An Improved Fixing Solution for Methylene Blue Preparations

A method is described which combines the writer's hot celloidin technic¹ with a form of the clearing-before-cutting procedure. The method requires only 16–17 days and yields a block which may be cut in any microtome, the sections being as thin as those afforded by paraffin with comparable material. The advantages of celloidin over paraffin, listed in the writer's earlier paper, are retained in the present method which, altho consuming more time than the hot process, requires less skill and gives superior results.     

Improved Fixation in Vitally Stained Methylene Blue Preparations

The present paper reports that ammonium molybdate dissolved in physiological saline for amphibian and mammalian tissues, and in sea water for squid tissues, forms a fixing solution that greatly reduces cellular distortion in vitally stained permanent methylene blue preparations.     

Staining Nerve Fibers with Methylene Blue. An Evaluation of Variables Used in An Immersion Technic

A study of the effects of osmotic pressure, pH, the presence of dextrose, acetate, pyruvate or lactate, and agents affecting cell permeability during supravital staining by methylene blue was made by means of an immersion technic. Mesentery and intestine of dogs and cats were used. Penetration of the dye was limited to the mesentery and more superficial layers of intestine. Conditions which facilitated the characteristic differentiation of of nerve fibers were: continuous oxygenation of the staining solution, pH about 5.6 stabilized by phosphate buffer, and the presence of small amounts of acetate and lactate. Young animals' tissue stained better than old. Methylene blue was a much more effective staining agent than less completely methylated thionins.     

Brilliant Cresyl Blue as A Stain for Chromosome Smear Preparations

Brilliant cresyl blue is substituted for carmine in the acetocarmine technic. A mixture of one part of 1% toluidine blue and three parts 0.75% brilliant cresyl blue dissolved in 45% acetic acid gives good results. Propionic acid may be used instead of acetic acid for more rapid penetration.     

A Rapid HCl/Toluidine Blue Squash Technic for Plant Chromosomes

Fresh or pretreated root tips are simultaneously fixed and hydrolysed in 5 N HC1 for 15 min at room temperature. After washing they are macerated on a slide in a drop of 0.05% toluidine blue made up in McIlvaine citric acid-Na₂HPO, buffer at pH 4.0. Pressure on the cover slip completes the squash preparation. It is made permanent by removing the cover slip on dry ice, air drying and mounting in Euparal.     

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.     

Methylene Blue for Rapid Identification of the Parathyroids

Rapid identification of parathyroid tissue has been rendered possible by preoperative intravenous infusion of methylene blue before exploration of the neck. The technique has been used on 17 patients with thyroid and parathyroid disorders. In all cases one or more of the parathyroids have been demonstrated with histological confirmation, but with greater experience almost all have been shown readily. This has resulted in an appreciable reduction in operating time, and the method should help to reduce the high incidence of clinical hypoparathyroidism after total thyroidectomy.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

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.     

A Nile Blue Staining Technic for the Differentiation of Melanin and Lipofuscins

When paraffin sections are stained in 0.05-.01% Nile blue in 1 % sulfuric acid, washed thoroughly in water and mounted in aqueous media, lipofuscins color deep blue, melanins dark green, myelin and red cells lighter greens and background pale green. If, immediately after staining, the preparations are at once extracted with acetone with or without a 1% sulfuric acid rinse, melanins remain dark green, mast cells color purple, lipofuscins and background decolorize and nuclei may stain light green.     

A Nile Blue Staining Technic for the Differentiation of Melanin and Lipofuscins

When paraffin sections are stained in 0.05-.01% Nile blue in 1 % sulfuric acid, washed thoroughly in water and mounted in aqueous media, lipofuscins color deep blue, melanins dark green, myelin and red cells lighter greens and background pale green. If, immediately after staining, the preparations are at once extracted with acetone with or without a 1% sulfuric acid rinse, melanins remain dark green, mast cells color purple, lipofuscins and background decolorize and nuclei may stain light green.     

A Basic Fuchsin and Alkalinized Methylene Blue Rapid Stain for Epoxyembedded Tissue

Sections 1 μ thick of epoxy-embedded, OsO₄-fixed tissues were stained with 4% aqueous basic fuchsin at 70 C for 1 min, rinsed well and destained, also at 70 C, for 1 min. A 2% aqueous methylene blue solution, alkalinized to pH 12.5 by mixing 1 N NaOH with the dye on the slide in the proportion of about 2:1, was then allowed to act for 2 min at 23-27 C. The stain was rinsed off the slide, and the preparation air dried before applying a mounting medium and cover glass. The mounting medium consisted of immersion oil sealed with epoxy household cement. Stains had not faded after 1 yr. The method is simple, rapid (total time 4-5 min), and provides sharp contrast between cellular and connective tissue components.     

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.     

Reduced Methylene Blue as a Stain for Crustacean Nerves

Effective in situ staining of crustacean nerves was achieved with leuco methylene blue reduced with either ascorbic acid or sodium hydrosulfite (Na₂S₂O₄). A stock solution of methylene blue, 0.4% (ca. 0.001 M), and the reductants, ascorbic acid or sodium hydrosulfite (0.01 M), were prepared in van Harreveld's crayfish physiological solution. Methylene blue stock solution was mixed with either of the reductants in the approximate ratio of 1:10, v/v, and titrated to the end point. Ascorbic acid reduction is light catalyzed and requires intense illumination during titration. The cleared or leucomethylene blue stock solution is suitable for immediate use as a working nerve stain. With either reductant, the working solution oxidizes on standing in air, but can be titrated repeatedly without loss of staining properties. Dissected nerve trunks or tissue were immersed in the working stain for 20 min at room temperature and the staining process observed until suitable contrast developed. Excess dye was decanted and the tissues flooded with crayfish physiological solution. Contrast could sometimes be enhanced by flooding the stained area with 1% hydrogen peroxide in van Harreveld's solution. When permanent mounts were prepared, tissues were dehydrated with tertiary butyl alcohol in preference to ethyl alcohol series. For anatomical and neurophysiological studies of nerve distribution in crustaceans, the alternative use of either ascorbic acid or sodium hydrosulfite, as reductants for methylene blue, was preferable to the more complicated rongalit-technique and characterization of neural elements was fully as satisfactory.