Studies on the Preparation and Recoloration of Fuchsin Sulfurous Acid

Some of the factors affecting the recoloration of Schiff's Reagent (fuchsin sulfurous acid or FSA) by formaldehyde have been studied spectrophotometrically to determine the optimal conditions for the reaction of this reagent with aldehydes.

Of the various reducing agents utilized in the preparation of the leuco dye from basic fuchsin, sodium sulfite and bisulfite proved to be the most satisfactory for obtaining in the reagent maximal sensitivity to recoloration with minimal quantitative variation of results.

The relative proportions of reducing agent and basic fuchsin present in die leuco dye determine its sensitivity to recoloration. Under the conditions of the present experiments, greatest reagent recoloration was obtained when the leuco dye contained 0.01 mole of sodium bisulfite and 0.001 mole of basic fuchsin per 100 ml., a ratio of 10/1.

The recoloration of a given amount of FSA is related to the amount of aldehyde and the temperature of the reaction.

The present experiments indicate the desirability of standardizing the composition of FSA and the conditions under which it is used, if the results of different investigators are to be readily reproduced or compared.     

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.     

Further Experiments with the Masson Trichrome Modification of Mallory's Connective Tissue Stain

Several dyes, notably ponceau 2R, azofuchsin 3B, nitrazine yellow, and Biebrich scarlet may replace imported “ponceau de xylidin” in the Masson ponceau acid fuchsin mixture. Of these Biebrich scarlet appears to be the best and may be used without acid fuchsin.

A mixture of equal parts of 5% solutions of phosphomolybdic and phosphotungstic acids is much superior to either acid alone and gives adequate mordanting in 1 minute at 22°C.

With the fast green modification, times in plasma and fiber stains can be reduced to 2 minutes each. With anilin blue a 4-minute plasma stain is required. One-minute final differentiation in 1% acetic acid is adequate.

Primary mordanting of formalin material may be accomplished by 5 minutes in saturated aqueous mercuric chloride or 2 minutes in saturated alcoholic picric acid. Three minutes washing in running water is required after these mordants.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

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.     

Basic Fuchsin as an Indicator in Endo's Medium

The writer has made an investigation of various samples of basic fuchsin for use in the Endo medium for differentiating the bacteria of the colon-typhoid group. Various different concentrations of the fuchsin samples have been used in making the media. The conclusions are as follows:

American made fuchsins differ markedly in their alcohol solubility properties. They contain materials which are very readily soluble in 95% alcohol, but which are precipitated by sodium sulphite.

This precipitation may be prevented by increasing the dilution of the fuchsin in alcohol.

In order to secure more dependable results in the use of decolorized basic fuchsin as an indicator in Endo Agar, it is advisable to test the fuchsin in different dilutions in alcohol in order to secure a completely decolorized solution. It is also advisable to carefully test those fuchsins which decolorize only in high dilutions with a known organism in Endo agar before relying on it as a satisfactory indicator for the presence of sewage organisms.     

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.     

A Rapid Silver-on-the-Slide Method for Nervous Tissue

A progressive silver staining method is described, which permits microscopic examination of the sections during the staining process. After formaldehyde fixation, dehydration and embedding in paraffin or celloidin, fine fibers and synaptic endings may be demonstrated. After formaldehyde fixation and mordanting in 3% K₂Cr₂O₇, myelinated fibers and mitochondria are specifically stained.

The unique feature of this method is, that the silver solution (0.5% protargol) is mixed with the reducing solution: 1.6% Rochelle salts, containing traces of Ag NO₃, MgSO₄, and K₂S (U.S.P.). The sections are placed directly into this mixture, which is then warmed to 45-55° C. Sections are removed when progressive staining is completed, washed in water, dehydrated and mounted.

In the fiber stain, nerve fibers and synaptic endings are dark brown or black, and nuclear chromatin is deep brown, against a pale yellow background. When the myelin sheath procedure is followed, the fiber bundles are deep brown, and the intensity of the staining remains the same for specific tracts, aiding in their identification.     

A Consideration of French's Test of Basic Fuchsin for Endo's Medium

In describing a method of testing for the return of color in decolorized fuchsin for use in Endo Medium, French states that variations in hydrogen ion concentration fail to influence the appearance of color in this medium.

Duplications of this test were made using alcoholic and aqueous solutions of fuchsin and both sodium sulfite and sodium bisulfite as decolorizing agents.

In the decolorized alcoholic solutions of fuchsin the color failed to reappear when formalin was added, but a small amount of a weak solution of lactic acid caused the color to return.

