Autoradiographic thymidine-3H activity after successive steps of the Feulgen reaction.

Autoradiographs were prepared of sections of the ovary of Dytiscus marginalis labelled with thymidine-3H after each successive step of the Feulgen reaction and after treatment with each separate component of the Schiff's reagent. Results of grain counts over ovarian nurse cells showed that losses of thymidine-3H activity occur not only during hydrolysis but also during the successive steps of the Feulgen reaction. It is suggested that the latter decrease in radioactivity may depend on the extraction of fragments of apurinic acid from the sections. An emulsion desensitizing effect has also been observed in sections stained with basic fuchsin alone; this effect appears, however, to be strongly counteracted by the metabisulphite present in the Schiff's reagent.     

Feulgen nucleal reaction. I. Quantitative extraction of fuchsin from Feulgen-stained nucleoprotein

A new extraction method for the quantitative determination of the fuchsin contained in a Feulgen-stained nucleoprotein sample has been introduced. The method is based on the following facts: (1) Treatment of a Feulgen-stained nucleoprotein sample with hot acid or alkali brings about a splitting of the linkage between fuchsin-SO(2) and the hydrolyzed nucleic acid moiety of nucleoprotein through aldehyde groups. (2) It also effectuates the formation of fuchsin from the liberated fuchsin-SO(2). (3) The fuchsin is made colorless by the treatment, but is restored to its original pink colored state when the pH of the acidic or alkaline medium is adjusted to 4.6. (4) The fuchsin, either pink colored or decolorized by alkali, can be extracted from an aqueous phase by amyl alcohol. A linear relationship was found to exist between the amount of fuchsin extracted by the FEM from a Feulgen-stained nucleoprotein sample and its DNA content. This relationship holds over a wide range of DNA concentration. From experiments utilizing this method, knowledge may be gained about the mechanism of the Feulgen reaction in situ which can lead to an improvement of the reaction in the field of cytochemistry.     

FEULGEN NUCLEAL REACTION : I. QUANTITATIVE EXTRACTION OF FUCHSIN FROM FEULGEN-STAINED NUCLEOPROTEIN

A new extraction method for the quantitative determination of the fuchsin contained in a Feulgen-stained nucleoprotein sample has been introduced. The method is based on the following facts: (1) Treatment of a Feulgen-stained nucleoprotein sample with hot acid or alkali brings about a splitting of the linkage between fuchsin-SO₂ and the hydrolyzed nucleic acid moiety of nucleoprotein through aldehyde groups. (2) It also effectuates the formation of fuchsin from the liberated fuchsin-SO₂. (3) The fuchsin is made colorless by the treatment, but is restored to its original pink colored state when the pH of the acidic or alkaline medium is adjusted to 4.6. (4) The fuchsin, either pink colored or decolorized by alkali, can be extracted from an aqueous phase by amyl alcohol. A linear relationship was found to exist between the amount of fuchsin extracted by the FEM from a Feulgen-stained nucleoprotein sample and its DNA content. This relationship holds over a wide range of DNA concentration. From experiments utilizing this method, knowledge may be gained about the mechanism of the Feulgen reaction in situ which can lead to an improvement of the reaction in the field of cytochemistry.     

Additional Schiff-Type Reagents for Use in Cytochemistry

Twenty-four new Schiff-type reagents were discovered in a survey of 140 different dyes. These dyes include acid fuchsin, acridine yellow, acriflavine hydrochloride, azure C., Bismarck brown R, Bismarck brown Y, celestine blue B, chrysoidine 3R, chrysoidine Y extra, cresyl violet, crystal violet, gentian violet, methylene blue, neutral violet, phenosafranin, phosphine GN, proflavine, toluidine blue O, and toluylene blue. Positive results obtained with crystal violet and a few samples of methylene blue are considered due to impurities. Various chemical extractions, aldehyde blocking reagents, and enzymatic treatments were used to verify the aldehyde specificity of the above dye-SO₂, reagents as well as azure A, brilliant cresyl blue, neutral red, safranin O, and thionin which have been mentioned by other workers. These reagents were tested in the Feulgen reaction for DNA and the PAS reaction for polysaccharides. Absorption curves were obtained from individual nuclei stained for DNA. The absorption peaks ranged from 450 mμ, to 630 mμ. depending on the dye studied. The Feulgen reaction could be followed by the PAS reaction or vice versa in mouse intestine using reactive dyes of complementary colors. The evidence indicates that a potential Schiff-type reagent must have at least one free NH₂ group on the dye molecule.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

Feulgen-positive staining of the cell nuclei in fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae

The Feulgen reaction and the staining of preparations with two DNA-specific fluorochromes, Hoechst 33258 and 4',6-diamidino-2-fenilindol (DAPI), were used to study the preservation of DNA in the fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae, Paramyrtacicarpus plurilocularis and Paramyrtaciphyllum agapovii collected in Yakutia (Siberia, Russia). It was shown that chromatin structures of the fossilized plants form stable red-purple complexes with the Schiff's fuchsin sulphuric acid reagent in situ . This coloration is specific for DNA, in particular, for the deoxyribose residues. It means that the cell nuclei of these 53–55 Myr old plants preserve a part of the deoxyribose backbone of DNA molecules. On the other hand, there was no, or only a very weak, staining of the cell nucleus with fluorochromes DAPI or Hoechst 33258, which specifically bind to the double-stranded DNA and do not bind to either the single-stranded DNA or RNA molecules. The stainability of fossil plant cell preparations with alcian blue shows that there are also polysaccharides containing carboxyl groups in the cell walls of fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 315–321.     

