A conjugate of cellulase with fluorescein isothiocyanate: a specific stain for cellulose.

A fluorescent technique has been developed for in situ staining of cellulose. The staining agent in conjugate of cellulase and fluorescein isothiocyanate (FITC). Application of this agent does not disturb intercellular or intracellular substances. The technique depends on the specific binding of the fluorescent labeled enzyme to its substrate. The stain has been tested on cell-free noncellulose polysaccharides similar to cellulose and does not stain them. The technique has been used to localize cellulose during the life cycle of Dictyostelium discoideum with results that correspond to previous work using other methods.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

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.     

A New Fluorescent Test for Cell Vitality Using Calcofluor White M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysisdeplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.     

A Quick and Standardized Giemsa Stain for Wet-Fixed Cytological Material

The Giemsa stain is one of the most widely used staining techniques in cytology, especially in hematology. A standardized Romanowsky-Giemsa staining procedure using pure cationic azure B (C.I. 52010) and anionic eosin (C.I. 45380) has been described by Wittekind et al (1982). A revised standard Giemsa staining procedure was recently published (Wittekind and Kretschmer 1987). Usually the Romanowsky-Giemsa stain is applied to air dried and methanol fixed cytological material, e.g. blood smears and bone marrow films (ICSH 1984).     

冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色

冬虫夏草和蛹虫草作为重要的药用真菌,得到广泛的重视,迄今已有大量的研究报道。然而,作为细胞学研究的基础处理方法,其菌丝隔膜和细胞核染色却缺乏必要的研究。荧光染色是一种程序简便、快速灵敏的染色方法。本研究选用DAPI、PI、Calcoflour White和刚果红等4种染料,对冬虫夏草、蛹虫草菌丝的隔膜和细胞核进行单独与组合染色实验,通过显微观察比较得出较好的染色方法。结果表明DAPI对冬虫夏草和蛹虫草菌丝的细胞核染色效果都较好,而Calcoflour White对两者的细胞壁染色效果较好且隔膜清晰。DAPI与Calcoflour White两者进行冬虫夏草菌丝组合染色的效果为最佳,但在蛹虫草菌丝染色中效果不太稳定。对蛹虫草菌丝较好的组合染色是DAPI与刚果红的组合,但其染色结果需要在激光共聚焦显微镜下观察。     

EVIDENCE FOR GLUCAN COMPONENTS IN THE PRIMARY ZYGOTE WALL OF CHLAMYDOMONAS MONOICA

The primary zygote wall of C. monoica is transient and is released from mature zygospores. The fluorochromes aniline blue and primulin, used in other systems to detect β-1,3 glucans, stain the primary wall intensely. Two β-1,3 glucan synthases have been identified in higher plants: a calcium-dependent synthase produced in response to wounding and induced by chitosan, and a magnesium-dependent enzyme, associated with pollen development and unresponsive to chitosan. Chitosan has no effect on C. monoica primary wall synthesis or staining properties. We are presently testing for the effect of magnesium and/or calcium depletion on primary wall synthesis. Aniline blue and primulin do not stain purified cellulose fibers, while the fluorochrome Calcofluor does. Calcofluor also stains the primary wall intensely. For all fluorochormes tested, fluorescence is first detected in motile quadriflagellate zygotes. Aniline blue staining maximizes quickly, while Calcofluor staining continues to intensify until primary wall release. Dinitrobenzonitrile, a specific inhibitor of cellulose synthesis in plants, has no effect on primary wall synthesis in C. monoica. Addition of glucanase or cellulase to partially purified primary walls results in wall thinning and loss of staining. Using electron microscopy, we are evaluating the effects of these enzymes on primary wall ultrastructure. Further studies are needed to determine whether all three fluorochromes are recognizing the same polysaccharide component (a β-1,3 glucan or a β-1,3; β-1,4 mixed glucan), or whether Calcofluor staining indicates the presence of a distinct component containing β-1,4 linkages, such as cellulose or a xyloglucan.     

A Clearing Technique for Detecting the Fungal Endophyte Acremonium sp. in Grasses

Leaf tissue of tall fescue Festuca arundinacea Schreb., hard fescue Festuca ovina L., red fescue Festuca rubra L. and perennial ryegrass Lolium perenne L. was stained with rose Bengal or aniline blue to detect the presence of the fungal endophyte Acremonium sp., Specimens were cleared using methyl salicylate, an optical clearing agent, and viewed using bright field microscopy. Tissue was presenred as dried tissue or stored in 70% aqueous ethyl alcohol before staining and clearing. Tissue was observed at 2, 4 and 12 weeks following clearing to check for stain retention. Staining with rose Bengal was inferior to aniline blue when followed by the clearing agent methyl salicylate. Fungal mycelia stained lighter with rose Bengal and were more difficult to detect than mycelia stained with aniline blue. The results illustrate the usefulness of combining staining and methyl salicylate clearing for detecting fungal endophytes.     

The Use of Alizarin Red S To Detect and Localize Calcium in Gametophyte Cells of Ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca²⁺ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca²⁺. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca²⁺ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

A fluorescent antibody staining technique to detect bacterial adherence to urinary tract epithelial cells

A fluorescent antibody technique has been devised to assess specifically the adherence of Escherichia coli in vitro to uroepithelial cells from healthy women and bacterial adherence in vivo to cells from women with symptomatic urinary tract infection. Similar values can be obtained using methylene blue as the bacterial stain, but this depends on the experience of the observer. The results indicate that E. coli adherence to uroepithelial cells is a factor in the infection process. We suggest that uroepithelial cells from patients with symptoms of a urinary tract infection whose urine has a low bacterial count (less than 10(3) cells/ml) could be examined for the presence of adherent uropathogens, which may be indicative of an infection. Although the fluorescent staining technique possibly would be expensive, the results would be specific and reliable. Other diagnostic and research applications suggest themselves as in studies of bacterial colonization of mucosal tissues or plastic catheters, where conventional light microscopy and radiolabelling methods are not effective.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.     

A RAPID SIMPLE TECHNIQUE UTILIZING CALCOFLUOR WHITE M2R FOR THE VISUALIZATION OF DINOFLAGELLATE THECAL PLATES1

The fluorescent stain Calcofluor White M2R readily binds to cellulose and other β-linked glucans (Hughs and McCully 1975). We have found the stain to readily bind to the thecal plates of armored dinoflagellates. Considerable detail is revealed about plate structure in both living and preserved specimens at the light microscopic level. This simple rapid technique should prove useful for the tabulation and study of dinoflagellate thecae.     

An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.     

A New Stain for Pollen Fertility Studies

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P. H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.     

Color Differential Staining of Nor-Associated Heterochromatic Segments Using Acridine Orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 × SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoicordum fragrans. Fluorescence on other heterocnromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

Conjugates of Chitinase with Fluorescein Isothiocyanate or Lissamine Rhodamine as Specific Stains for Chitin In Sztu

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.