Spectrophotometric Characteristics and Assay of Biological Stains

I. The chemical and spectrophotometric methods adopted in the Stain Commission laboratories for the certification of stains are described. Spectrophotometric examination is by the Beckman spectrophotometer in which the color density at the absorption maximum is determined as well as a ratio of color densities designed to detect abnormal symmetry of the absorption curve. A somewhat modified titanous chloride assay is described; eerie sulfate is used to determine the normality of the unstable titanous chloride, and the oxidant solution, which is stable, is standardized against Bureau of Standards arsenious oxide. The general approach used for re-study of the stains by these methods and the criteria necessary for adoption of the spectrophotometric method of assay are described.

II. This report gives the details of the spectrophotometric examination and assay of the individual stains in the thiazin group, namely methylene blue, thionin, methylene violet, toluidine blue O, and azures A, B, and C. The spectrophotometric method of assay was adopted for the first three named; the others showed too large a variation among samples to permit adoption of this simpler type of assay. In the case of methylene violet it was necessary to do nitrogen analyses to relate purity of the dye and color density. Complete absorption curves for typical samples of each of the above stains are presented.     

Spectrophotometric Characteristics and Assay of Biological Stains III. The Xanthenes

In this continuation paper of the work on the chemical and spectrophotometic characteristics of commercial stains, data on the xanthene dyes are presented. In the xanthene group of dyes, it has been found possible to assay pyronin B, eosins B and Y and ethyl eosin by spectrophotometric means. Phloxine B, rose Bengal, and erythrosin B are assayed by die color acid precipitation method. Typical absorption curves are given for these dyes as well as representative spectral and assay data.     

Improving Biological Dyes and Stains: Quality Testing Versus Standardization

This paper discusses the impact of both standardization and quality testing of dyes and stains in biology and medicine. After a brief review of why standardized dyes and stains are not presently available commercially, two types of testing and ways of improving dye quality are described. National or international organizations could be established to define standardization of dyes and stains. Standardization would be specifically defined as a list of physico-chemical parameters such as elaborated in this paper. Commercial batches of comparable quality may be labeled by the supplier as “standard dye.” a procedure currently performed by the European Council for Clinical and Laboratory Standardization (ECCLS). Also recommended to improve dye quality is commercial dye testing by independent laboratories with subsequent certification for use. This sort of quality control is currently carried out in the United States by the Biological Stain Commission (BSC). The advantages and disadvantages of both techniques and the use of image analysis for the definition of standards are discussed. A combination of both the BSC testing protocols and the ECCLS standards should be established for extended quality control of biological dyes and stains.     

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.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Absorption Ratios of Biological Stains

Absorption ratios are supplied for most of the dyes used as biological stains.

The use of these ratios will enable the analyst to identify the stains conveniently, and will also frequently afford information of considerable value respecting their purity.     

Progress in the Standardization of Stains the Standardization of Eosin and Related Dyes

Decolorizers which are distinctly acidic or basic in their chemical nature give abnormally high decolorization in the Gram stain for bacteria. Acidic substances yield more regular results. Ideally an “inert” decolorizer should be used, but ordinarily such substances will not dissolve the dye or dye-mordant precipitate from the smear. The most practical substances seem to be those so very slightly acidic in character as to be practically inert, such as acetone or alcohol, or a mixture of such substances.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Progress in the Standardization of Stains Research on the Chemistry of Dyes

One of the services that the Stain Commission has been able to perform for biologists is in assisting them to identify dyes of unknown composition with which desired staining effects have been secured. This enables them both to obtain new supplies of the same dyes and to publish their results in such a form that other biologists may duplicate them.