Quantitative cytochemical determination of desoxyribonucleic acid with the Feulgen nucleal reaction

The possibility of using the Feulgen nucleal reaction for a quantitative cytochemical estimation of desoxyribonucleic acid (DNA) was investigated. The intensity of the reaction in nuclei was determined by absorption measurements with the microscope. The accuracy of such measurements was tested by comparison with measurements on the same material with a Beckman spectrophotometer. The values obtained with the microscope agreed within a few per cent with those obtained with the Beckman spectrophotometer. Furthermore, the errors introduced by uneven distribution of absorbing material, by variations in the numerical aperture of the system, and by variation in the area used on the phototube were investigated empirically. The following variables were studied with regard to their effect on the intensity of the Feulgen reaction: type of fixation, time of hydrolysis after acetic acid-alcohol and formalin fixation, time of staining in leucobasic fuchsin, method of preparation of leucobasic fuchsin. The intensity of the Feulgen reaction in liver and erythrocyte nuclei of various vertebrates, fixed in acetic acid-alcohol, was then compared with the DNA content of these nuclei as determined by chemical analysis on a known number of nuclei. The intensity of the reaction was found to be proportional to the DNA content of the nuclei, if nuclei of similar structure and DNA concentration were compared. In nuclei of different structure and DNA concentration (i.e. liver and erythrocyte nuclei), fixed in acetic acid-alcohol, the intensity of the Feulgen reaction was, however, not proportional to the DNA content. This difficulty was overcome by isolating nuclei in sucrose and by fixing them in formalin. Uniform distribution of DNA and therefore uniform coloring after the Feulgen reaction were thus obtained. In such nuclei with uniform distribution of absorbing material the Feulgen reaction was found to be proportional to the DNA content of nuclei, even if they differed greatly in their DNA concentration. The Feulgen nucleal reaction is not quantitative in an absolute sense. For absolute determinations nuclei of known DNA content must be treated together with the unknown material to serve as standard. From these data it therefore appears possible to determine cytochemically relative amounts of DNA in cellular structures by measuring their absorption after treatment with the Feulgen nucleal reaction.     

Effectiveness Of Sch1ff Variants In The Periodic-Schiff and Feulgen Nucleal Technics

The presence of additional S02 in Schiff reagents above that required for bleaching of the basic fuchsin has no significant effect on the behavior of such reagents in performing the periodic-Schiff or Feulgen nucleal reactions. Schiff reagents made from aqueous solutions containing less than 0.125% basic fuchsin will not demonstrate in these reactions all the tissue elements stained by stronger solutions. Schiff reagents made from aqueous solutions containing more than 0.5% basic fuchsin will in time precipitate considerable amounts of leucofuchsin. Fresh activated charcoal discriminates much more sharply between the yellow contaminant which occurs in basic fuchsin when the fuchsin has been converted to the leuco state than it does when the fuchsin is in the colored condition. Precipitation of leucofuchsin occurs at pH 3 or slightly above in Schiff reagents of generally useful concentration, but at lower levels down to the pH of 10 N HC1, hydrogen ion concentration does not influence their histochemical effectiveness.     

Staining properties of aldehyde fuchsin analogs.

This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.     

The Feulgen reaction after glutaraldehyde fixation.

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH4 in 1% NaH2PO4 for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCl for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

Double Staining for Comparative Measurements in Squash Preparations

Comparative measurements of nuclei or chromosomes following different treatments are seldom made on squash preparations, since variations which arise during preparation of the slides may easily mask genuine treatment differences. This drawback may be overcome by making use of dyes which, when substituted for basic fuchsin in Schiff's reagent, will give a Feulgen-type reaction with chromatin. By selecting dyes of contrasting colours, it is possible to intermingle cells from different treatments in the same squash preparation, and to perform comparative measurements on adjacent cells.

Suitable dyes which contrast well with basic fuchsin are toluidine blue, or azure A (which stain chromatin blue) and chrysoidin yellow (which stains chromatin yellow). These dyes are made up and used in the same manner as ordinary Feulgen reagent.

