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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.

Azure Stains↗

Section preparation of human marrow for light microscopy.

Preparation of human marrow sections has been studied systematically in order to facilitate accurate identification of marrow cells. Both of the methods developed involve embedding marrow cores in methyl methacrylate. In one, acrolein fixation is followed by staining of deplasticized sections with eosine-y followed by azure II; in the other, neutrophilic forms are identified by their esterase-specific reactivity in marrow fixed with neutral-buffered formalin. These preparations are suitable for quantitative studies of marrow cellularity.

Acrolein↗

Light and scanning electron microscopic observations on the two contrasting types of sister chromatid differential staining after ultraviolet light irradiation.

Chinese hamster cells were grown with 50 microM 5-bromodeoxyuridine (BrdU) during the penultimate S phase to obtain chromosomes with the TB-TT chromatid constitution. Chromosome preparations made by the airdrying method were used to study the sister chromatid differential staining (SCD) resulting from ultraviolet (UV) irradiation followed by Giemsa staining by light and scanning electron microscopy (SEM). When chromosomes irradiated with UV light (253.7 nm, 5.2 J/m2/s) for more than 5 h were stained with 1% to 4% Giemsa in phosphate buffered saline (PBS) or in distilled water, the resulting SCD invariably belonged to the B-light type in which the TB-chromatid stained lightly. SEM observations of these chromosomes suggested that the B-light SCD was due to the selective photolysis of the TB-chromatid. On the other hand, when chromosomes were irradiated for only 10 min, and stained with 1% Giemsa in PBS, they showed a B-dark type SCD in which the TB-chromatid stained darkly. However, when chromosomes irradiated for 10 min were stained with 4% Giemsa in PBS or 1% Giemsa in distilled water, the resulting SCD again belonged to the B-light type. These findings indicate that when the irradiation dose is small, the resultant SCD is not a simple reflection of selective photolysis in the TB-chromatids and the type of SCD depends not only on the concentration of Giemsa but also on the salinity of the staining solution.

Animals↗

A modified technique for combination of cytochemistry and radioautography of haemic cells.

Cultured mouse bone marrow and human blood cells were labelled with 3H-thymidine, smeared, and stained, using peroxydase reagents or the May-Grünwald Giemsa procedure. The slides were dipped in a approximately 12% solution of polyvinylchloride, bleached by a photographic fixer and processed for liquid emulsion radioautography. This method yielded technically very satisfactory radioautographs of pre-stained smears.

Animals↗

Comparative colorimetry in cytophotometric measurements of azure B-eosin Y-stained and Giemsa-stained blood cell smears.

The advantages of the Commission Internationale de l'Eclairage (CIE) color measurements and the use of the standardizable purified azure B-eosin Y stain, as compared to the commonly used commercial Giemsa stain, were studied. Color measurement and analysis according to the CIE standards allows the comparison of color measurements from different slides stained by either the same or different procedures. Moreover, measurements from different cytophotometric systems can be compared. The results can also be directly correlated with human visual subjective color estimates and thus used to quantify the human observations. The color information in the air-dried cell specimens, which differs from the color characteristics of the components of the staining solution, was measured and analyzed.

Azure Stains↗

A survey of transforms of the CIE 1931 chromaticity diagram with some new non-linear transforms and histological illustrations of their utility.

Surveys are presented of the published data on colour matching, and of the published transforms of the CIE 1931 chromaticity diagram. All transforms are given in the form of standardized equations and tables of coefficients. Both linear and non-linear transforms are considered, although the latter are given special attention. The concept of a uniform chromaticity space (UCS) is discussed, and the published transforms are assessed for uniformity using the colour matching data. 2 series of new, non-linear transforms are presented, both based on 2-dimensional polynomial equations, and both derived using iterative optimization techniques. The 1st series attempts to approximate Farnsworth's (1961) diagram. The 2nd uses Nutting's colour matching points directly. The coefficients of these transforms are tabulated. It was found that increasing the number of coefficients in the 2-dimensional polynomial, beyond 15 each for x and y, does little to increase the uniformity of the resulting transform. The usefulness of UCS's in assessing stain performance is illustrated by examples drawn from the areas of azure B/eosin staining of blood cells and Papanicolaou staining of epithelial cells from the uterine cervix.

Blood Cells↗

Dotted chromosomes produced in sodium phosphate solution supersaturated with NaHCO3.

Dotted chromosomes were consistently produced in both BrdU and non-BrdU substituted Chinese hamster cells after treatment with 1.0 M Na-phosphate solution, adjusted to pH 9.0 with a supersaturating amount of NaHCO3, and at a temperature of 80--95 degrees C. -- A series of changes in chromosome morphology was produced as the temperature of the solution was progressively increased. In BrdU-treated cells, G-banding and differentially stained sister chromatids were sequentially produced prior to the appearance of dots. In non-BrdU treated cells, only G-banding was produced before dot formation. In general, the patterns of dots correspond to the G-banding patterns. --Chromatids, with uni- or bifilarly BrdU substituted DNA or with normal DNA, required differential temperatures for the production of dots. Since the temperature required for dot formation was always slightly higher than that required for producing differentially stained chromatids, this phenomenon can be used as an important indicator for determining the optimal temperature required for revealing differentially stained chromatids.

Animals↗

Microwave-assisted staining procedures in routine histopathology.

The use and practicability of microwave-assisted staining procedures in routine histopathology over more than three years has been evaluated. A domestic microwave oven was used to speed up the following staining procedures: Haematoxylin-Eosin (for frozen sections), Romanowsky-Giemsa, Periodic acid-Schiff (PAS), Ziehl-Neelson, Papanicolaou, Feulgen and Grocott--stain on buffered formalin fixed sections or cytologic smears. These staining procedures can be made highly reproducible providing; (1) Staining vessels are placed in the same position inside the oven; (2) Accurate timing in seconds is observed. Microwave-assisted staining procedures are equal to or even superior to those of the standard methods. Staining times can be reduced to 2%-10% of the conventional staining procedures. The basic staining protocols are presented.

Azure Stains↗