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.     

Ultrastructure of the bone marrow of the salamander Plethodon glutinosus (Caudata: Plethodontidae)

The unusual lymphogranulopoietic bone marrow of the large lungless salamander Plethodon glutinosus was examined by light and electron microscopy. Developing neutrophils, eosinophils, and fat cells were found in large numbers, while lymphocytes of various sizes, plasma cells, plasmablasts, macrophages, pigment cells, and fibroblasts were present in more moderate numbers. Basophils were observed only rarely. Macrophages were found in extravascular locations and did not appear to be associated directly with the walls of the blood vessels supplying the marrow. Both neutrophils and eosinophils seemed to arise from small precursor cells whose ultrastructural features bore a resemblance in some ways to those of mammalian myeloblasts described by Bainton and Farquhar ('66). Developing neutrophils and eosinophils seemed to produce only single populations of specific cytoplasmic granules, rather than both primary (azurophilic) and secondary (specific) inclusions, as are produced typically by mammalian granulocytes. Both eosinophilic and neutrophilic granules were formed in association with Golgi complexes; and eosinophilic granules were much larger, more densely stained, and more regularly rounded in shape than the inclusions of developing neutrophils. Peroxidase activity was associated with the specific granules of neutrophils but seemed to be lacking in the granules of eosinophils. The specific granules of eosinophils were especially unusual because they contained irregularly shaped, lightly stained cores which occasionally displayed a distinctly crystalline substructural organization. The specific granules of basophils also possessed a prominent crystalline organization. The overall appearance of the marrow of Plethodon suggests that it functions not only as a valuable source of neutrophils, eosinophils, and cells of the lymphoid series, but also as a part of the phagocytic system of the animals and as an important repository for fat.     

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.     

Vital and Calcofluor staining ofCosmarium and its protoplasts

Summary Intact cells, protoplasts, primary and secondary walls ofCosmarium species were stained with 13 vital stains and with Calcofluor. The intracellular contents of viable cells and protoplasts were not stained with any of the test dyes although the primary and secondary walls often were stained. Protoplasts disintegrated after short periods in acid stains but did survive for up to 24 hours in several dilute basic dyes. Once the protoplast membrane was damaged, the nuclei and cytoplasm were brightly stained with most dyes, whereas the vacuoles remained unstained. Calcofluor was particularly useful in determining the porous structure and pattern of the primary walls.     

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.     

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.     

Metal Chelates as Reversible Stains for Detection of Electroblotted Proteins: Application to Protein Microsequencing and Immunoblotting

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.     

A Modification of Mayer's Hemalum

A survey of histological literature shows that Delafield's hematoxylin is one of the most widely used histological stains. Personal interviews with workers in this field elicit comments which seldom appear in print. Most workers confess that it usually requires several unsuccessful attempts to make a usable stain. The experienced technician is no more successful than the self-taught beginner. I have examined, at various schools, preparations stained with so-called Delafield's stain and the range of colors and degrees of muddiness found were surprising. A somewhat greater degree of certainty and uniformity is desirable in a stain which is to be used by classes in elementary histology. Mayer's hemalum has not enjoyed the popularity which it deserves perhaps because the formula given in a widely used text is incorrect. This was found to be a source of discouragement to self-taught students in some smaller colleges.     

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.     

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.     

Population Dynamics and Identification of Endophytic Bacteria Antagonistic Toward Plant-Pathogenic Fungi in Cotton Root

The antagonistic potentials of endophytic bacteria isolated from the roots of six cotton cultivars at different developmental stages were determined in vitro toward three pathogens: Verticillium dahliae Kleb V107 and V396 and Fusarium oxysporum f.sp. vasinfectum (F108). The populations of antagonistic endophytic bacteria (AEB) toward V107, V396, and F108 at the flowering and maturation stages were significantly higher than those at the seedling stage were. More AEB were found to be antagonistic toward pathogens V396 and F108 than V107. Results from the multivariate analysis of variance showed that the populations of AEB were significantly different for the main factors of cultivars, stages, and their interactions. Based on 16S rDNA sequence analysis, the 39 AEB isolates that antagonized V107, V396, and F108 (BAEB) consisted of seven genera, in which the genus of Enterobacter (17 out of 39) and Pantoea (14 out of 39) were predominant among the BAEB isolates. Characterized by BOX-PCR fingerprints, these 39 BAEB isolates represented 35 different cluster types. To explore the antagonistic mechanisms, the agar diffusion method was used to detect cell-wall-degrading enzyme activity and siderophore secretion. Nearly half of these BAEB isolates showed protease and chitinase activity, while all 39 BAEB isolates excreted siderophores. However, pectinase, cellulase, and xylanase activity were hardly detected. A germination experiment revealed that nine of the 39 BAEB isolates significantly improved the vigor index of the cotton seedlings.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

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.