Samples of cells from the two treatments to be compared are fixed, washed and hydrolysed in 1 N HCl at 60 C. One sample is stained in regular Feulgen reagent, the other in the contrast dye, then both are macerated and thoroughly mixed on the same slide in a single drop of 45% acetic acid. A coverslip is added, and the preparation flattened to the required amount and made permanent after dry-ice removal of the cover. This technique may also be utilised for comparative grain counts in autoradiography, provided that the contrast dye does not cause chemical fogging of the film.     

Studies on the Quantitative Nature of the Feulgen Stain

Bacterial suspensions were stained with Schiff's reagent according to the procedure suggested in essence by Dondero et al. (1954). Cell suspensions, Schiff's reagent, supernatant fluids and stained cells were analyzed by a micro-Kjeldahl procedure in an effort to quantitate the Feulgen reaction. The concentration of the bacterial suspension, type of fixative, time of hydrolysis and pH of cells and dye were varied and the effects analyzed quantitatively. While the cells were often stained deeply as determined by visual observation, the quantity of dye nitrogen in the cells was not large enough to be measured with the procedure employed. Significant quantitative results were obtained consistently only when the pH of the Schiff's reagent was raised. Feulgen reactions with solutions of formaldehyde and with solutions of DNA were also analyzed quantitatively after removing the colored compounds with charcoal. The analyses indicated that the DNA solution and the formaldehyde solution reacted differently with the dye.     

The Feulgen Reaction after Glutaraldehyde Fixation

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH₄ in 1% NaH₂PO₄ for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCI for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

The Feulgen reaction 75 years on

 The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ. Accepted: 13 January 1999     

Feulgen Reaction for Thymonucleic Acid

The importance of thymonucleic acid in tissues is discussed briefly. The technic of the Feulgen reaction which has been employed in photometric histochemical observations in tumors is described. The evidence for the specificity of the Feulgen reaction is reviewed and additional experimental observations are reported. The staining of tissues by the Feulgen reaction is compared with that of hematoxylin, basic fuchsia, and fuchsin-sulfurous-acid reagent in which the color had been developed by the addition of formaldehyde. The stains were compared with respect to (1) the selective staining of the cytologic components of the tissues, (2) the staining of tissues following varying intervals of acid hydrolysis and (3) the photometric determination of the fading of the stained tissue by a carbon arc light. The photometric apparatus employed is suitable for the study of many problems on the staining of tissues. Staining by the Feulgen reaction is different from that of both the basic fuchsin from which the fuchsin-sulfurous-acid was prepared and from that of the product of the fuchsin-sulfurous-acid which had reacted with an aldehyde. Under carefully controlled conditions, the Feulgen technic is a relatively specific histochemical reaction for thymonucleic acid.     

Combined Schiff Procedures

The usefulness of the PAS reaction in histological investigation is frequently enhanced when it can be combined with other procedures. This is frequently difficult to accomplish due to adverse interaction of the more numerous reagents necessary for combined techniques. Details are given for methods of combining PAS staining with iron hematoxylin, direct silver, and aldehyde fuchsin and for the use of the Feulgen reaction to provide nuclear staining for Sudan black preparations.     

Plain Water as a Rinsing Agent Preferable to Sulfurous Acid After the Feulgen Nucleal Reaction

After staining for the Feulgen nucleal reaction with Schiff's reagent, slides were immediately submerged in running distilled or tap water and washed for 30 sec or longer. Rapid and complete removal of residual Schiff's reagent from the stained tissue will give preparations which show all details characterizing the nucleal reaction, and which are more durable in storage than those processed with the customary washing in a solution of SO₂. Care must be taken to insure that all parts of the slides are thoroughly washed and that, on the surface of the sections, no spilled adhesive or other interfering coating retards the washing. Standardization of the procedure for quantitative DNA determination may be facilitated by this modification.     

On the history of basic fuchsin and aldehyde-schiff reactions from 1862 to 1935

Summary The nature of products formed by aldehydes and Schiff's reagent, whether they are sulfonic or sulfinic acid compounds, has been the subject of much discussion. It seems therefore timely to review early studies of aldehyde-Schiff reactions, including the history of pararosanilin and related dyes. Dyes of the basic fuchsin group have been studied extensively since 1862, and their triphenylmethane structure was established in 1878. The currently used structural formulas were introduced around the turn of the century. Reactions of basic fuchsin with aldehydes, with and without addition of SO₂, were investigated by Schiff in the 1860's i.e. before the structure of these dyes was known. In 1900 Prud'homme showed that the reaction products of basic fuchsin, sodium bisulfite and formaldehyde are alkylated and sulfonated derivatives of the parent compound; further chemical studies indicated attachment of the sulfonic acid group to the carbon atom of the aldehyde. Prud'homme's findings were repeatedly confirmed during the following decades. Wieland and Scheuing were apparently unaware of these studies and introduced the sulfinic acid theory in 1921; furthermore, they considered substitution at two amino group of Schiff's reagent essential for formation of the colored compound. However, later chemical and spectroscopic studies showed no evidence of-N-sulfinic acids but supported the sulfonic acid theory of Prud'homme.     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.