The Standard Romanowsky-Giemsa Stain in Histology

A new and technically simple Romanowsky-Giemsa (RG) stain is proposed as a standardized technique for use in histology. An RG stock solution (pure azure B 7.5 g/l, eosin Y as eosinic acid 1.2 g/l in dimethylsulfoxide) is diluted to form the working solution with HBPES-buffer, pH 6. Staining time is 30-90 min after formolcalcium solution (or 2-4 hr after formaldehyde-organic acid mixtures). The resulting overstained sections are to be differentiated. A tannic acid-acetic acid combination in an isopropanol-water mixture was found to give optimum results within 100 sec. Subsequent dehydration is in isopropanol only. The staining pattern obtained is polychrome. The distribution of colors in detail is influenced by the modes of pre- and posttreatment. Of practical interest is the development of green and greenish blue colors on collagen fibrils which contrast strongly against the pink of sarcoplasm. For this and other reasons, this RG stain version seems suitable to replace the trichrome Gomori-type trichrome stains under appropriate processing conditions.     

Standard Specimens for Stain Calibration: Application to Romanowsky-Giemsa Staining

Standardized specimens with reprodcible 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.     

A Quick-Mixed Aluminum Hematoxylin Stain

Two stock solutions are composed as follows: A) aluminum sulfate, sodium iodate and acetic acid in aqueous propylene glycol and B) hematoxylin in pure propylene glycol. When combined in specified proportions the stock solutions yield aluminum-hematein dissolved in nontoxic propylene glycol. The ready-to-use stain, prepared in small volumes as needed, performs well in paraffin sections of plant tissues.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

A Mysterious Malady: the Malachowski-Wright-Giemsa Stain to the Rescue

Abstract

A patient entered hospital with a puzzling absolute monocytosis. Admitting blood smears had been stained with Diff-Quik, a Romanowsky stain. When additional smears were stained using a standard Malachowski-Wright-Giemsa method, the reason for the monocytosis became abundantly clear.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

Confocal laser Scanning Microscopy of Mitochondria within Microspore Tetrads of Plants Using Rhodamine 123 as a Fluorescent Vital Stain

The present study demonstrates that rhodamine 123 penetrates the callose walls surrounding plant microspores before they are released from tetrads. The stain accumulates in active mitochondria due to the electrical potential across the mitochondria1 membrane. Accumulation of dye does not occur in mitochondria of fixed cells and fades quickly when mitochondrial activity is inhibited by exposure to carbonyl cyanide m-chlorophenyl hydrazone. Rhodamine can be used as a viability test for microspores still within tetrads, thus making it possible to determine when during development genes leading to pollen sterility are expressed. Rhodamine 123 is excited by blue (550 nm) light and can thus be used with confocal laser scanning microscopy. Anthers of Nicotiana tabacum, Oenothera villari-cae, Silene dioica and Lycopersicum esculentum were studied here.     

A Modified Hortega-Globus Stain is Superior to Bielschowsky and Bodian Stains for Demonstrating Neuritic Plaques

The quantitative assessment of the age-dependent number of neuritic plaques is essential for the diagnosis of Alzheimer type dementia. This study reports the superiority of a modified Hortega-Globus stain compared to Bielschowsky and Bodian stains applied to samples obtained from ten brains of patients with a clinical history of progressive dementia. In two of ten cases only the modified Hortega-Globus stain allowed confirmation of the diagnosis of senile dementia of the Alzheimer type (SDAT). The counts of neuritic plaques in sections stained by other methods were not sufficient to establish the histological diagnosis of SDAT. These results indicate that the choice of the most sensitive staining method is critical for the correct histopathologic diagnosis of the Alzheimer type dementia.     

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).     

Zinc-Specific N-(6-Methoxy-8-Quinolyl)-Para-Toluenesulfonamide as a Selective Nontoxic Fluorescence Stain for Pancreatic Islets

Separation of the endocrine from the exocrine pancreatic tissue by fluorescence activated sorting has been limited by the lack of an ideal fluorescent label for islet tissue. Our studies indicates the zinc-specific stain N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), has characteristics ideal for use as a fluorescent label for islet tissue. Dispersed rat pancreas cells stained with TSQ produced bright blue fluorescence when excited by UV light [peak emission wavelength at 480 nm. maximal excitation at 365 nm). The fluorescence was specific for islet tissue as confirmed by counterstaining with the islet-specific stain dithizone and there was minimal background staining of exocrine tissue. Stained tissue remained brightly fluorescent for 2 hr. with some fading by 4 hr. Injection of TSQ into rats at a concentration sufficient to produce staining of islets produced no toxicity discernible at 4 months. The viability of isolated rat islets stained with TSQ was maintained as shown by supravital staining, in vitro secretion of insulin, and reversal of diabetes after transplantation of stained islets into diabetic syngeneic recipients.     

