Methylene blue, 191

Methylene blue is probably the most frequently used viable stain in the wine industry. The dye exists in two redox-dependent states. The colorless, or leuccpformy represents the reduced state as would be seen in cases where the dye is taken up (and reduced) by viable yeasts which then appear colorless on a blue background. In contrast, dye reduction is not seen in dead yeast where the colored (oxidized) form concentrates intracellularly. Such yeast appear blue-black. 

Although useful as a viability dye, methylene blue rapidly becomes toxic to microorganisms therefore, preparations should be examined within 10 min of preparation. 

Methylene blue (0.01% w/v) Dissolve 0.3 g of methylene blue (methylthionine chloride) in 30 mL of 95% ethyl alcohol and 100 mL citrate buffer (pH 4.6). 

Citrate buffer (pH 4.6) In approximately 90 mL of distilled water, dissolve 2.4 g NaH-citrate and 2.1 g NaH2 citrate. Bring to a final volume of 100 mL. 

Methylene blue, the viable stain, is substantially more dilute than methylene blue, the redox indicator used in reducing sugar titra-metric analyses. The latter is, in fact, rapidly toxic to yeasts, leading to erroneously high cell mortality. Where both procedures are carried out in the same lab, it is wise to clearly indicate the dye s purpose. 

Methylene blue is a heterocyclic aromatic chemical compound with the molecular formula CigHigNaSCl. It has many uses in a range of different fields such as biology and chemistry. 

Methylene blue exists in two redox-dependent states reduced and oxidized. The reduced state (fewco-form) is colorless whereas the oxidized form is blue. If a viable yeast cell takes up the dye, methylene blue is reduced to the colorless form so that the cell appears colorless (or white) against the blue background. Thus, cells that reduce the dye to the colorless form are assumed to be viable, whereas dead yeast do not reduce the dye and appear blue or black. 

Although traditionally used in brewing and winemaking industries, some researchers have argued against the application of methylene blue as a means to determine yeast viability. For example, O Connor-Cox et al. (1997) noted that the dye does not stain all dead cells in a sample, a fact that can lead to overestimating yeast viahUity. 

Although several forms of methylene hlue are available, the preparation used for biological work is methylene hlue chloride. Methylene hlue thiocyanate, a salt used as a redox indicator in milk testing, should not be used as a biological stain. Because methylene blue rapidly becomes toxic to microorganisms, preparations should he microscopically examined within 10 min. 

Prepare a citrate buffer by dissolving 2.4g disodium hydrogen citrate (Na2HC6H507) and 2.1 g sodium dihydrogen citrate (NaH2CeH507) in a minimal amount of distilled water and dilute to lOOmL. Adjust pH to 4.6 if necessary. 

Dissolve 0.3g methylene blue chloride in 30mL of 95% v/v ethanol. 

This is one of the so-called simple stains, and the version usually used requires Loeffler s methylene blue. This is made by adding a saturated solution of methylene blue in ethanol (300 ml) to one litre of 0.01% aqueous KOH. Smears are stained for three minutes and then washed with water. 

These dyestuffs are far more stable than the indamines and indophenols, and, unlike these, do not give quinone when treated with acid. For this reason they are capable of practical application, one of them, methylene blue, discovered by Caro, being used on a very large scale. The dyestuffs of this class have a violet or blue shade. 

The base, C12H9N3S, forms a black crystalline powder or needles with a greenish reflex. It dissolves in alcohol, forming a reddish-violet solution, and in ether, forming an orange solution. 

Hydrochloride, Ci2H9N3S,HCl, forms beetle-green needles, soluble in water with a violet colour. 

Lauth s Violet gives a green solution with concentrated sulphuric acid, and on dilution the colour changes through blue to violet. On reduction it yields paradiamidothiodiphenylamine [36]. 

Isothionine is an isomeric dyestuff formed by oxidation of a diamidothiodiphenylamine of unknown constitution [36]. 

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An example of the time effects in irreversible adsorption of a surfactant system is shown in Fig. XI-8 for barium dinonylnapthalene sulfonate (an oil additive) adsorbing on Ti02 (anatase). Adsorption was ineversible for aged systems, but much less so for those equilibrating for a short time. The adsorption of aqueous methylene blue (note Section XI-4) on TiOi (anatase) was also irreversible [128]. In these situations it seems necessary to postulate at least a two-stage sequence, such as... 

One hundred milliliters of an aqueous solution of methylene blue contains 3.0 mg dye per liter and has an optical density (or molar absorbancy) of 0.60 at a certain wavelength. After the solution is equilibrated with 25 mg of a charcoal the supernatant has an optical density of 0.20. Estimate the specific surface area of the charcoal assuming that the molecular area of methylene blue is 197 A. ... 

Dye adsorption from solution may be used to estimate the surface area of a powdered solid. Suppose that if 3.0 g of a bone charcoal is equilibrated with 100 ml of initially 10 Af methylene blue, the final dye concentration is 0.3 x 10 Af, while if 6.0 g of bone charcoal had been used, the final concentration would have been 0.1 x Qr M. Assuming that the dye adsorption obeys the Langmuir equation, calculate the specific surface area of the bone charcoal in square meters per gram. Assume that the molecular area of methylene blue is 197 A. ... 

