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.     

Basic Fuchsin-Crystal Violet; A Rapid Staining Sequence for Juxtaglomerular Granular Cells Embedded in Epoxy Resin

A basic fuchsin-crystal violet staining sequence for demonstration of juxtaglomerular granular cells in epoxy-embedded tissues is rapid and results in slides with excellent contrast and intensity. Procedure: Cut sections 0.3-0.6 μ thick. Hydrate through xylene and alcohol to water. Stain in modified Goodpasture's stain (basic fuchsin, 1; aniline, 1; phenol, 1; 30% alcohol, 100) for 20-30 sec; rinse in tap water; stain in modified Stirling's (crystal violet, 5; alcohol, 10; aniline, 2; water, 88) for 20-30 sec; rinse in tap water and dry on a hotplate; mount in a synthetic resin. Granular cells of the juxtaglomerular apparatus are stained an intense dark blue by the crystal violet. Arterial elastic membranes and collagen are pale blue. Other structures are shades of red.     

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.     

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.     

Chemical structures and staining mechanisms of Weigert's resorcin-fuchsin and related elastic fiber stains

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsin, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are cationic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachloroferrate anion (FeCl4-) and not as Fe or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, the observed staining of elastic fibers is only as expected.     

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.     

Chemical Structures and Staining Mechanisms of Weigert'S Resorcin-Fuchsin and Related Elastic Fiber Stains

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsia, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are canonic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachlorofemte anion (FeCI₄^-) and not as Fe⁺⁺⁺ or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, die observed staining of elastic fibers is only as expected.     

A Differential Stain Favorable to the Diagnosis of Neisserian Infection

A differential Gram stain has been evolved which incorporates the combined features of the original Gram and Pappenheim methods. National Aniline crystal violet and new methyl green and pyronin are the dyes preferred. The iodine mixture of Kopeloff and Beerman is a satisfactory mordant and Merck's pure technical acetone is an excellent differentiating agent. A system is established by means of the dyes and reagents which form a physicochemical equilibrium, provided pure dyes are employed, and the technic is carried out with precision. Gram-positive bacteria are coated by means of buffered crystal violet solution and the iodine-sodium hydroxide solution precipitates the crystal violet from other substances. The dye-iodine precipitate is readily dissolved by pure acetone. Iodine green, a pure derivative of crystal violet has the effect of noninterference in the technic and has selective action upon nuclear substance. Pyronin has affinity for Neisserian organisms primarily and acts as an inert substance upon most other proteins, (except cytoplasm of eosinophils, lymphocytes, plasma cells, and endothelial cells). The following technic is recommended:

Stain air-dry films 3 to 5 minutes in a 1% solution of crystal violet in 10 parts of Clark and Lubs' phosphate buffer of pH 6.6 to 7.0 and 90 parts water. Decant and flush with 2% iodine in N/10 NaOH. Decant and decolorize in acetone 10 seconds or less. Air dry and counterstain 1 1/2 to 2 minutes with methyl-green-pyronin (2 parts 2% aqueous methyl green National with one part 0.3% aqueous pyronin yellowish). Wash and air dry. Oil of Bergamot is preferable to xylene as a clearing agent. Best results are obtained if each slide is handled separately as for staining blood films.     

Rapid Staining of Nuclei and Chromosomes in Root Tips

Feulgen reagent quickly heated to and maintained at 60 C just before immersion of plant material, basic fuchsin in acid alcohol at room temperature, and pinacyanol at room temperature will stain hydrolyzed root tip nuclei and chromosomes in one minute or less. This technic, coupled with fast fixation, can be utilized when uncertainties exist as to when to begin sampling plant meristem cells for mitoses or when time does not allow for standard fixation and Schiff staining.     

Dye Complexes Produced in Gram Staining

Complexes of crystal violet and iodine containing (a) one molecule and four atoms, and (b) one molecule and two atoms respectively, were prepared and their solubility in alcohol (95%) determined. Both complexes were only slightly soluble in alcohol, the former being less soluble than the latter. The solubility decreased with decreasing concentration of alcohol, and also on storage. Complexes of malachite green, basic fuchsin and Victoria blue B with iodine were also prepared, and complexes of each dye with picric acid. Two complexes of different composition could be obtained from crystal violet and picric acid, and from Victoria blue and iodine. Complexes with basic fuchsin were much more soluble in alcohol (95%) than complexes with the other dyes tested. Dye-mordant complexes have some of the properties of organic charge-transfer complexes.     

