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.     

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.     

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.     

革兰氏染色三步法与质量控制

革兰氏染色(Gram stain),是细菌学中一个经常使用和十分重要的方法,自从1884年微生物学家Gram氏发明著名的革兰氏染色法以后,100多年来虽然经过后来学者的几次改进,但都仍然沿用着Gram氏原来的四步法,基本原理也没有改变。最近Allen氏对Ziehl-Neelsen抗酸菌染色法的改进,是一个良好的启示,使我们开始了革兰氏染色三步法的研究并取得了成功。现将我们建立的革兰氏染色三步法与质量控制报告如下。 1 材料和方法 1.1 结晶紫染色液 甲液:结晶紫2g;95％乙醇20ml。 乙液:草酸铵0.8g;蒸馏水80ml。 甲乙二液先分别溶解,然后混合在一起,过滤除去残渣后装入滴瓶中备用。     

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.     

Gross Differential Staining of the Hypophysis

The lobes of the hypophysis of many mammals can be differentiated by staining either the entire gland for 5–8 hr, or gross 1–2 mm slices of the gland for 3–5 min in a mixture of acid fuchsin (National Aniline, certified Andrade indicator) and methylene blue (Fisher certified reagent). For best results, the staining mixture contains 0.8–1.0% acid fuchsin and 0.2–0.4% methylene blue, both made up in 10% formalin adjusted to pH 6.70–6.75 with phosphate buffer. The anterior lobe stains a light blue, the intermediate lobe a dark blue, the posterior lobe a light pink and the capsule a dark pink.     

Staining Bacteria and Yeasts with Acid Dyes

Various acid dyes prove satisfactory for the routine staining of bacteria. Those used are acid fuchsin, anilin blue w. s., fast acid blue R, fast green FCF, light green, orseilline BB, erythrosin, phloxine and rose bengal. Acid fuchsin, fast green, anilin blue, and orseilline are especially recommended. Phenolic solutions of the dyes, acidified with acetic acid, with the addition of ferric chloride to those containing acid fuchsin, anilin blue, fast green or light green, are used. Procedures are given in detail for staining or demonstrating vegetative cells, resting and germinating spores, capsules, sheaths and glycogen in bacteria; germinating and conjugating spores of yeast; and for counterstaining after acid fast or Gram staining. The principal advantages of using acid dyes are better differentiation, and less tendency for slime amd debris to take the dye.     

Nucleolar Definition in Hemalum and Eosin Staining

This investigation was designed to clarify why routine hemalum and eosin staining so frequently demonstrates nucleoli in a distinctive purple to red shade. Participation of eosin in the effect suggests that nucleolar basic protein is being stained, and this fact has been confirmed with bromphenol blue. Dye affinities of the nucleolus have been further studied by successively substituting basic and acid fuchsin for the pyronin in the standard Unna-Pappenheim mixture. At pH 4.8 it appears that the nucleolus has both acid and basic groups freely available for staining.     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

The Effect of Preliminary Treatment (Fixing Fluids) on Staining Properties of the Tissues

As was reported in a previous paper,¹ staining properties depend on the chemical composition of the tissues and on the strength of the dyes themselves. Applying mixtures of basic and acid dye on tissues (methylene blue, eosin Y) at different pH-values, it is possible to find differences in the isoelectric points of the nuclei and cytoplasm of different tissues. For example, the nucleus of polymorphonuclear cells of the blood consists of the most acid protein, with an isoelectric point around pH 2.5, while the nucleus of lymphatic tissues has an isoelectric point of about pH 4.0, and that of connective tissue about pH 3.4.

With a knowledge of the above, a constant method of staining at various pH-values was used to study the effect of different fixing fluids on the staining properties of the tissues. In this way it was found that many fixing fluids gave very stable compounds with tissue proteins, and that they almost permanently change the chemical composition (i.e. the staining properties of the tissues). In some instances, these changes can be easily explained from the regular chemical standpoint. For example, formalin forms inert compounds with amino groups of the amino acids of proteins and in this way it makes the tissue proteins more acid, i.e. it moves the isoelectric point of the proteins toward a lower pH-value. The same is true in the case of the polivalent acids. The bivalent heavy metals such as mercury, on the contrary, it is assumed, combine with carboxyi groups of amino acids and in this way move the isoelectric point of the proteins toward a higher pH.     

