results. Some precipitation of azures, due to heat after 5 mo, adversely affected the quality of staining solutions, while cooler storage conditions were most favorable. Spectrophotometric and chromatographic studies indicated that the J.S.B. solution 1 was composed of blue and purple components, corresponding to higher methylene azures with methylene blue and thionin with allied products respectively.     

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.