Demonstration of Urinary Eosinophils in Schistosoma haematobium: a Comparative Study among Three Different Stains

Three stains, Hansel's stain, alkaline erythrocin B (AEB) and naphthalene black (NB), were used to demonstrate eosinophils in the urine of patients infected with Schistosoma haematobium. Hansel's stain was superior to the other two stains; it stained eosinophils bright red and their nuclei faint blue, and they were easily differentiated from neutrophils, lymphocytes, macrophages and epithelial cells. The method using AEB took longer than Hansel's stain and 10% of the specimens were lost during staining with this method. Like eosinophils, the neutrophils took up NB stain and their nuclei stained poorly with the counterstain.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

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.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

Nuclear Staining of Colletotrichum Gloeosporioides F. SP. Malvae Conidia with Fluorescent and Nonfluorescent Stains

Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores.     

Immunohistochemical demonstration of Trypanosoma evansi in tissues of experimentally infected rats and a naturally infected water buffalo (Bubalus bubalis)

Trypanosoma evansi was demonstrated by an immunohistochemical technique in formalin-fixed paraffin-embedded tissues of experimentally infected rats. Trypanosoma evansi was visible readily, nuclei were stained darkly, the cytoplasm was stained moderately, and the cell membranes were delineated clearly. The parasites were present in small- to large-sized blood vessels of all organs, in extravascular spaces of ventricles and neuropil of the brain, and in interstitial tissues of the lung and testes. This method also stained nuclei but not cytoplasm or cell membranes of Trypanosoma congolense, and did not stain Trypanosoma theileri. In a water buffalo (Bubalus bubalis) with nonsuppurative meningoencephalitis, the presence of T. evansi could not be demonstrated by conventional histological stains. However, the trypanosomes were recognized readily in the Virchow-Robin spaces and neuropil of the brain by the immunohistochemical method.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Nuclear death in living Tetrahymena: the case of the haploid nuclei

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by "apofluor", a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. "Apofluor" includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the "viable" nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by "apofluor", and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by "apofluor" it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.     

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.     

Alternative staining methods for Lowicryl sections.

A number of stains and stain combinations have been identified that, when used with the hydrophilic resin Lowicryl K11M, produce marked improvements over aqueous uranyl and lead salts (UA-Pb) in terms of low granularity, specificity, and range of components contrasted. Three test specimens, tobacco mosaic virus (TMV), starfish sperm, and cultured mouse fibroblasts, were used to evaluate stain characteristics. UA-Pb showed a preference for nuclei acids, which were stained specifically by osmium ammine-B at pH 1.5. A number of stain combinations in which UA was followed or preceded by salts containing barium, manganese, tungsten, molybdenum, and vanadium provided excellent staining of protein-containing components, each stain combination being unique in terms of the degree to which specific components were discriminated. These stains were particularly effective for visualizing internal components of the nucleus where a number of fibrillar and particulate structures not seen with UA-Pb were well contrasted.     

Association of mast cells with Warthin's tumor in fine needle aspirates of the salivary gland

OBJECTIVE: To determine the significance of the presence of mast cells in Warthin's tumor by evaluating the occurrence of these cells in cellular and immunohistochemical preparations. STUDY DESIGN: Specimens derived from five cases of FNAC were examined. A total of four slides from five cases were prepared from each: two air-dried smears were stained with May-Grünwald-Giemsa (MGG) stain and two with Hansel's stain. The other two were alcohol fixed and stained using the Papanicolaou method. The smears were evaluated for the presence of mast cells, especially associated with oxyphilic cells. In order to investigate the location of mast cells, we also counted those cells by means of immunohistochemistry using anti-mast cell monoclonal antibody AA1. RESULTS: The Hanselstained cellular sample from Warthin's tumor contained numerous mast cells, associated mainly with large, oxyphilic cell sheets. The number of AA1-positive cells (mast cells) stained with immunohistochemistry was greater in epithelial component than in lymphoid stroma. CONCLUSION: Mast cells in a salivary gland aspirate might be indicative of Warthin's tumor; therefore, MGG-stained slides offer the advantage of ease of preparation, particularly when the typical cytologic features are not present.     

Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.     