Alcoholic solutions of fuchsin failed to decolorize in sodium bisulfite solutions until a few drops of NaOH were added. The color, then, reappeared immediately.

Solutions of peptones to which fuchsin had been added were substituted for the original fuchsin solution. Alcoholic and aqueous solutions of fuchsin were added to equal amounts of a 1% peptone solution. The peptone solutions varied in their hydrogen ion concentration and the results showed that those which were neutral decolorized readily while the more acid solutions were but partially decolorized.

Fuchsin decolorized according to results found in this test, was not satisfactory in the Endo medium, especially in the case of the aqueous solutions of fuchsin.

Experiments which were carried on by other workers and checked with this method all indicated that some acid is necessary to secure the restoration of color.     

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.     

The Masson Trichrome Staining Methods in Routine Laboratory Use

A resume of Masson's trichrome staining methods is given, with detailed directions for carrying out all of his procedures. The results obtained thru their use in a routine laboratory are discussed at length, as well as the fact that they also work very well on tissues fixed in ways other than those he prescribes, and stained with chemicals and dyes other than those he uses. The fact is stressed, however, that the closer one adheres to his precepts, the better will be the results.

The stains described include bis hematozylin-phloxine-saffron, his iron-hematozylin-ponceau-anilin-blue, his variants of this stain (of which the light green stain is excellent), his metanil yellow and his modification of the familiar Van Gieson technic. All these stains are based on familiar laboratory methods, improved and rendered trichrome, so that they present no great obstacles in technic.

Of the methods cited, the writer prefers the “light green” procedure. Sections are prestained in Regaud's iron-hematoxylin, followed by a mixture of ponceau de xylidine and acid fuchsin. This is followed by mordanting in phosphomolybdic acid and the sections are finally stained in light green. The results are very precise and pleasing and afford immediate orientation as the connective tissue is green, the nuclei black or dark purple, the cytoplasm of the cells is in varying tones of red. The method may be used after fixation in almost any good medium; altho the results are not as brilliant as those obtained after one of Masson's prescribed fixations, it is believed that they are even then superior to those following the routine hematoxylin-eosin method.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

Demonstration Of Golgi Zones By Three Technics For Carbohydrates

The carbohydrate of the Golgi apparatus of several organs of rats, rabbits, and frogs was selected as the principal test material for the behavior of three different technics: 1) periodic acid with colored fuchsin; 2) “direct” chromic acid piperazine silver; 3) periodic acid with leucofuchsin.

Parallel sections of organs in which positive reactions were observed, were treated before staining with a series of reagents to characterize them as glycoprotein.

The results obtained by the three technics under any constant set of conditions were essentially identical in all cases. It is concluded that discrepancies that may have been noted up to now are due to several factors, probably the most important being the tissue's physiological status and the influence of fixation. The study shows that HIO₄, -fuchsinl and chromic acid silver methods are, at least empirically, as valid as HIO₄, -leucofuchsin technics.

Considering the differences in the oxidative mechanism of chromic and periodic acids and other data, the possibility of two different chemical pathways leading to the same final result is discussed.

It has been found that colored fuchsin, as well as its leuco form, can be used in the histochemical demonstration of aldehydes after periodic acid treatment (Arzac, 1948). In a later report (Amc, 1950), a series of reactions were obtained with colored fuchsin which differed in several ways from the results of others using Hotchkiss' method. For example, Gersh (1949) reported the presence of probable glycoproteic granules in the Golgi apparatus of rabbit and guinea pig's intestine. Leblond (1950) also found positive Golgi reactions in different cells of male excretory ducts and in other organs of the rat. Such reactions had not been observed with the colored fuchsin technic in any of the two above-mentioned occasions.

Since the latter investigators used different fixatives, which might have caused the discrepancies, the experiment described below was undertaken to study: (a) the influence of fixation on the final re-actions elicited by HI04-fuchsin (colored and leuco-form) and chromic acid piperazine silver methods; (b) the results obtained in the demonstration of Golgi zones of several rat's, rabbit's and frog's organs by these methods.     

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.     

Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

Aldehyde fuchsin staining, direct or after oxidation: problems and remedies, with special reference to human pancreatic B cells, pituitaries, and elastic fibers.

