An investigation of aldehyde fuchsin staining of unoxidized insulin

Aldehyde fuchsin is a standard stain for the secretion granules of pancreatic B cells. The participation of either insulin or proinsulin in aldehyde fuchsin staining is in dispute. There is some evidence that permanganate oxidized insulin is stained by aldehyde fuchsin. Aldehyde fuchsin staining of unoxidized insulin has not been investigated adequately despite excellent staining results with tissue sections. Unoxidized insulin and proinsulin suspended by electrophoresis in polyacrylamide gels were fixed with Bouin's fluid and placed in aldehyde fuchsin for one hour. Because the unoxidized proteins were not stained by aldehyde fuchsin, it was concluded that neither insulin or proinsulin are responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections. A series of controlled experiments was undertaken to test the effects of fixatives, oxidation and destaining procedures on aldehyde fuchsin staining of insulin, proinsulin and other proteins immobilized in polyacrylamide gels. It was demonstrated that only oxidized proteins were stained by aldehyde fuchsin and that cystine content of the proteins had no apparent relation to aldehyde fuchsin staining. It was concluded that neither insulin nor proinsulin is likely to be responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections.     

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Aldehyde Pararosanilin (True Aldehyde Fuchsin) Stains Purified Insulin, Oxidized or Not, Following Adequate Fixation with Either Formalin or Bouin'S Fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from “basic fuchsin” whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC.

Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content.

We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam® sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

The demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins

The most distinctive property of aldehyde fuchsin is its staining of certain nonionic proteins and peptides in unoxidized cells and tissues. These substances include granules of pancreatic islet B cells, elastic fibers and hepatitis B surface antigen. Aldehyde fuchsin made from two different basic fuchsins, each certified by the Biological Stain Commission and labelled C.I. (Colour Index) No. 42500 (pararosanilin), did not stain pancreatic B cells at all. Stain Commission's records and retesting showed that each of the "faulty" basic fuchsins was not pararosanilin, but rosanilin, whose Colour Index number is 42510. These basic fuchsins were labelled with the wrong Colour Index number when packaged. Additional basic fuchsins were coded by V.M.E. and tested by R.W.M. for their capacity to make satisfactory aldehyde fuchsins. Only certain of these aldehyde fuchsins stained unoxidized pancreatic islet B cells. The same aldehyde fuchsins stained elastic fibers strongly. Each basic fuchsin whose aldehyde fuchsin was judged satisfactory proved to be pararosanilin. Aldehyde fuchsin solutions made from other basic fuchsins stained elastic fibers only weakly and did not stain pancreatic B cells at all in unoxidized sections. Each basic fuchsin whose aldehyde fuchsin was unsatisfactory proved to be rosanilin. It appears that only aldehyde fuchsin made from pararosanilin stains unoxidized pancreatic B cell granules dependably. We found that basic fuchsins from additional lots of Commission-certified pararosanilin and rosanilin were also labelled with incorrect Colour Index numbers when packaged. Steps were taken to prevent recurrences of such mislabelling which has made it difficult until now to correlate differences in the properties of pararosanilin and rosanilin. A table is provided of all basic fuchsins that have been certified by the Biological Stain Commission since 1963 when they began the practice of subdesignating basic fuchsins according to whether they are pararosanilins or nonpararosanilins. The consumer can readily determine from the certification number on the label the correct subdesignation of any Commission-certified basic fuchsin listed here. Until now, mislabelling of some lots of pararosanilin as rosanilin and vice-versa has confused and frustrated the users of basic fuchsins in other applications such as the carbol fuchsin staining of tubercle bacilli and certain cytochemical tests, e.g. esterase and acid phosphatase, that utilize hexazotized pararosanilin as a coupling reagent. Consumers experiencing trouble with any Commission-certified dye should look to the Biological Stain Commission for help. This is an important reason for purchasing, whenever possible, only Biological Stain Commission certified dyes.     

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.     

Effects of fuchsin variants in aldehyde fuchsin staining

Aldehyde fuchsin stains pancreatic B cell granules, hypophyseal basophils, goblet cell mucins, gastric chief cells, hyaline cartilage, and elastica. Neither the chemical structure of aldehyde fuchsin nor its staining mechanism is known. This study was undertaken to clarify the role of the fuchsin component of aldehyde fuchsin in its staining reaction. The major findings of this investigation include: 1) single N-methylation of the fuchsin molecule abolishes staining of unoxidized pancreatic B cells, although it does not prevent reaction of fuchsin with paraldehyde; 2) aldehyde fuchsin is probably a Schiff base condensation product of pararosaniline and acetaldehyde; 3) a Schiff base structure alone cannot account for aldehyde fuchsin staining of unoxidized pancreatic B cells; 4) a fully potent aldehyde fuchsin is possibly a Tris-Schiff base derivative of pararosaniline.     

