Methylene Violet (Bernthsen), by Zinc-Alkali-Chlorate Hydrolysis of Methylene Blue

Though Bernthsen's methylene violet (MV) is a common constituent of polychrome methylene blue, the hydrolytic oxydation of methylene blue to yield azure-free MV has been considered a difficult chemical reaction since the time of Bernthsen, who used Ag₂O in the hydrolysis. MV is qualitatively distinguished from azures by Bernthsen's criteria and the author's new tests: (1) light-excited isomeric change, (2) reactivity to acidity, (3) reaction with KCIO, and (4) reaction with Na₂SO₃ of azures in CHCI₃, while MV gives none. But MV shows (5) indicator properties at pH 4, while azures do not. For practical hydrolysis, treat methylene blue (10 parts by weight) and KCIO₃ (1 part) with 1-2 N NaOH to convert methylene blue to a mixture of MV and azures. Then dilute the solution, add a Zn salt and NaHCO₃ in excess of the amount needed to convert the NaOH to Na₂CO₃. Boil the solution gently for 1-2 hr. The end point of the reaction is found by pipetting a drop of reactant into 3% acetic acid in a test tube, adding CHC1₃ and extracting. The acetic layer should then be almost colorless while the CHC1₃ is colored intensely cherry red. After cooling, the precipitated dye is filtered and dried. This procedure gives good yields of a dye which meets the criteria given by Bernhsen. The peak of the absorption curve in solution, pH 4-11, is at 624 mμ (Bernthsen 625 mμ) and in acid solution, pH 0-4, 588 mμ (Biological Stains, 1953; 580 μ). The dye contains so little azures, that purification of the MV fraction obtained from the reaction mixture is unnecessary when it is used in the Wright-type Romanowsky stain. The remarkable staining effect of MV is its power to bring out red azurophil granules of monocytes and lymphocytes when used with eosinated thiazins in Wright's stain.     

Subsidiary Dyes in Methylene Blue

Suitable tests have been devised for the detection of azure B (trimethyl thonin) and methylene violet in methylene blue. All samples of methylene blue examined have been found to contain appreciable proportions of azure B.     

Subsidiary Dyes in Methylene Blue

Suitable tests have been devised for the detection of azure B (trimethyl thonin) and methylene violet in methylene blue. All samples of methylene blue examined have been found to contain appreciable proportions of azure B.     

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.     

Quirks of dye nomenclature. 8. Methylene blue,azure and violet

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.     

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.     

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.     

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.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

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.     

A Comparison of the Crystal Violet Nuclear Stain with Other Technics

The crystal violet nuclear stain was compared with the acid Giemsa and thionin-SO₂ stains, and it was found that the three technics revealed nuclear structures which were identical. Various methods of fixation and hydrolysis were tested and it was concluded that the crystal violet gave more uniform results if used without fixation or hydrolysis. The effects of these treatments on the other technics 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.     

Staining of Keratin and Keratohyalin with the Reactive Dye Levafix Red Violet E-2BL

Demonstration of keratin in Zenker-fired skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-PBL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fired sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaC1, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

Methylene blue as an antimalarial agent

Methylene blue has intrinsic antimalarial activity and it can act as a chloroquine sensitizer. In addition, methylene blue must be considered for preventing methemoglobinemia, a serious complication of malarial anemia. As an antiparasitic agent, methylene blue is pleiotropic: it interferes with hemoglobin and heme metabolism in digestive organelles, and it is a selective inhibitor of Plasmodium falciparum glutathione reductase. The latter effect results in glutathione depletion which sensitizes the parasite for chloroquine action. At the Centre de Recherche en Santé de Nouna in Burkina Faso, we study the combination of chloroquine with methylene blue (BlueCQ) as a possible medication for malaria in endemic regions. A pilot study with glucose-6-phosphate dehydrogenase-sufficient adult patients has been conducted recently.     

