Article

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

Histochemistry 01/1987; 86(3):331-6.
Source: PubMed

ABSTRACT 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).

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    ABSTRACT: 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).
    Histochemie 04/1987; 86(3):331-336. DOI:10.1007/BF00490267 · 2.93 Impact Factor
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    ABSTRACT: Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky--Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky--Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.
    The Histochemical Journal 05/1988; 20(6-7):329-34. DOI:10.1007/BF01002725
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    ABSTRACT: Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.
    Histochemie 12/1988; 91(1):77-80. DOI:10.1007/BF00501915 · 2.93 Impact Factor

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