Several variations of the chemical method are in use. In the one described below, a freshly prepared Fehling s solution is standardised by titrating it directly against a standard solution of pure anhydrous glucose when the end-point is reached, I. e., when the cupric salt in the Fehling s solution is completely reduced to cuprous oxide, the supernatant solution becomes completely decolorised. Some difficulty is often experienced at first in determining the end-point of the reaction, but with practice accurate results can be obtained. The titrations should be performed in daylight whenever possible, unless a Special indicator is used (see under Methylene-blue, p. 463). 

This enzyme, sometimes also called the Schardinger enzyme, occurs in milk. It is capable of " oxidising" acetaldehyde to acetic acid, and also the purine bases xanthine and hypoxanthine to uric acid. The former reaction is not a simple direct oxidation and is assumed to take place as follows. The enzyme activates the hydrated form of the aldehyde so that it readily parts w ith two hydrogen atoms in the presence of a suitable hydrogen acceptor such as methylene-blue the latter being reduced to the colourless leuco-compound. The oxidation of certain substrates will not take place in the absence of such a hydrogen acceptor. 

Make up a methylene-blue solution by grinding 0 1 g. with water and making up to i litre with water. 

Place 5 ml. of milk in each of two test-tubes A and B. Boil the milk in B thoroughly for 2 minutes to destroy the enzyme, and cool. Then in each test-tube place i ml. of the acetaldehyde solution and i ml of the methylene-blue solution. Mix gently by inverting the tubes avoid shaking with air. Now place A and B in a water-bath maintained at 40-50. After a time (usually about 20 minutes) the dye in A is completely decolorised except at the surface, while B is unaffected. 

Grote s reagent is useful for the determination of 2-aminothiazoie in blood and wine (145), This thiazole may be extracted from its aqueous solution and then titrated in nonaqueous medium (MeOH) with HCIO4 in the presence of a mixed methyl red-methylene blue indicator (146). 

With short periods of irradiation (with high-pressure mercury lamps) under oxygen in chloroform containing methylene blue as a sensitizer, variously substituted 2-arylthiazoles are converted in the corresponding 2-aryloxazoles (823). 

Brown and Lin reported a quantitative method for methanol based on its effect on the visible spectrum of methylene blue. In the absence of methanol, the visible spectrum for methylene blue shows two prominent absorption bands centered at approximately 610 nm and 660 nm, corresponding to the monomer and dimer, respectively. In the presence of methanol, the intensity of the dimer s absorption band decreases, and that of the monomer increases. For concentrations of methanol between 0 and 30% v/v, the ratio of the absorbance at 663 nm, Asss, to that at 610 nm, Asio, is a linear function of the amount of methanol. Using the following standardization data, determine the %v/v methanol in a sample for which Agio is 0.75 and Ag63 is 1.07. 

The methylene blue and resazurin reduction methods indirectly measure bacterial densities in milk and cream in terms of the time interval required, after starting incubation, for a dye—milk mixture to change color (methylene blue, from blue to white resazurin, from blue through purple and mauve to... 

The direct microscopic count determines the number of viable and dead microorganisms ia a milk sample. A small amount (0.01 mL) of milk is spread over a 1.0 cm area on a microscope sHde and allowed to dry. After staining with an appropriate dye, usually methylene blue, the sHde is examined with the aid of a microscope (oil immersion lens). The number of bacterial cells and clumps of cells per microscopic field is determined and, by appropriate calculations, is expressed as the number of organisms per milliliter of sample. 

The quaHtative determination of water-soluble perchlorates by precipitation using methylene blue yields a violet precipitate (105). Using potassium, mbidium, or cesium salts for precipitation from ethanol—water solutions can serve as a quaHtative determination of perchlorates (106). 

There are many colorimetric methods used for trace analysis of peroxides using reagents such as ferrous ion, leuco base of methylene blue, yy -diphenylcarbohydrazide, titanium(IV), iodide ion, and Ai,A7-dimethyl- -phenylenediamine. The latter two are the most commonly used reagents... 

Thus, the presence of a thiamine ring in Cl Sulfur Blue 9 was conclusively proved. The thiamine ring is the fundamental chromophore that accounts for the high color value of both the sulfur dye and Methylene Blue [61-73-4] including their abiUty to form pale yeUow leuco forms on reduction. Methylene Violet (15) is obtained from Methylene Blue (16) by hydrolysis in boiling alkah. 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Methylene Blue [61-73-4] CI Basic Blue 9 (CI 52015), (26), is the classic thiazine dye still in use today. This dye was first reported in 1876 (1). 

Synthesis. The method of synthesis for Methylene Blue described in reference 14 is stiU the stepwise method of choice for thiazine dyes. /V,/V-Dimethy1-y-pheny1ene diamine [99-98-9], CgH22N2, reacts with sodium thiosulfate [7772-98-7] to form the thiosulfonic acid which condenses with /V, /V-dimetby1 ani1 ine [121 -69-7], CgH N, in the presence of sodium dichromate [10588-01-9] to the indamine, then with copper sulfate [18939-61 -2] and sodium dichromate to Methylene Blue (26). 

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