Borax Methylene Blue: A Spectroscopic And Staining Study

Borax methylene blue is quite stable at room temperatures of 22-25 C. At 30 C polychroming is slow; during 50 days in a water bath at this temperature the absorption peak moves from 665 to 656 nm. At 35 C, the absorption peak reaches 660 nm in 7 days, 654 nm in 14. At 60 C polychroming is rapid, the absorption peak reaching 640-620 nm in 3 days. When the pH of the borax methylene blue solutions, normally about 9.0, is adjusted to pH 6.5, the absorption peak remains at 665 nm even when incubated at 60 C for extended periods.

When used as a blood stain 0.4 ml borax methylene blue (1% methylene blue in 1% borax), 4 ml acetone, 2 ml borax-acid phosphate buffer to bring the solution to pH 6.5, and distilled water to make 40 ml, with 0.2 ml 1% eosin added just before using, an excellent Nocht-Giemsa type stain is achieved after 30 minutes staining. The material plasmodia P. falciparum, P. vivax, and P. berghei stain moderate blue with dark red chromatin and green to black pigment granules.

The study confirms Malacnowski's 1891 results and explains Gautier's 1896-98 failure to duplicate it.     

Studies on Polychrome Methylene Blue II. Acid Oxidation Methods of Polychroming

The action of K₂Cr₂O₇, Ag₂O, KMnO₄, HgO and NaIO₃ in polychroming methylene blue is explored. The last two have no action in neutral or acid methylene blue solutions. With the other three reagents the amount of polychroming, as measured by the shift in the absorption spectrum, is roughly proportional to the amount of oxidant used. Various lots of methylene blue produce similar products with similar proportions of K₂Cr₂O₇. With similar quantities of this reagent similar products are produced by polychroming at 100°, 80°, 70° or 60° C. At 100° C. the action of K₂Cr₂O₇ or of Ag₂O appears to be completed in 15 minutes. In K₂Cr₂O₇ polychroming, H₂SO₄ can be substituted for HCl, and subsequent BaCO₃ neutralization removes the salts formed and prevents accidental alkali polychroming. K₂Cr₂O₇ polychroming produces products with narrower absorption bands than alkali polychroming.     

Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

The actual mechanism of the differentiation of lactose-fermenting and non-lactose-fermenting organisms on eosin-methylene-blue medium is not reported in the literature. The present study is an attempt to elucidate this problem.

The color of colon forms on E.M.B. agar was found to depend on two factors: (1) the reaction of eosin with methylene blue to form a dye compound of either acidic or neutral nature, and (2) the production, by lactose-fermenting colonies, of a sufficiently low pH so that this dye compound is taken up by individual cells of the colony. Non-lactose-fermenting organisms are not colored because the compound is not taken up in alkaline reaction.

An explanation is offered to account for the occasional blue colonies found on E.M.B. medium. It is suggested that these colonies form a relatively high pH and thus cause slight dissociation of the compound. This dissociation would allow independent staining of the colonies by 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.     

Borax methylene blue: a spectroscopic and staining study.

Borax methylene blue is quite stable at room temperatures of 22-25 C. At 30 C polychroming is slow; during 50 days in a water bath at this temperature the absorption peak moves from 665 to 656 nm. At 35 C, the absorption peak reaches 660 nm in 7 days, 654 nm in 14. At 60 C polychroming is rapid, the absorption peak reaching 640-620 nm in 3 days. When the pH of the borax methylene blue solutions, normally about 9.0, is adjusted to pH 6.5, the absorption peak remains at 665 nm even when incubated at 60 C for extended periods. When used as a blood stain 0.4 ml borax methylene blue (1% methylene blue in 1% borax), 4 ml acetone, 2 ml borax-acid phosphate buffer to bring the solution to pH 6.5, and distilled water to make 40 ml, with 0.2 ml 1% eosin added just before using, an excellent Nocht-Giemsa type stain is achieved after 30 minutes staining. The material plasmodia P. falciparum, P. vivax, and P. berghei stain moderate blue with dark red chromatin and green to black pigment granules. The study confirms Malachowski's 1891 results and explains Gautier's 1896-98 failure to duplicate it.     

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.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

Reactions of Hemoglobiniferous Cells to Aced and Basic Dyes under Varying Conditions of H-Ion Activity

1. An attempt has been made to apply Loeb's concept of the amphoteric nature of proteins for the discrimination of suspected hemoglobiniferous substances from known hemoglobiniferous substances according to their reactions to acid and basic dyes with reference to the isoelectric point of hemoglobin (pH 6.8).

