Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears

Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).     

Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

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.     

The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.     

Understanding Romanowsky staining

Summary Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of toxic granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).To whom offprint should be sent     

A convenient zymogram stain for cellulases

The release of dye from phosphoric acid-swollen cellulose azure by endocellulases and exocellulases is the basis of a technique for locating these enzymes on gels.     

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Microspectrophotometric Studies of Romanowsky Stained Blood Cells. III. The Action of Methylene Blue and Azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. I. Staining Properties of Commercial Samples and Their Component Dyes

SYNOPSIS. Studies on the composition of commercial Giemsa stain and its effect upon staining quality are reported. These studies were supplemented by observations on the preparation of the components of Giemsa stain and their staining properties in aqueous solution, in Nocht's solution, and in laboratory prepared Giemsa stains containing one azure component. Five groups of commercial batches were differentiated on the basis of their staining reactions on thick and thin films of bovine blood containing Babesia bigemina and B. argentina. Spectrophotometric and chromatographic analysis showed that four groups differed in the proportions of the thiazine components present, while the fifth-group did not appear to be Giemsa stain. Comparison of their staining effects with those obtained with each component in laboratory prepared stains indicated that the major effects of commercial batches on both blood cells and parasites were due to the thiazine component or components in highest proportions, with satisfactory staining of protozoa associated with those batches containing high proportions of methylene blue and azure B and low proportions of the remaining thiazine components.
The function of each component of Giemsa stain is defined and the need for the proper balancing of thiazine eosinates with free azure is shown. Close correlation was obtained between analysis by spectrophotometry and chromatography and direct staining tests when samples initially with low MX values were re-examined spectrophotometrically after removal of their methylene violet content. The existence of a leuco form of eosin is reported and its possible significance to the Romanowsky effect is discussed.     

Acid alcoholic brilliant cresyl blue stain for gastric and other mucins

Summary Some but not all samples of brilliant cresyl blue (6-methyl-7-dimethylamino-2-phenoxazin chloride) under C. I. No. 51010 in Conn's Biological Stains when dissolved at 1% level in 50–70% alcohol containing 1% concentrated (12 N) hydrochloric acid, stain (in 30 min) a wide variety of human and laboratory animal mucins blue black on an almost unstained background. The mucoprotein of the gastric surface epithelium and of the peptic gland neck cells of several species reacts strongly. A 16 hr 60° C methylation in 0.1 M methyl-sulfuric acid in methanol is required to block the staining of these gastric and some intestinal mucins, while 1–2 hr intervals suffice to prevent the staining of mast cells, cartilage and metachromatic sulfomucins generally. Saponification (1% KOH/70% alcohol, 20min) does not restore staining in either location group, indicating that sulfate mucins are probably reacting in both.Most other basic dyes fail to stain mucins from acid alcohol solutions: azure A, toluidine blue, resorcin blue, orcein, resorufin, azoresorufin brown, azolitmin, lacmoid, gallocyanin, Nile blue, methylene green, pararosanilin, crystal violet, Victoria blue R. Some staining occurred with one of three lots of Victoria blue B, with two lots of Victoria blue 4 R and with one lot each of Bernthsen's methylene violet, elastin violet PR and elastin purple PP.The stain may be preceded by the Feulgen reaction to give red nuclei, or followed by a brief collagen stain in an alcoholic acid fuchsin (0.05–0.1%), picric acid (1.5%) solution.Presented before the Symposium of the Histochemische Gesellschaft in Hamburg, 28. September 1968.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

On the mechanism of Verhoeff's elastica stain: A convenient stain for myelin sheaths

Summary Verhoeff (1908) recommended an iron-hematein formula containing Lugol's solution for demonstration of elastic tissue; sections are differentiated until desired staining patterns are obtained. Verhoeff's stain colored a variety of tissue structures and showed higher substantivity for myelin sheaths than for elastin. Addition of HCL or omission of Lugol's solution decreased or abolished coloration of pseudo-elastica and thus enhanced selectivity for elastin. Substitution of Fe⁺⁺ for Fe⁺⁺⁺ abolished dye binding by elastin.A review of chemical data indicated interaction of components of Lugol's solution with the dye. Hematein and Fe⁺⁺⁺ form a variety of cationic, anionic and non-ionic chelates; the ratio of these compounds changes with time. Dye binding apparently occurs mainly via van der Waals forces and hydrogen bonds.Verhoeff's elastica stain is definitely not specific for elastin and is inferior to orcein and resorcin-fuchsin because of the required differentiation with its inherent bias to produce patterns which conform to expectations. However, Verhoeff's elastica stain is far superior to other metal-hematein technics for myelin sheaths. The combined Verhoeff-picro-Sirius Red F3BA stain can be performed in 30 min and does not require differentiation. It is therefore suggested to reclassify Verhoeff's elastica stain as a method for myelin sheaths.     

