Ethyl Violet. A Selective Dye for the Isolation of Gram-Negative Bacteria

Minimal inocula of Gram-negative and positive bacteria were seeded into tryptose broth containing varying concentrations of dyes. Three dyes were used, namely crystal violet, brilliant green and ethyl violet. Growth rates were determined for 2, 4 and 6 hours incubation. All three dyes were equally effective in inhibiting Gram positive bacteria. Ethyl violet showed markedly less toxicity toward Gram negative bacteria than did either crystal violet or brilliant green.     

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.     

Effect of pH on the antimicrobial activity of some triphenylmethane dyes.

Four common dyes were tested as inhibitors of four types of bacteria over the pH range 5.0-9.0. Inhibition of the gram-negative types, Salmonella anatum and Enterobacter aerogenes, was markedly affected by the pH of the medium. These organisms tolerated concentrations of crystal violet and ethyl violet about 100-fold higher at pH 5.0 than at pH 9.0. Above pH 7.0 brilliant green (BG) and malachite green (MG) were precipitated as their respective carbinols and lost their inhibitory properties with these two organisms. Two gram-positive types, Staphylococcus aureus and Bacillus cereus, were more sensitive to dyes and results were less affected by pH. The carbinol forms of MG and BG were nearly as inhibitory to these organisms as the ionized forms.     

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.     

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。     

Removal of basic dyes from aqueous medium using a novel polymer: Jalshakti

Studies were carried out to remove basic dyes such as safranine T, methylene blue, crystal violet, light green, brilliant milling violet and patent blue VS from their aqueous solutions using biodegradable polymeric absorbent material, viz., Jalshakti (JS). Results showed that 93% safranine T, 98% methylene blue and 84% crystal violet were adsorbed on JS relative to their initial concentration (10 mg L(-1)). The optimum pH was found to be 6.0+/-0.5 and smaller size of particle of JS resulted better adsorptive removal of the dyes. IR spectroscopic and potassium ion release studies revealed that basic dyes were selectively removed through adsorption-ion-exchange mechanism involving carboxylic groups and K+ ions of JS.     

Methods for the Standardization of Biological Stains: Part IV

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission, in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

A Modification of the Technic of Handling Chick Embryos

In this paper are given methods for determining the suitability of certain dyes of the triphenylmethane group for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission, in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Malachite green, brilliant green, light green SF yellowish, fast green FCF, basic fuchsin (rosanilin and pararosanilin), acid fuchsia, methyl violet, crystal violet, gentian violet, methyl green and anilin blue. For each of these dyes, methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Extractive biodecolorization of triphenylmethane dyes in cloud point system by Aeromonas hydrophila DN322p

The biological treatment of triphenylmethane dyes is an important issue. Most microbes have limited practical application because they cannot completely detoxicate these dyes. In this study, the extractive biodecolorization of triphenylmethane dyes by Aeromonas hydrophila DN322p was carried out by introducing the cloud point system. The cloud point system is composed of a mixture of nonionic surfactants (20 g/L) Brij 30 and Tergitol TMN-3 in equal proportions. After the decolorization of crystal violet, a higher wet cell weight was obtained in the cloud point system than that of the control system. Based on the results of thin-layer chromatography, the residual crystal violet and its decolorized product, leuco crystal violet, preferred to partition into the coacervate phase. Therefore, the detoxification of the dilute phase was achieved, which indicated that the dilute phase could be discharged without causing dye pollution. The extractive biodecolorization of three other triphenylmethane dyes was also examined in this system. The decolorization of malachite green and brilliant green was similar to that of crystal violet. Only ethyl violet achieved a poor decolorization rate because DN322p decolorized it via adsorption but did not convert it into its leuco form. This study provides potential application of biological treatment in triphenylmethane dye wastewater.     

The Mechanism of the Gram Reaction. III. Solubilities of Dye-Iodine Precipitates and Further Studies of Primary Dye Substitutes

Solubilities of dye-iodine precipitates in alcohol and in aqueous safranin solution were determined by direct solubility methods and by photocolorimetric methods. It was found that, increasing precipitate solubility in alcohol or safranin solution gave decreasing differentiation between Gram-positive and Gram-negative bacteria. Dyes which did not stain the cells well as a primary stain did not give good Gram stains, regardless of the solubilities of their precipitates. Some dyes (typified by methylene blue) which gave relatively alcohol-insoluble iodine precipitates gave inferior Gram differentiation because these precipitates were readily soluble in the safranin counterstain.

Solubilities of precipitates of crystal violet and various iodine substitutes were determined photocolorimetrically. The ability of a substance to replace iodine in the Gram stain correlated with its ability to give a precipitate which was only slightly soluble in alcohol and relatively insoluble in aqueous safranin solution.

It was concluded that the usual Gram reagents are not truly specific for the differentiation. Any dye and mordant could be used if the dye was deeply colored, stained the cells well, and if the precipitate of dye and mordant was only slightly soluble in alcohol and relatively insoluble in the counterstain. These factors, combined with those influencing differences in cell membrane permeability, constitute the most important factors in the Gram stain differentiation.

Studies were made concerning the ability of dyes to substitute for crystal violet in the Gram procedure. Of 29 dye samples reported on here for the first time none proved to be good substitutes for crystal violet.     

Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium

Biodegradation of crystal violet (N,N,N',N',N',N'-hexamethylpararosaniline) in ligninolytic (nitrogen-limited) cultures of the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance of crystal violet and by the identification of three metabolites (N,N,N',N',N'-pentamethylpararosaniline, N,N,N',N'-tetramethylpararosaniline, and N,N',N'-trimethylpararosaniline) formed by sequential N-demethylation of the parent compound. Metabolite formation also occurred when crystal violet was incubated with the extracellular fluid obtained from ligninolytic cultures of this fungus, provided that an H2O2-generating system was supplied. This, as well as the fact that a purified ligninase catalyzed N-demethylation of crystal violet, demonstrated that biodegradation of crystal violet by this fungus is dependent, at least in part, upon its lignin-degrading system. In addition to crystal violet, six other triphenylmethane dyes (pararosaniline, cresol red, bromphenol blue, ethyl violet, malachite green, and brilliant green) were shown to be degraded by the lignin-degrading system of this fungus. An unexpected result was the finding that substantial degradation of crystal violet also occurred in nonligninolytic (nitrogen-sufficient) cultures of P. chrysosporium, suggesting that in addition to the lignin-degrading system, another mechanism exists in this fungus which is also able to degrade crystal violet.     

Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium.

Biodegradation of crystal violet (N,N,N',N',N',N'-hexamethylpararosaniline) in ligninolytic (nitrogen-limited) cultures of the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance of crystal violet and by the identification of three metabolites (N,N,N',N',N'-pentamethylpararosaniline, N,N,N',N'-tetramethylpararosaniline, and N,N',N'-trimethylpararosaniline) formed by sequential N-demethylation of the parent compound. Metabolite formation also occurred when crystal violet was incubated with the extracellular fluid obtained from ligninolytic cultures of this fungus, provided that an H2O2-generating system was supplied. This, as well as the fact that a purified ligninase catalyzed N-demethylation of crystal violet, demonstrated that biodegradation of crystal violet by this fungus is dependent, at least in part, upon its lignin-degrading system. In addition to crystal violet, six other triphenylmethane dyes (pararosaniline, cresol red, bromphenol blue, ethyl violet, malachite green, and brilliant green) were shown to be degraded by the lignin-degrading system of this fungus. An unexpected result was the finding that substantial degradation of crystal violet also occurred in nonligninolytic (nitrogen-sufficient) cultures of P. chrysosporium, suggesting that in addition to the lignin-degrading system, another mechanism exists in this fungus which is also able to degrade crystal violet.     

Particle beam liquid chromatography-mass spectrometry of triphenylmethane dyes: application to confirmation of malachite green in incurred catfish tissue

Eight triphenylmethane dyes (malachite green, leucomalachite green, gentian violet, leucogentian violet, brilliant green, pentamethyl gentian violet, N′,N′-tetramethyl gentian violet and N′,N″-tetramethyl gentian violet) have been characterized by particle beam liquid chromatography-mass spectrometry. The electron ionization spectra obtained of these dyes by this technique exhibit similar fragmentation, with the formation of phenyl and substituted phenyl radicals, and loss of alkyl groups from the amines. It was observed that the six cationic dyes are reduced in the mass spectrometer source to form the corresponding leuco compounds. This technique was evaluated for the confirmation of malachite green and leucomalachite green in incurred catfish (Ictalurus punctatus) muscle tissue.     

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 70 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 60 mA/cm² and 34 3C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.     

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 7.0 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 +/- 60 mA/cm2 and 34 degrees +/- 3 degrees C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.     

禾本红酵母Y－5对结晶紫、孔雀石绿的吸附

研究了培养时间、初始pH、温度对禾本红酵母Y-5吸附结晶紫、孔雀石绿的影响,并对吸附剂的解吸和循环利用等进行考察.结果表明： 在染料浓度为50 mg·L^-1、pH 7.0、摇床转速150 r·min^-1、30 ℃、吸附10 h时,红酵母对结晶紫、孔雀石绿的吸附率分别达峰值,为93.8%和87.7%;解吸后的菌体对结晶紫、孔雀石绿的吸附率分别为85.5%和78.5%,说明菌体对染料的吸附是可逆的,循环利用效果良好,菌体可再生和循环利用;禾本红酵母Y-5对结晶紫、孔雀石绿的脱色机理为吸附作用,染料多被吸附在红酵母表面的羟基（-OH）上,其吸附染料快速、高效、可逆,在染料废水处理上具有潜在的应用价值.     

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.     

Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme

The ability of the white-rot fungus Lentinula (Lentinus) edodes to decolorize several synthetic dyes was investigated using solid state cultures with corn cob as substrate. Cultures, containing amido black, congo red, trypan blue, methyl green, remazol brilliant blue R, methyl violet, ethyl violet and Poly R478 at 200 ppm, were completely decolorized after 18 days of incubation. Partial decolorization was observed in the cultures containing 200 ppm of brilliant cresyl blue and methylene blue. High manganese peroxidase activity (2600 U/g substrate), but very low lignin peroxidase (<10 U/g substrate) and laccase (<16 U/g substrate) activities were detected in the cultures. In vitro, the dye decolorization was markedly decreased by the absence of manganic ions and H2O2. These data suggest that manganese peroxidase appear to be the main responsible for the capability of L. edodes to decolorize synthetic dyes.     

Stain Solubilities Part 1. Data in the Literature

The rather meager data found in the literature concerning the solubilities of the dyes used as biological stains is reviewed. Solubility data have been found concerning the following dyes: picric acid, martius yellow, crystal ponceau, methyl orange, tropaeolin O, orange II, Bismarck brown, Congo red, auramine, malachite green, fuchsin, methyl violet, gentian violet, crystal violet, methyl green, diphenylamine blue, aurin, corallin, phenolphthalein, flluorescein, eosin Y, iodo-eosin, methylene blue, alizarin, indigo carmine, and carmine. Much of this information is of questionable reliability. The writer is investigating the matter and his original data are to appear in subsequent papers.