2. The substances in the cytoplasm of known hemoglobiniferous cells (red blood corpuscles, normoblasts and erythroblasts) of the lymph nodes, spleen and bone marrow of the albino rat, when suspended in buffered dye-sucrose solutions, retain eosin on the acid side of pH 7.0, but the substances of the Russell bodies, suspected of being hemoglobiniferous, do not retain eosin at all; and the cytoplasm of the plasma cells, also alleged to be slightly hemoglobiniferous, only retains eosin on the acid side of pH 6.4.

3. The only basic dye used which did not precipitate in buffer solutions was methylene blue. This did not react with hemoglobin in accordance with Loeb's concept, because it did not penetrate mature red blood corpuscles and in those immature erythrocytes which it did penetrate it was precipitated by the reticulum.

4. Therefore, from the results obtained with the acid dye, it is tentatively concluded that the substance in the Russell bodies and in the cytoplasm of the plasma cells are not hemoglobiniferous because they do not react as do the substances in known hemoglobiniferous cells with reference to the isoelectric point of hemoglobin.

5. More investigation, however, must be carried out on both fresh and fixed material before a final unequivocal answer can be made to this problem.     

Adsorption of cationic dye from aqueous solutions by date pits: Equilibrium,kinetic, thermodynamic studies,and batch adsorber design

The retention profile of methylene blue from aqueous solutions onto the solid adsorbent date pits has been investigated in a batch system. The characterization and adsorption efficiency for methylene blue was evaluated using date pits. Fourier Transform Infra-Red, Scanning Electron Microscope, Brunauer–Emmett–Teller analysis were performed to determine the characteristics of the material. The effect of contact time, initial dye concentration, adsorbent dosage, temperature, and solution pH were investigated. The adsorption was found to increase with increasing time, decreasing concentration of dye, decreasing temperature and increasing dosage up to equilibrium values which was 20 min, 25°C, and 0.1 g adsorbent, respectively. The adsorption was favorable at high and low pH (pH 3, pH 7). The adsorption equilibrium data were best fitted by Freundlich isotherm. The adsorption kinetics was found to follow the pseudo second order kinetic model. Thermodynamic parameters such as free energy, enthalpy, and entropy were calculated and found to be ?4.6 kJ/mole, ?7.9 kJ/mole, and ?11.8 kJ/mole, respectively. The thermodynamic parameters of the uptake of methylene blue onto the date pits indicated that, the process is exothermic and proceeds spontaneously at low temperature. A single stage batch adsorber was designed for adsorption of methylene blue by Date Pits based on optimum isotherm.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

The Preparation of Vital Neutral Red

Neutral red iodide suitable for vital staining was prepared by condensing nitrosodimethylanilin hydrochloride with m-toluylenediamine and the indamine, toluylene blue, was obtained. This was subjected to air oxidation and converted to the eurhodine, neutral red. The purification of this dye was brought about by converting it into its comparatively insoluble stannous chloride double salt, filtering, dissolving in water, and precipitating the neutral red iodide with potassium iodide solution. This was re-dissolved in water, reprecipitated with potassium iodide solution and crystallized from 95% ethanol. The uncrystallized dye was also found satisfactory for vital staining. Several other preparations of neutral red iodide were made, using a somewhat different procedure than that given above, and it was generally found that satisfactory stains were obtained only when the preparation was free from toluylene blue.

The chloride of the color base was prepared by continuing the air oxidation of the toluylene blue until a test sample indicated its complete conversion into neutral red. The color was salted out with sodium chloride and crystallized from 95% ethanol. Both the crystallized and uncrystallized products were found to be excellent stains.     

Metal Contaminants in Commercial Thiazine Dyes

The iron, potassium, sodium and zinc contents of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (GI. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidme blue (C.I. 52040) have been determined by atomic absorption spectrophotometry.

The metal concentrations varied widely in the 38 samples examined—iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

Differential in Toto Staining of Bone, Cartilage and Soft Tissues

The following method for staining bone and cartilage allows study of the gross cleared specimen and does not injure the tissues for subsequent microscopic study: Fix in 10% neutral formalin; bleach thoroughly in 3% H₂O₂ in sunlight. Wash in distilled water. Stain bone 24 hours in 0.01 g. of Biebrich scarlet in 100 ml. of distilled water. Destain in 95% alcohol until soft tissues and cartilage are colorless. Stain cartilage 24 hours in a pH2 buffer solution of 2.1g. of citric acid per 100 ml. of water with 0.001 g. of methylene blue. Destain in pH2 buffer solution until soft tissues are pale. Dehydrate in two changes of 95% alcohol in preparation for clearing. (This completes the destaining and may remove too much stain from the cartilage if destaining in the pH2 solution has been carried too far.) Place in Groat's clearing fluid and cover loosely so that the alcohol may evaporate, or remove the alcohol in vacuo. Groat's Mixture No. 19 is usually satisfactory.