Structural Effects on Arthrobacter Methylene Hydroxylase Activity

Arthrobacter 4-44-2 (ATCC 25581), capable of subterminal oxidation of n-hexadecane to 2-, 3-, and 4-alcoholic and ketonic products, was examined for the ability of this methylene hydroxylase capability to be induced and repressed and for structural relationships influencing methylene function oxidation. Induction was best carried out by use of n-alkanes from 10 to 16 carbons in length and was especially strong with methylcyclohexane among cyclic compounds tested. Induction was not observed with several related alcohols, 1-unsaturated compounds, or methoxy and ethoxy compounds tested. After induction, n-alkanes 14 and 16 carbons in length were transformed to the corresponding internal oxidation products; however, no activity was observed with even-carbon alkanes of shorter chain length. Hexadecene-1 and all alcohols tested, including cyclododecanol, were transformed to corresponding ketonic or aldehydic products. Cyclic compounds tested, including cyclododecane, were not oxidized by induced cells, suggesting that a methyl group plays a role in orientation of the substrate for the methylene hydroxylation but that the methyl function was not as critical after completion of the hydroxylation step regardless of structural configuration. Acetate strongly repressed induction of n-hexadecane methylene hydroxylase activity. Inducibility of methylene hydroxylase activity was confirmed by use of cell-free systems with methylcyclohexane as an inducer. A stimulation of methylene hydroxylase activity by addition of reduced pyridine nucleotides and ferrous ion was indicated.     

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.     

The Site of Action of the Crystal Violet Nuclear Stain

Enzymatic treatment of bacterial cells prior to staining revealed that the crystal violet nuclear stain reacts with protein components of the nucleus as contrasted to the desoxyribonucleic acid specificity of some nuclear stains.     

A Pigment-producing Spoilage Bacterium Responsible for Violet Discoloration of Refrigerated Market Milk and Cream

A psychrophilic strain of bacteria identified as Chromobacterium lividum was established as the causative agent of an outbreak of violet discoloration in refrigerated, pasteurized retail milk and cream.

The organism was rod-shaped, gram-negative, and produced viscid colonies with abundant violet pigment on Tryptone glucose yeast extract agar. Growth was abundant at 4 C but none occurred at 37 C. Growth in milk was characterized by a dark violet ring at the surface after a few days, and the deep violet color gradually extended through the product in older cultures. Some proteolysis occurred. The pigment appeared to be similar to that of other known species of Chromobacterium and assisted in identification of the genus of the causative organism.

The isolated strain of C. lividum was destroyed by exposure to 56 C for 5 min which suggested postpasteurization contamination as the source of the spoilage organism in commercial milk and cream.

     

Methylene blue as an antimalarial agent

Abstract

Methylene blue has intrinsic antimalarial activity and it can act as a chloroquine sensitizer. In addition, methylene blue must be considered for preventing methemoglobinemia, a serious complication of malarial anemia. As an antiparasitic agent, methylene blue is pleiotropic: it interferes with hemoglobin and heme metabolism in digestive organelles, and it is a selective inhibitor of Plasmodium falciparum glutathione reductase. The latter effect results in glutathione depletion which sensitizes the parasite for chloroquine action. At the Centre de Recherche en Santé de Nouna in Burkina Faso, we study the combination of chloroquine with methylene blue (BlueCQ) as a possible medication for malaria in endemic regions. A pilot study with glucose-6-phosphate dehydrogenase-sufficient adult patients has been conducted recently.     

An Improved Crystal Violet Method with Alkaline Differentiation for Juxtaglomerular Granules

In the previous alkaline crystal violet method for selectively demonstrating juxtaglomerular (JG) granules (Harada 1971), the staining solution was found to be unstable. Subsequent testing has shown that the alkali is equally effective if applied after a nonalkalized aqueous solution of crystal violet has been applied for the staining, thus allowing stable stock solutions of the staining reagents to be used. The new procedure is as follows:

Sections of 4 μ thickness from adult mouse kidney fixed in phosphate-buffered 10% formalin were cut from paraffin-embedded material and attached to slides with albumen adhesive. They were deparaffinized, hydrated, and washed in tap water.