Successful production of aldehyde fuchsin (AF) having the unique properties described by Gomori depends on each of many critical variables. AF made from basic fuchsins which contain mainly rosanilin (C.I. 42510) do not stain properly-fixed pancreatic B cells, pituitary basophils, or elastic fibers in unoxidized sections. AF made from basic fuchsins containing mainly pararosanilin (C.I. 42500) stains these entities strongly. Substances stained by AF without oxidation fall into two classes: 1) nonacidic peptides and proteins, most of which contain half-cystines, and 2) polyanions, particularly when sulfated. Group 2 substances stain rapidly, Group 1 substances stain slowly. Many modifications of aldehyde fuchsin have been described. Modified aldehyde fuchsins (MAFs) differ in the kind of aldehyde and in the amount of aldehyde and hydrochloric acid used in their formulation; they differ also in the temperature and duration of the ripening necessary before they can be used. If microsections are first oxidized by acid permanganate or other oxidant, MAF staining of pancreatic B cells, pituitary basophils and other substances containing cystines is speeded and intensified. Most modified methods prescribe oxidation, but the author's does not. The chemical basis, final result and potential side-reactions of oxidation methods (OXMAF) differ from those of direct methods (DIMAF) such as the author's. DIMAF staining is slower but inherently simpler and less destructive. The time required for optimal staining with DIMAF depends on the potency of the stain, which in turn depends on how the stain was made and its age. Detection of DIMAF--reactive peptides and proteins may be hampered by the strong staining of polyanions. This can be remedied if the polyanions are first stained with Alcian blue (AB) or other durable basic dye of contrasting color resistant to acid ethanol. Experiences with the AB-DIMAF staining of pancreatic B cells, pituitaries and elastic fibers in formalin-fixed human tissues are detailed. Proper control of the variables which affect MAF will insure useful and reliable results either directly or after oxidation. Authors and editors are urged to be more careful hereafter to distinguish the results of DIMAF from those of OXMAF methods. Published reports should always specify the parameters that affect the properties of MAF. In OXMAF methods the steps intervening between oxidation and staining should be spelled out. Such care should help dispel the confusion and uncertainty which cloud the use and reputation of aldehyde fuchsin at present. This unique dye deserves wider and wiser use.     

Acid Fuchsin as a Connective Tissue Stain After Phosphomolybdotungstic Mordanting

Certain acid fuchsias stain connective tissue deep red after phosphomolybdotungstic mordanting in a modified Masson procedure, others are entirely unsatisfactory for mis purpose. Spectrophotometric examination gives no reliable criteria for separation of acid fuchsins satisfactory for this purpose from unsatisfactory ones. Sulphonation of basic fuchsin with 3.5 to 4 parts of 25-30% fuming H₂SO₄ to 1 part of dye gives a satisfactory product at temperatures as low as 65 to 70°C. in 30 minutes, while use of 5 to 7.5 parts of acid at this and at higher and lower temperatures gives unsatisfactory products. Satisfactory products may be produced with 15% fuming H₂SO₄ in similar quantities, and even with concentrated H₂SO₄, but some unconverted basic fuchsin remains with both and, with the latter, lower quantities give unsatisfactory products. Brief chemical studies indicate that oversulphonation may occur in the manufacture of acid fuchsin and that this is just as deleterious as undersulphonation.     

The Oxidation Products of Methylene Blue

The mechanism of the oxidation of methylene blue varies with the conditions. The formation of trimethyl thionin (azure B) and of asymmetrical dimethyl thionolin (azure A) is followed under alkaline conditions by that of dimethyl thionin (methylene violet) and under acid conditions by that of monomethyl thionin (named by authors azure C).

Simple and practical methods are given for the preparation of azure A and azure C. The latter product, which has not been obtained from methylene blue hitherto, has valuable staining properties as a nuclear and bacterial stain in tissue and may also be employed satisfactorily as a substitute for azure A in the MacNeal tetrachrome formula as a blood stain or substitute for the Giemsa stain.

Azure B has no particular merit in staining.

Azure C proves to be a very valuable stain. A procedure is given for its use with eosin Y and orange II as counterstains, by which it is possible to demonstrate bacteria in tissue and at the same time the cytological elements of the tissue.     

Demonstration of Axial Elements of Sex Vesicles of Spermatocytes Using Coomassie Brilliant Blue

The axial element of sex chromosomes in the sex vesicle of rat and mouse spermatocytes has been visualized under the light microscope by the dye Coomassie brilliant blue (CBB). After staining in the CBB solution for 3-10 minutes, the axial elements appeared as darkly stained threads in the sex vesicles, whereas in controls stained with Giemsa or carbol fuchsin, the sex vesicles were usually uniformly stained. The axial elements are best seen when chromosome preparations were made by the flame drying technique. In rat spermatocytes the staining quality could be further improved by a brief treatment with trypsin solution (0.025%).

The CBB staining procedure is simple and easily controllable. The results suggest that the CBB stained material is protein in nature and is more resistant to trypsin digestion than other nuclear proteins.