Histology and histochemical enzyme‐staining patterns of major immune organs in Epinephelus malabaricus

The histological architecture of major immune organs in the Malabar grouper Epinephelus malabaricus was investigated. The novel characteristics such as melanomacrophage centres (MMCs) appeared in the thymus and lymphopoietic tissue formed as foci in the head kidney. Leukocyte distribution in organs was identified by enzyme histochemistry. β‐glucuronidase (BG) reactive cells in the cortex region of the thymus were botryoidally aggregated. Both acid phosphatase (AcP) and BG reactive cells concentrated within the specialized lymphopoietic foci in the head kidney, suggesting that the foci might be functional. Primitive histological characters of immunity were observed in the spleen. Although leukocyte aggregation was demonstrated in the spleen, additional enzyme histochemistry indicated that the aggregate might not be the equivalent of white pulp found in other vertebrates. The histological evidence did not support intestinal involvement in the immune system: there was no demonstrable gut associated lymphoid tissue. The limited distribution in the cortex and medulla boundary and the condensed format of the lymphocytes suggests a functional role for the MMC in the thymus of E. malabaricus . The MMC appearance in the thymus of a teleost was unusual.     

Histology,ultrastructure, and pigmentation in the horny scales of growing crocodilians

Alibardi L. 2011. Histology, ultrastructure, and pigmentation in the horny scales of growing crocodilians. —Acta Zoologica (Stockholm) 92 : 187–200. The present morphological study describes the color of hatchling, juvenile, and adult crocodilian skin and the origin of its pigmentation. In situ hybridization and immunostaining indicate that crocodilian scales grow as an expansion of the proliferating epidermis of the hinge region that form thin lateral rings. In more central areas of growing scales, new epidermal layers contribute to increase the thickness of the stratum corneum. The dark pigmentation and color pattern derive from the different distribution of epidermal and dermal chromatophores. The more intensely pigmented stripes, irregular patches and dot‐like spots, especially numerous in dorsal scales, derive from the incorporation of the eumelanosomes of epidermal melanocytes in differentiating beta cells of the epidermis. Dermal melanophores, mainly localized in the loose upper part of the dermis, also contribute to the formation of the dark or gray background of crocodilian scales. The eumelanosomes of dermal melanophores determine the darkening of the skin pattern in association with the epidermal melanocytes. Iridophores are infrequent, while xantophores are present in the species analyzed with a sparse distribution in the superficial dermis among melanophores. The presence of xantophores and of the few iridophores in areas where epidermal melanocytes are absent appear to determine the brown or the light yellow‐orange background observed among the darker regions of crocodilian scales.     

News from the Biological Stain Commission No. 14

Abstract

In the 14^th issue of News from the Biological Stain Commission (BSC) the BSC’s International Affairs Committee presents information from the meetings of ISO/TC 212/WG 3, In vitro diagnostic products, and from the final plenary meeting of ISO/TC 212, Clinical laboratory testing and in vitro diagnostic test systems, held on 17-19 October 2011 in Las Vegas, NV. Information is also presented from the 26th meeting of CEN/TC 140, In vitro diagnostic medical devices, held on 5 December 2011 in Berlin, Germany.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

How formalin affects the outcome of routine and special stains

The discovery of formaldehyde for preserving tissue structures produced a new dimension in microscopy. Preserving structure and morphology became important; therefore, identifying a proper fixing agent for particular structures, chemical entities, and tissues, also became important. The methods for demonstrating tissue structures evolved and were implemented with careful observation and documentation of the results and outcomes. Formalin was incorporated into many techniques, and provided helpful results in many cases and hindrances in others. The effects of formalin on the outcomes of routine and special staining techniques are reported here.     

How formalin affects the outcome of routine and special stains

The discovery of formaldehyde for preserving tissue structures produced a new dimension in microscopy. Preserving structure and morphology became important; therefore, identifying a proper fixing agent for particular structures, chemical entities, and tissues, also became important. The methods for demonstrating tissue structures evolved and were implemented with careful observation and documentation of the results and outcomes. Formalin was incorporated into many techniques, and provided helpful results in many cases and hindrances in others. The effects of formalin on the outcomes of routine and special staining techniques are reported here.     

古尼虫草组织学研究

用光学显微镜,扫描电子显微镜对不同成熟度的古尼虫草进行了组织学观察,结果表明,古尼虫草子囊顶部有增厚的幅状结构,包囊子囊壳的菌丝致密而纤细,子座菌髓管状菌丝粗壮而疏松,形成很多的中空结构,古尼拟青霉分生孢子梗顶端膨大,形成分生孢子,分生孢子属外生芽殖型。     

改良革兰染色法在显示石蜡切片真菌中的应用

目的建立真菌的病理切片改良革兰染色法。方法选取确诊真菌感染的组织蜡块,切若干空白片,通过苏木素-伊红染色（HE）、高碘酸-无色品红染色（PAS）、六胺银染色（GMS）、改良革兰染色后,比较改良革兰染色法的染色效果。结果改良革兰染色法的染色效果好,该法具有易操作、染色效果稳定等优点。结论改良革兰染色法能够清晰地显示出组织切片中的真菌,并且染色效果稳定,在检测组织中的真菌时可发挥重要的辅助诊断价值,具有广泛的应用前景。     

The Papanicolaou Stain for Negri Bodies in Paraffin Sections

Paraffin sections ot the hippocampus (Amnion's horn) from brains of dogs and cows, fixed in sublimate-alcohol (HgCl₂, sat. aq., 1 vol.; absolute alcohol, 2 vol.) were stained by Papanicolaou's (1942) method for vaginal smears. Negri bodies were stained a bright rose color, with nucleoli dark blue. Even though the orange G were omitted, good staining of Negri bodies was obtained. Eliminating this dye simplifies the technic without impairing its effectiveness in the diagnosis of rabies, but the complete staining gives a somewhat more colorful and brighter histologic picture.