For a combined stain, first stain bone, as above, and then apply the cartilage stain.

Seal jars with an ordinary liquid wood glue such as LePage's.     

Staining Methods For Studying Ingredient Distribution In Baked Cakes

A method is described for preparing cake crumb for sectioning and staining. Previous to embedding, the fat was stained and fixed by exposing small blocks of cake to the fumes from a 5%, freshly-prepared, aqueous solution of osmic acid (OsO₄). This was followed by dehydration in ethyl alcohol and tertiary butyl alcohol, removal of air under vacuum and infiltration with paraffin.

Sections were cut 20 and 9Op thick and mounted with water.

Wax was removed by immersion in xylene. The sections were rehydrated in a series of ethyl alcohol dilutions, from concentrated to dilute, then transferred to distilled water.

Protein was then stained pink by immersion of the slides in an acidified 0.04% water solution of eosin Y, or starch was stained blue with a dilute aqueous solution of iodine. Ten grams iodine and 10 g. KI were dissolved in 25 ml. distilled water. This stock solution was diluted for use one to two hundred times.

The relationship between protein and starch was demonstrated by staining the sections with eosin, differentiating in 50% alcohol and staining with iodine.

When slides of cake crumb were prepared in this way, the fat was stained black, the protein bright pink and the starch granules a dark blue.     

A Consideration of French's Test of Basic Fuchsin for Endo's Medium

In describing a method of testing for the return of color in decolorized fuchsin for use in Endo Medium, French states that variations in hydrogen ion concentration fail to influence the appearance of color in this medium.

Duplications of this test were made using alcoholic and aqueous solutions of fuchsin and both sodium sulfite and sodium bisulfite as decolorizing agents.

In the decolorized alcoholic solutions of fuchsin the color failed to reappear when formalin was added, but a small amount of a weak solution of lactic acid caused the color to return.

Alcoholic solutions of fuchsin failed to decolorize in sodium bisulfite solutions until a few drops of NaOH were added. The color, then, reappeared immediately.

Solutions of peptones to which fuchsin had been added were substituted for the original fuchsin solution. Alcoholic and aqueous solutions of fuchsin were added to equal amounts of a 1% peptone solution. The peptone solutions varied in their hydrogen ion concentration and the results showed that those which were neutral decolorized readily while the more acid solutions were but partially decolorized.

Fuchsin decolorized according to results found in this test, was not satisfactory in the Endo medium, especially in the case of the aqueous solutions of fuchsin.

Experiments which were carried on by other workers and checked with this method all indicated that some acid is necessary to secure the restoration of color.     

The use of pleopods for shell water circulation and respiration by hermit crabs

The use of the pleopods was investigated in two species of Diogenid hermit crabs, Dardanus arrosor and D. calidus. A transparent glass shell was used to observe the movements of the pleopods in hermit crabs. The movements consisted of periodic, irregular beating, which generated an irregular flow of the water within the shell.

A strong, regular beating movement was elicited when there was detritus or faeces in the shell, as was occupation of a new shell. Injection of water with a low oxygen concentration into the shell failed to induce any variation in the pleopod beating frequency (PBF), while the injection of water with a high CO₂ concentration induced a sudden PBF increase. When pH was varied and CO₂ concentration held at normal atmospheric level, there was a change in PBF. However, CO₂ variation alone, at constant pH, did not trigger any visible reaction. This suggests that there is a receptor for pH, which acts independently of the CO₂ concentration.

The function of males hermit crabs pleopods has always been obscure. However, at least in Dardanus, they are actively involved in water circulation within the shell for shell-cleaning and probably for ventilation.     

The Azure Dyes: Their Purification and Physicochemical Properties. I. Purification of Azure A

A method is described for the separation of azure A from commezcial samples of polychrome methylene blue. Up to 300 mg of the pure dye may be isolated in this way. The method is based on chromatography using columns 90 cm high, 7 cm in diameter, loaded with 3 g of polychrome methylene blue. The absorbent is silica gel, the eluent a mixture of acetic and formic acid.

Poor solubility of the dye acetate in water necessitates dissociation of the acetate by alkalinization and subsequent conversion of the dye to the chloride with diluted Ha. Demethylation that occasionally occurs during this procedure is negligible. Pure azure A does not spontaneously demethylate under ordinary conditions.     

Inhibition of oxidative DNA damage in vitro by extracts of brussels sprouts

Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light.

The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC₅₀ values were 6-20 μg/ml corresponding to the extract of 5-20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C.

Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable.     

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.