Method of counterstaining preparations for immunofluorescent studies of the pituitary]

To elucidate nonfluorescent structural elements of the hypophyseal parenchyma for immunofluorescent investigations, properties of some dyes most commonly applied for hypophysis staining have been studied. Such dyes as paraldehide-fuchsin, light green, orange G, chromotrop 2R, hematoxylin, eosin, fuchsin, azocarmin possess their own intensive luminescence and block immunofluorescence completely. Some other dyes (trypan blue, bromthymol blue, aniline blue, malachite green, methyl green) though not blocking immunofluorescence, they do not reveal hypophyseal cellular elements distinctly enough. Good results have been obtained with 0.3% water solution of toluidine blue, 0.5% solution of methylene light blue, methylene blue, as well as with Gram--Weigert's staining and with gallocyanin after Einarson. For special staining of corticotropocytes, the authors recommend 0.1% solution of bromphenol blue in barate buffer, pH 8.2.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Improvements in dehydration and cement line staining for methacrylate embedded human bone biopsies

Undemineralized methacrylate embedded bone biopsies and other bone specimens can be processed much more rapidly by application of acidified 2,2-dimethoxypropane (DMP) dehydration, which requires two hours, than by traditional graded ethanol dehydration, which requires at least four days. This shortened processing time is valuable when biopsy results are urgently needed to detect osteomalacia or to determine bone features prior to possible parathyroidectomy. We have processed over 200 bone specimens with DMP and have compared DMP dehydration to graded ethanol dehydration in 11 biopsies in which two plugs were available from the same patient. DMP dehydration does not compromise the following: tetracycline retention, Goldner's stain, acid phosphatase localization or histochemical identification of aluminum. Cement lines, which provide a record of past remodelling, are useful in clinical interpretation of bone biopsies. We have adapted two stains, toluidine blue and methylene blue/basic fuchsin, for improved cement line identification. Five-micrometer sections individually demineralized in acetate buffer prior to cement line staining show best results with toluidine blue at pH 5.5 and with methylene blue/basic fuchsin at pH 2.5-3.5.     

Acid alcoholic brilliant cresyl blue stain for gastric and other mucins

Summary Some but not all samples of brilliant cresyl blue (6-methyl-7-dimethylamino-2-phenoxazin chloride) under C. I. No. 51010 in Conn's Biological Stains when dissolved at 1% level in 50–70% alcohol containing 1% concentrated (12 N) hydrochloric acid, stain (in 30 min) a wide variety of human and laboratory animal mucins blue black on an almost unstained background. The mucoprotein of the gastric surface epithelium and of the peptic gland neck cells of several species reacts strongly. A 16 hr 60° C methylation in 0.1 M methyl-sulfuric acid in methanol is required to block the staining of these gastric and some intestinal mucins, while 1–2 hr intervals suffice to prevent the staining of mast cells, cartilage and metachromatic sulfomucins generally. Saponification (1% KOH/70% alcohol, 20min) does not restore staining in either location group, indicating that sulfate mucins are probably reacting in both.Most other basic dyes fail to stain mucins from acid alcohol solutions: azure A, toluidine blue, resorcin blue, orcein, resorufin, azoresorufin brown, azolitmin, lacmoid, gallocyanin, Nile blue, methylene green, pararosanilin, crystal violet, Victoria blue R. Some staining occurred with one of three lots of Victoria blue B, with two lots of Victoria blue 4 R and with one lot each of Bernthsen's methylene violet, elastin violet PR and elastin purple PP.The stain may be preceded by the Feulgen reaction to give red nuclei, or followed by a brief collagen stain in an alcoholic acid fuchsin (0.05–0.1%), picric acid (1.5%) solution.Presented before the Symposium of the Histochemische Gesellschaft in Hamburg, 28. September 1968.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

Microspectrophotometric Studies of Romanowsky Stained Blood Cells. III. The Action of Methylene Blue and Azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

A histochemical comparison of methylene-blue/acid fuchsin with hematoxylin and eosin for differentiating calcification of stromal tissue

Benign and malignant connective tissue tumors consist of a fibrous component that contains varying amounts of one or more types of bone or other calcified tissue. Diagnosis of these connective tissue tumors often poses challenges for pathologists, because it is difficult to differentiate the organic matrix of osteoid from hyalinized stroma. To establish a definitive diagnosis, it sometimes is advantageous to demonstrate histologically by special staining either the type of calcification or the presence or absence of calcification. We compared the efficacy of methylene blue-acid fuchsin (MB-AF) to hematoxylin and eosin (H-E) for connective tissue tumors suspected to contain calcifications and to devise an optimal staining technique for calcification that would be specific, simple, and cost- and time-effective. We examined 50 benign and 45 malignant connective tissue tumors that were suspected to contain calcifications. Sections were stained with H-E and MB-AF and evaluated. MB-AF stained bone pink, which contrasted with blue soft tissue. After MB-AF staining, osteoid was faint pink in a blue stromal background. Osteoid was not visualized in H-E stained sections; it was stained the same shade of pink as stromal tissue. Dystrophic calcification and cementum could be identified equally well using either staining technique, but contrast was better after H-E staining. MB-AF staining of bone was comparable to H-E staining and could be used effectively to stain bone and osteoid. MB-AF is a simple, single step procedure. It also stains cementum blue with faint blue rimming and dystrophic calcification bluish-pink, but it cannot be used as a specific stain for types of calcification other than bone and osteoid.     

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

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

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.