Effect of Temperature on Argyrophil Impregnation: Development of A High Temperature Rapid Argyrophil Procedure

Out studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

The light microscopic demonstration of hydroperoxidase-positive Phi bodies and rods in leukocytes in acute myeloid leukemia.

Unique fusiform or spindle-shaped particles (Phi bodies) and rods with hydroperoxidase (catalase and/or peroxidase) activity are present in human granulocyte precursors only in acute myelogenous leukemia (AML). These newly recognized particles are much more numerous and prominent than Auer rods. They may be rapidly and readily identified using the microscope in marrow or peripheral blood films when the procedures recommended in this paper for fixation, incubation for hydroperoxidase demonstration in 3,3'-diaminobenzidine (DAB)/H2O2 medium, copper salt treatment and counterstaining (optional) with the Papanicolaou method are employed. Films prepared in the same manner but treated with benzidine/H2O2 medium for myeloperoxidase did not reveal these particles. We believe that Phi bodies are pathognomonic of AML since they are almost invariably present in AML patients with active disease. Their presence serves to distinguish AML from acute lymphocytic leukemia and from chronic granulocytic leukemia in blast crisis. Since the particles disappear in disease remission and reappear upon relapse, the recommended procedure is not only useful in diagnosis but in guiding therapy. When a very rapid diagnosis is needed, it is not necessary to counterstain the preparations, but the nuclei, cytoplasm and plasmalemma can readily be observed in the granulocyte precursors when they are counterstained by the Papanicolaou method. This treatment does not diminish the clarity of the Phi bodies and rods which stain by virtue of their peroxidatic activity. This cytochemical diagnostic procedure should be considered for adoption by hematology laboratories.     

Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.     

Large-scale purification of myeloperoxidase from HL60 promyelocytic cells: characterization and comparison to human neutrophil myeloperoxidase

A large-scale purification procedure was developed for the isolation of myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils.     

Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS

When collection of ejaculated sperm samples is not possible, as is the case with wild species, the epididymides of sacrificed wild males become the only possible source of spermatozoa. Mature cauda epididymal spermatozoa display characteristics similar to those of ejaculated sperm cells. The present work proposes a sperm staining technique suitable for the morphometric evaluation of red deer epididymal sperm using a new computerized system. Epididymides from wild animals were extracted no later than 2h post mortem. After epididymal sectioning, sperm samples were collected, cooled to and equilibrated at 5 degrees C, and frozen in liquid nitrogen. Before staining, sperm samples were thawed for 20s at 37 degrees C, and used for the preparation of slides. Three different sperm stains were tested: Hemacolor, Diff-Quik, and Harris' Hematoxylin. Morphometric analyses of sperm samples were performed using the morphologic module of the ISAS. Two hundred spermatozoa per sample and stain were captured at random and analyzed. Sperm morphometric values were significantly affected by the staining technique used. Moreover, significant differences were observed between animals. In our study, Diff-Quik could be considered to be the best sperm staining method, as it provided the highest percentage of well automatically analyzed cells by the ISAS, and discriminates better between animals. This sperm staining technique also proved to be a useful method for characterizing and discriminating between sperm samples of different animals.     

Carotenoid and Pterin Pigment Localization in Fish Chromatophores

The classical sulfuric acid method for the histochemical detection of carotenoids has been adapted to give a reliable cytological localization of these compounds in fish chromatophores. This procedure consists mainly in fixing skin fragments in glutaraldehyde and dehydrating in a 50% solution of glycerin followed by exposure to air. It is essential that the preparations permit direct contact of the sulfuric acid with the pigment cells. Under these conditions, carotenoid containing cells stain green or blue. When associated with the extraction of the carotenoids by acetone, the procedure permits the distinction between pterin and carotenoid in fish chromatophores.     

The Histochemical Demonstration of Hemoglobin in Blood Cells and Tissue Smears

The demonstration of intracellular hemoglobin in permanent preparations has long been a problem. The affinity of hemoglobin for iron hematoxylin is well known but this stain also colors yolk, chromatin, and other structures and is therefore not a reliable criterion. The presence of hemoglobin has been associated with an acidophil cytoplasm which stains a characteristic color, but a careful inspection of living cells in early hematopoetic or embryological stages demonstrates that hemoglobin is present in the erythrocytes which are quite basophilic. In the course of some research on the blood of embryonic frogs it became desirable to demonstrate the presence of hemoglobin in cells by means of a specific staining reaction.     

