The Feulgen Reaction After Hydrolysis at Room Temperature

Among the cytochemical methods for demonstrating desoxyribonucleic acid, the hydrochloric-acid-Schiif reaction has been valuable since Feulgen reported it in 1924. A difficulty in that technic is that the section may come loose from the slide; this is caused by hydrolysis at 60° C. When sections were hydrolyzed by 1, 3, 5 or 6 N HCl at room temperature for 15 minutes, adequate hydrolysis and the strong development of color occurred with 5 N HCl. Similarly successful results were obtained with 5 N nitric acid hydrolysis for 10 minutes. Both procedures appear to be as practical as hydrolysis in 1 N HCl at 60° for 4-6 minutes.     

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.     

The Specificity of the Feulgen Reaction for Thymonucleic Acid

The results of experiments on the specificity of the Feulgen reaction for thymonucleic acid do not substantiate the observations of Carr. The staining is not localized in the nucleus because of the destruction of cytoplasmic constituents following acid hydrolysis or because of the absorbing power of chromatin, since the cytoplasm and nucleolus can still be stained by numerous dyes. The effects of factors such as the acid hydrolysis and sulfurous acid washing baths upon the cytologic distribution of dye were studied on tissues stained with (1) fuchsin-sulfurous-acid (Feulgen) reagent, (2) fuchsin-sulfurous-acid reagent colorized by the addition of formaldehyde, (3) basic fuchsin in one-tenth normal HCl, and (4) basic fuchsin in distilled water. Under comparable conditions, important differences between these stains were found in the effects of preliminary hydrolysis; rapidity of staining and destaining; extractability of dye from tissues by water, alcohol, and sulfurous acid solution; rate of fading from exposure to light; localization of stain in tissues; and differences in hue. After treating tissues with desoxyribonuclease, an enzyme which acts only upon thymonucleic acid, cells do not stain with the Feulgen technic. Following removal of nucleic acid from chromatin by hydrolysis, attempts to demonstrate an absorption of thymonucleic acid upon the residual nuclear protein were unsuccessful.

The evidence for and against the specificity is discussed. In agreement with most other investigators, on the basis of the evidence in the literature as well as these experiments, it is concluded that when properly controlled the Feulgen reaction is relatively specific for thymonucleic acid.     

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.     

Some Evidence for the Specificity of the Feulgen Reaction

Carr has attacked the specificity of the Feulgen reaction on three grounds: that the chromosomes are adsorbents capable of regenerating the color of the Schiff reagent; that selectivity for the nucleus depends on destruction of cytoplasm by acid hydrolysis preceding staining; and that the reaction is not blocked by SO₂ water, as he says it should be if staining occurs by a chemical reaction. The first point was tested by staining chromosomes treated with nuclease. They were Feulgen negative, but their protein basis remained intact. The second point was tested by hydrolyzing fixed tissues, washing off solutes, drying, and comparing weight loss with controls. As differences were negligible, the fixed cytoplasm must not have been made soluble by hydrolysis. Carr's third point was not tested experimentally. It is concluded that these objections to specificity of the Feulgen reaction are not valid.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

Feulgen Cytophotometry of Pine Nuclei; Effects of Fixation, Role of Formaline

The effects of 5 fixatives: FAA, Carnoy's, Craf III, formalin and glutaraldehyde were analyzed for use in quantitative Feulgen cytophotometry of pine embryo tissues. Craf III and glutaraldehyde had serious deficiencies because they depressed the absorption peak, severely interfered with DNA extraction and in the case of glutaraldehyde there was considerable cytoplasmic dye-binding. Neutral 10% formalin gave good tissue fixation but did not permit the degree of enzymatic or acid extraction of DNA as did Carnoy's solution. Haupt's adhesive, with the usual 4% formalin as a hardener, at temperatures of 45-56 C completely prevented the enzymatic extraction of nuclear DNA by DNase and also greatly increased the resistance of the DNA to mineral acid hydrolysis. Denaturation of DNA by formalin appeared to be responsible for these results. Absorption was linearly related to both section thickness and DNA concentration per nucleus.     

Feulgen DNA stainability of bone tumors after demineralization

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen-stained sections from archival tissue was also studied. Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability. In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one-third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.     

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 nucleal reaction

Summary Electrophoretic means of separation revealed the presence of as many as five reaction products in Schiff-apurinic acid reaction at the maximum. They differed not only in their absorption maxima, but also in their ratios of apurinic acid phosphorus to fuchsin moiety. Some considerations on the reaction mechanism to account for the occurrence of these multiple reaction products have been made. The stoichiometry of Schiff-apurinic acid reaction was studied with respect to the main product responsible for the presentation of reaction color. A reaction product consisting of six or eight atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be formed, provided that the reagent of infinite concentration is used. From theoretical view point, a reaction product consisting of four atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be expected with the reagent of infinite concentration, provided that apurinic acid retains essentially the nucleotide sequence of its parent desoxyribonucleic acid except for some modification of the original purin nucleotide groups to react as aldehyde moieties, and provided that the reaction proceeds at a constant rate irrespective of the concentrations of the reagent.     

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.