Report from the President the Biological Stain Commission: Its Goals, Its Past and Its Present Status

This is a brief overview of the goals, evolution, and present status of the Biological Stain Commission. The main function of the Commission is the testing and certification of dye batches intended for biological applications. The testing is supported by charges made for batch testing and by the sale of certification labels affixed to individual dye containers. Submission of dyes for testing is voluntary, depending on the cooperation of the companies who sell them and the consumers who buy them. The supportive role of the University of Rochester School of Medicine and Dentistry—both past and present—is not well known and should be. Increasingly federal regulations affect the production, availability, and cost of dyes. Commission income from the sale of labels has decreased in recent years. Continuation of its work requires changes that will produce more income. Much dye is now sold in solutions instead of dry powders. The value of using Stain Commission certified dyes whenever possible is illustrated by the case of basic fuchsin. Years ago this dye was a mixture. Most basic fuchsin now marketed consists mainly of either pararosanilin (Colour Index No. 42500) or rosanilin (C.I. No. 42510). The Biological Stain Commission discovered that some certified batches of both pararosanilin and rosanilin sold as “basic fuchsin” had incorrect C.I. numbers on the labels. Sometimes that caused failure of the aldehyde fuchsin stain. Unless made with pararosanilin, aldehyde fuchsin does not stain pancreatic islet B-cells, elastic fibers, and hepatitis B surface antigen in unoxidized sections. Mislabelling by packagers may interfere with other applications of pararosanilin and rosanilin. The Commission acted to publicize and correct this problem. Biological Stain Commission publications help educate microscopists and histotechnologists about dyes and their best use. Stain Commission representatives from member scientific societies provide valuable input about changes in the availability and quality of such dyes as hematoxylin and others; they also provide useful feedback to their societies about dye problems. Each new generation of biologists and histotechnologists should be taught the importance of using only Stain Commission certified stains when available. They should be taught also to notify the Stain Commission whenever they experience problems with any certified dye.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.     

The role of paraldehyde in the rapid preparation of aldehyde fuchsin

Preparation of aldehyde fuchsin normally requires ripening for 3 to 5 days. By using a 5-fold excess of paraldehyde a fully potent aldehyde fuchsin can be prepared in 24 hr at room temperature. Aldehyde fuchsin prepared by both normal and accelerated ripening afforded comparable results, including selective staining of unoxidized pancreatic B cells. Dried aldehyde fuchsin prepared form pararosaniline and reconstituted in acid alcohol has spectrophotometric properties different form the ripened strain. Reconstituted aldehyde fuchsin stains unoxidized B cells adequately only if staining time is extended. Excess paraldehyde added to reconstituted aldehyde fuchsin retards decomposition but does not produce a normal stain by spectrophotometric standards. Warming of aldehyde fuchsin solutions to accelerate ripening has been shown to produce deleterious effects and should be avoided.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Comparison of historic Grübler dyes with modern counterparts.

Seventeen Grübler dyes produced in Germany between 1880 and 1939 were examined in this study. These dyes were: fuchsin-bacillus, diamond fuchsin, fuchsin S acid, rubin S, safranin O water soluble, safranin yellowish water soluble, methyl eosin, Sudan III, scarlet R, auramine, orange G, aniline blue, pyronin, carmine, lithium carmine, hematein and aurantia. Spectrophotometry and staining characteristics were used to determine the maximum absorbance and efficacy of each dye in common staining techniques. The spectral curves and staining characteristics of these dyes compared well with modern dyes used as controls. Fuchsin bacillus and diamond fuchsin are synonyms for basic fuchsin. Fuchsin S acid and rubin S are synonyms for acid fuchsin. The scarlet R sample was the same as the Sudan III. The two safranins were the same. The basic fuchsin samples were unsuitable for preparation of Schiff's reagent. Both basic fuchsin and pyronin samples were less concentrated than modern counterparts. It is noteworthy that the dyes worked well after up to 100 years in storage, and this observation indicates that dyes can have a long shelf life when stored in cool, dry, air-tight conditions.     

Aldehyde-fuchsin: historical and chemical considerations.

The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.     

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.     

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.     

Aldehyde-fuchsin: Historical and chemical considerations

Summary The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.     

Standardization of the Feulgen-Schiff technique

Summary 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 m.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.     

A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes.     

The Mechanism of the Gram Reaction I. The Specificity of the Primary Dye

Dyes of all major types were tested for their suitability as the primary dye in the Gram stain. When a counterstain was not used, some dyes of all types were found to differentiate Gram-positive from Gram-negative organisms. When a counterstain was used, these dyes were found to vary greatly in their suitability. Those dyes found to be good substitutes for crystal violet were: Brilliant green, malachite green, basic fuchsin, ethyl violet, Hoffmann's violet, methyl violet B, and Victoria blue R. All are basic triphenylmethane dyes. Acid dyes were generally not suitable. Differences in the reaction of Gram-positive and Gram-negative cells to Gram staining without the use of iodine were observed and discussed but a practical differentiation could not be achieved in this manner. Certain broad aspects of the chemical mechanism of dyes in the gram stain are discussed.     

Influence of some aldehyde blocking agents on staining of depurinized DNA with cationic dyes.

Rat liver, spleen and Walker carcinosarcoma imprints were subjected to depurinizing Feulgen hydrolysis and then treated with blocking agents of aldehyde groups. Such blockators as sodium bisulfite and hydroxylamine which multiplay additionally anionic groups in DNA and intensify the reactions with cationic dyes, ensuring anisotropic staining. Hydrazine lowers the binding of carionic dyes to DNA, instead phenylhydrazine, completely blocks both aldehyde and phosphate groups. When the imprints were treated with 2.4-dinitrophenylhydrazine, aldehyde and phosphate groups of apurinic acid were blocked, and DNA staining by cationic dyes occurred only on account of nitrogroups of the blocking agents which have been used. The staining reaction of cationic dyes after the use of anionogenic blocking agents of aldehyde groups is prospective not only for revealing DNA but also for several other compounds with natural or potential aldo- and ketogroups. However the reaction with phenylhydrazine can serve as a staining without removal of DNA prior to staining as an optional procedure.