A Simplified Method for Staining Mast Cells with Astra Blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however, the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl₂ · 6H₂O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

A simplified method for staining mast cells with astra blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Differential Staining of Calcified Tissues in Plastic Embedded Microtome Sections by A Modification of Movat's Pentachrome Stain

Movat's pentachrome I stain has been adapted and modified as a stain for Undecalcified bone sections. After embedding in methyl methacrylate, this procedure yields consistently good results, with an excellent and colorful contrast between mineralized and unmineralized compartments of both cartilage and bone. in addition, osteoblats, osteoclasts, and other cells and tissue components can easily be differentiated. the staining properties of the lacunar wall surrounding the osteocytes are considered to reflect various states of osteocytic activity. the method is especially useful for the study of bone growth and bone repair, and as a stain for conventional histomorphometry and computer-assisted image analysis in bone biopsies.     

Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)]

Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.     

Differential staining of calcified tissues in plastic embedded microtome sections by a modification of Movat's pentachrome stain.

Movat's pentachrome I stain has been adapted and modified as a stain for undecalcified bone sections. After embedding in methyl methacrylate, this procedure yields consistently good results, with an excellent and colorful contrast between mineralized and unmineralized compartments of both cartilage and bone. In addition, osteoblasts, osteoclasts, and other cells and tissue components can easily be differentiated. The staining properties of the lacunar wall surrounding the osteocytes are considered to reflect various states of osteocytic activity. The method is especially useful for the study of bone growth and bone repair, and as a stain for conventional histomorphometry and computer-assisted image analysis in bone biopsies.     

HISTOCHEMICAL STUDIES ON MUCOPROTEINS IN NERVE CELLS OF THE DOG

Autonomic and sensory ganglion cells in the senile dog contain a deposition of PAS-positive substances which has been shown to be mucoprotein in nature. Data are presented to show that this PAS-positive mucoprotein can be demonstrated by metachromatic staining with toluidine blue after the mucoprotein is sulfated. This procedure indicates that mucoprotein is also present in a granular form in all nerve cells in both senile and young dogs. The evidence for this is further substantiated by the use of the aldehyde-fuchsin stain following both periodic acid oxidation and sulfation. The granular and non-granular deposition can be demonstrated by the periodic acid-aldehyde-fuchsin method due to the affinity of the aldehyde-fuchsin stain for aldehydes. It can be demonstrated following the sulfation-aldehyde-fuchsin method owing to the affinity of the stain for the sulfuric group. The evidence for this latter phenomenon has been reported by Scott and Clayton (6). It is concluded that mucoprotein is present in a granular form in all nerve cells in both senile and young dogs but is not concentrated enough in the latter to be demonstrated by the PAS method.     

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.     

Standardized thionin-eosin stain in bronchial cytology. A substitute for hematoxylin-eosin Y staining

A standardized thionin-eosinic acid stain was developed as a quick and highly reproducible staining method for bronchial cytology. Bronchial smears and paraffin-embedded sputum samples were stained with thionin-eosin and with the conventional hematoxylin-eosin Y. Spectral absorption characteristics and staining intensity of thionin-eosin-stained cells were investigated by means of cytophotometry. The staining pattern of thionin-eosin is very close to that of the hematoxylin-eosin stain; the contrast between nucleus and cytoplasm is significantly higher for thionin-eosin. Thionin-eosin can be used for "dye-fixation" of cytologic smears and tissue imprints. Blueing and differentiation (as for hematoxylin-eosin) is not required for thionin-eosin; thus, fixation and staining can be performed within two minutes. The spectral absorption characteristics of thionin-eosin allow reliable automated cytophotometric discrimination of cell nuclei and cytoplasm. The standardized thionin-eosin stain is recommended as a substitute for the hematoxylin and eosin stain in bronchial cytology.     

Evaluation of a new system for the fixation, concentration, and staining of intestinal parasites in fecal specimens, with critical observations on the trichrome stain

Proto-fix (Alpha-Tec Systems, Inc., Vancouver, WA) is a new single vial, environmentally safe, parasitology (pathogenic and nonpathogenic protozoans and helminths) fixative and transport solution. It is used in conjunction with a new concentration/sedimentation reagent, CONSED, (Alpha-Tec Systems, Inc. Vancouver, WA) as a replacement to the formalin-ethyl acetate (FEA) concentration procedure using Lugol's iodine. The newly adopted procedure was tested against the FEA concentration samples using split proficiency testing samples supplied by the American Association of Bioanalysts (AAB). Routinely, patient samples collected, fixed, and transported in Proto-Fix were processed and tested at Diagnostic Labs, Inc. (DLI), Phoenix, AZ. Detected parasites were documented using a video camera-printer system attached to the optical equipment. The quality of the fixative and stain were found to be superior to that of the FEA-Lugol's method and the yield of detected parasites was considerably higher. Eighty-five percent of 39 unknown parasite species tested were correctly detected using the Proto-fix-CONSED system compared to 46% using the FEA-Lugol's method. Of all the other methods and stains used at DLI, the trichrome stain (a popular modification of Gomori's trichrome stain for tissue sections) was found to be least reliable.     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.