Fluorescent Vital Stains for Complementary Labelling of Protoplasts from Trichoderma Spp

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.     

An Assessment of Routine Chromosomal Staining Procedures in Radioautography

Unlabeled human chromosome preparations were treated with commonly employed chromosome stains as follows: (I) they were stained, destained, coated with liquid emulsion, developed, fixed, and restained; (II) stained and coated directly; or (III) coated and then stained. Of the stains tested, the methylene blue-eosin type (Giemsa, MacNeal's, Wright's) was useful for application after coating, although a similar stain (eosin-Stevenel's blue) caused formation of a heavy precipitate in the emulsion when so used. None of these stains could be employed before coating, however, even though they were removed with acid alcohol prior to dipping, because they caused chemographic grain formation in the emulsion. Aceto-orcein and Feulgen could not be employed after coating because the procedures removed the emulsion from the slides. Safranin was also found to be ineffective for staining coated preparations due to chemical changes caused by the photographic processing. The only stain which did not cause chemography, and hence can be used before coating slides, is aceto-orcein. Since this stain fades during radioautographic processing and cannot be employed after coating, we recommend secondary use of one of the methylene blue-eosin type stains for revisualization of the chromosome spread.     

DNA fluorescent stain accumulates in the Golgi but not in the kinetosomes of amitochondriate protists.

Hindgut symbiotic trichomonads (uninucleate Caduceia versatilis, and multinucleate Stephanonympha sp. and Snyderella tabogae) from the dry-wood-eating termite Cryptotermes cavifrons (Kalotermitidae) accumulate DAPI (4,6diamidino-2-phenylindole) in the membranous sacs of the Golgi complex. This form of Golgi complex, typical of protists in the class Parabasalia, is called a parabasal body. Trichomonads contain organellar systems, mastigonts, that consist of four undulipodia (e.g. eukaryotic flagella and cilia), axostylar microtubules, a parabasal body and other structures. These cells bear from one (in the case of Caduceia) to hundreds (in the case of Snyderella) of mastigonts. These features are characteristic of their protist class (Parabasalia). The nuclei of all three species stained with DNA-specific stains: DAPI, SYTOX, acridine orange, propidium iodide, ethidium bromide and Feulgen, at optimal concentrations, but kinetosomes failed to stain at all. The nuclei, parabasal bodies and symbiotic bacteria (but no microtubular structures) fluoresced in glutaraldehyde-fixed cells stained with 1.45 microM DAPI. Parabasal bodies of Snyderella and Caduceia treated to remove lipids with Triton X-100, or treated with 5% trichloroacetic acid, lacked DAPI-fluorescence. I conclude that DNA, present as expected in nuclei and bacterial symbionts, is absent from and not associated with calonymphid kinetosomes. The reason for DNA-RNA stain accumulation in the Golgi cistemae is not clear.     

The Masson Trichrome Staining Methods in Routine Laboratory Use

A resume of Masson's trichrome staining methods is given, with detailed directions for carrying out all of his procedures. The results obtained thru their use in a routine laboratory are discussed at length, as well as the fact that they also work very well on tissues fixed in ways other than those he prescribes, and stained with chemicals and dyes other than those he uses. The fact is stressed, however, that the closer one adheres to his precepts, the better will be the results.

The stains described include bis hematozylin-phloxine-saffron, his iron-hematozylin-ponceau-anilin-blue, his variants of this stain (of which the light green stain is excellent), his metanil yellow and his modification of the familiar Van Gieson technic. All these stains are based on familiar laboratory methods, improved and rendered trichrome, so that they present no great obstacles in technic.

Of the methods cited, the writer prefers the “light green” procedure. Sections are prestained in Regaud's iron-hematoxylin, followed by a mixture of ponceau de xylidine and acid fuchsin. This is followed by mordanting in phosphomolybdic acid and the sections are finally stained in light green. The results are very precise and pleasing and afford immediate orientation as the connective tissue is green, the nuclei black or dark purple, the cytoplasm of the cells is in varying tones of red. The method may be used after fixation in almost any good medium; altho the results are not as brilliant as those obtained after one of Masson's prescribed fixations, it is believed that they are even then superior to those following the routine hematoxylin-eosin method.     

Identification of normal and leukemic granulocytic cells with merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.