细菌脱色酶TpmD对三苯基甲烷类染料脱色的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行有效脱色的脱色酶,命名为TpmD。该酶的亚基分子量为29.4kDa,等电点为5.6。该酶催化上述4种三苯基甲烷类染料脱色反应的适合温度为40~60℃,适合pH范围为5.5~9.0。动力学参数测定结果显示TpmD对结晶紫、碱性品红、灿烂绿及孔雀绿的Km值分别为24.3、40.65、4.2、68.5μmol-1.L-1,Vmax值分别为19.6、74.1、82.8、115.6μmol.L-1.s-1。结晶紫为该酶的最适反应底物。TpmD催化的脱色反应依懒于NADH/NADPH及分子氧的存在,显示该酶属于NADH/NADPH依赖型的氧化酶类。这是国内外首次关于细菌中三苯基甲烷类染料脱色酶酶学性质的描述。     

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.     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

The Effect of Organic Solvents on the Appearance of Bacterial Nuclei

Treatment of bacterial smears with organic solvents was found to produce a typical appearing nuclei which could lead to false interpretation of nuclear events. The crystal violet nuclear stain, which does not utilize organic solvents or acid hydrolysis, was used as a basis of comparison. Acid hydrolysis also was observed to affect the appearance of the nucleus. It is concluded that caution should be used in interpreting nuclear structure and activity in bacterial cells, especially when organic solvents or acid hydrolysis are involved in the staining technic.     

Improved Cytological Methods with Crystal Violet

It is well known that the crystal-violet-iodine technic usually provides excellent cytological preparations, the necessary skill for making such preparations is not difficult to acquire and critical examination of detail under the high powers of the microscope is generally possible. It is frequently complained, however, that the stain is not permanent and tends to fade more or less rapidly according to the exact details of the procedure followed. During the past ten years the author has tested many different brands of gentian violet, methyl violet, crystal violet and the related series of dyes in connection with experiments on the chemistry of chromatin, and certain points have been observed that might prove of service in ordinary staining technics.     

A Note on the Gram Stain

A modification of the Gram stain in which iodine-alcohol is substituted for 95% alcohol as a decolorizing agent has been found particularly useful in staining Gram-positive organisms in tissues and also for smears. The technic for tissue sections follows:

1. Apply nuclear stain.
2. Wash.
3. Stain in Hucker's gentian violet 2 to 3 minutes (i. e. 1 part Sat. Alc. Sol. crystal violet to 4 parts 1% Aqu. Sol. ammonium oxalte).
4. Wash in water.
5. Stain in Gram's iodine 5 minutes.
6. Wash in water.
7. Decolorize in 95% alcohol to which enough tincture of iodine has been added to give a mahogany color.
8. Counterstain.
9. Dehydrate and mount.

     

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.     

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。     

Dye Adsorption by Bacteria at Varying H-Ion Concentrations

Adsorption of hydrogen ions and dye cations by washed bacterial cells shows a reciprocal relationship. Apparently, H-ions and crystal violet ions are held by the cell at the same adsorption centers, and the influence of H⁺ on basic dye adsorption is one of direct competition or replacement The adsorption of H⁺ and acid fuchsin is similar in that an increase is noted as the pH of the suspension is lowered.     

Detailed Differentiation of Bacteria by Means of A Mixture of Acid and Basic Dyes at Different pH-Values

As previously reported by the author (1927), a mixture of methylene blue and eosin Y can be used for the differential staining of bacteria. It gives a fairly deep staining of bacteria at about pH 3 and above. Below pH 3 the eosin Y stains bacteria only a very pale pink; at such high H-ion concentration, the eosin is present as undissociated color acid, and for this reason not enough eosin is in solution to stain bacteria. To improve the staining at such reactions, the eosin was replaced by a stronger acid dye, namely acid fuchsin. The mixture of methylene blue medicinal Merck and acid fuchsin can be successfully used at a pH-value as low as 0.8. The method of staining by this new mixture is entirely the same as with the old mixture. It is sensitive enough to detect the difference in the isoelectric points: (1) of the single bacteria from the same pure culture, (2) of different strains of the colon and typhoid organisms. Some strains of the colon organism were found by this method with an isoelectric point at a pH-value as low as that of the Staphylococcus. Others, on the contrary, have their isoelectric point as high in the pH-scale as that of the typhoid organism. The new mixture can also be used for the study of the chemical composition of the different parts of bacterial body. Applying it at a definite pH-value, the author was able to stain differentially polar bodies of the typhoid group and of the diphtheria organism. This new mixture can be recommended in staining of B. diphtheriae as a substitute for Neisser's stain. It is interesting to note that polar bodies of the colon group consist of more alkaline protein than the body of the bacteria itself, i. e., they are stained by acid fuchsin. The polar bodies of the B. diphtheriae on the contrary are composed of more acid protein than the bacterial body; i. e., they are stained by methylene blue. The impossibility of detecting the above mentioned variations in the isoelectric points of bacteria using the Gram method is explained by the absence of pH variations in the latter technic. The differentiation of bacteria by the Gram stain depends chiefly on the varying stability of the compound formed (Gram-positive or Gram-negative bacteria plus gentian violet and iodine) in the presence of organic solvents, such as alcohol, acetone, etc.