Colorimetry for the Stain Technologist. III. The Specification of Color Difference

This paper illustrates the calculation of color differences, involving luminance as well as chromaticity components. Color differences have been calculated for a large number of stained histological objects. Four different color difference formulae have been used, namely, those associated with the FMC 2, U*V*W*, L*u*v* and L^*a^*b^* systems. Comparison has first been made between various hematological substrates after staining with two different azure B-eosin Y stains. Next, comparison is made for the same substrates after staining with one of the azure B stains and a methylene blue-eosin Y stain. Pairwise comparison is also made of various substrates from the epithelium of the uterine cervix after Papanicolaou staining. Finally, pairwise comparison documents color differences accompanying maturation for the erythroid and myeloid cell lines in azure B-eosin Y stained bone marrow. The limitations of current color difference formulae are discussed.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be ''Gram-positive,'' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be ''Gram-negative.'' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths.

Verhoeff (1908) recommended an iron-hematein formula containing Lugol's solution for demonstration of elastic tissue; sections are differentiated until desired staining patterns are obtained. Verhoeff's stain colored a variety of tissue structures and showed higher substantivity for myelin sheaths than for elastin. Addition of HCL or omission of Lugol's solution decreased or abolished coloration of pseudo-elastica and thus enhanced selectivity for elastin. Substitution of Fe++ for Fe+++ abolished dye binding by elastin. A review of chemical data indicated interaction of components of Lugol's solution with the dye. Hematein and Fe+++ form a variety of cationic, anionic and non-ionic chelates; the ratio of these compounds changes with time. Dye binding apparently occurs mainly via van der Waals forces and hydrogen bonds. Verhoeff's elastica stain is definitely not specific for elastin and is inferior to orcein and resorcin-fuchsin because of the required differentiation with its inherent bias to produce patterns which conform to expectations. However, Verhoeff's elastica stain is far superior to other metal-hematein technics for myelin sheaths. The combined Verhoeff-picro-Sirius Red F3BA stain can be performed in 30 min and does not require differentiation. It is therefore suggested to reclassify Verhoeff's elastica stain as a method for myelin sheaths.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. II. Changes in Erythrocytes Infected with Babesia bigemina and B. argentina

SYNOPSIS. Cytoplasmic stippling, intensification of the cell margin, and alterations in color, which have been reported in erythrocytes parasitized by Plasmodium falciparum in man, have been seen also in bovine erythrocytes parasitized by either Babesia bigemina or B. argentina. These changes appear to be identical in the human and bovine infections.
Tests with each component of Giemsa stain in simple aqueous solutions alone and in various combinations with eosin, together with tests with Giemsa stains containing one azure component, showed that demonstration of the changes depends on the presence of azure A and eosin and on prolonged staining times at pH 7.2 to 7.4. Specific tests suggested that the changes represent catabolic by-products of the parasites.     

Standardization of the Romanowsky staining procedure: an overview

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.     

Fresh Tissue Diagnosis in the Operating Room

A polychromatic stain giving color reactions closely simulating hematoxylin and eosin, without overstaining and obscuring the finer detail of the cell, has been developed in this laboratory. It is as follows: To 4 parts of 1% aqueous azure A (azure I) which has been previously filtered, add very rapidly one part of filtered 0.5% aqueous Erie garnet B. The mixture is immediately filtered to prevent precipitation. Occasional refiltering may be necessary if the mixture has been standing a month or more. Float tissue slices from a freezing microtome directly into distilled water; pick them up with a round glass rod and immerse into the stain which has been poured into a small section dish. The staining time varies with different tissues but 10 to 15 seconds usually gives the most satisfactory results. Lift sections from the stain, still on the same glass rod, float thru two changes of distilled water, and then transfer to a clean glass slide. Place a large drop of 40% glucose on a clean cover slip and immediately invert over the section. The mounted specimen is then ready for microscopic examination. In examining the tissues under the microscope it is best to employ an intense artificial light (an ordinary 40 or 60 watt bulb is sufficient). After mounting, one-half to one minute should elapse before examining the section to allow it to clear.     

Azure Stains

Two uses of methylene azure are suggested. This dye gives a very good nuclear stain after most fixations when preceded by weak NaOH; but eosin Y cannot be used as a counter-stain. Methylene azure also proves very useful in the Mallory eosinemethylene-blue technic, in which it can be substituted to advantage for polychrome methylene blue. The following three schedules are recommended:

2.5% aqueous phloxined˙ 15 minutes

0.1% aqueous azured˙ 1-30 minutes

2.5% aqueous phloxined˙ 15 minutes

Mixture in equal parts of 0.1% azure and 0.1% methylene blued˙ 30 minutes

2.5% aqueous phloxined˙ 1 minute

1.0% aqueous azured˙ 1-2 minutes

Of these the first two give rather better results; but when time is lacking the third is quite satisfactory.     

Newer applications of the histological stain prepared from Pterocarpus santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.