Staining of microsporidian spores with diamidine phenylindole

A number of microscopic techniques and dyes are available to diagnose microsporidian infections in invertebrate and vertebrate hosts. Among these, DNA-specific fluorochrome DAPI is widely used to stain DNA in prokaryotic and eukaryotic cells, alone or in combination with other histochemical or fluorescent dyes. Moreover, this dye also binds to membraneous structures and protein complexes. In our studies, DAPI was used to stain spores of microsporidia infecting orthopteran, coleopteran, dipteran and lepidopteran insect hosts. DAPI staining of diplokarya helped to discriminate the Nosema-like microsporidian spores from spore-shaped bodies lacking this characteristic staining. It was found, moreover, that non-DNA staining occurred in many cases and other components of the spores were stained: the exospore, the cytoplasm, the extruded polar filament and the polaroplast. Staining of these structures was feeble as compared to DNA and in most cases did not interfere with nuclear apparatus staining. Feebly stained cytoplasm and exospore clearly indicated unstained zone of endospore, making it easier to diagnose both mono- and diplokaryotic spores. Staining of extruded polar filament allowed to demonstrate viability and to observe some stages of extrusion process of microsporidian spores.     

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。     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

A rapid staining technique for the detection of the initiation of germination of bacterial spores

AIMS: We propose to apply the Wirtz-Conklin staining technique to evaluate spore germination. METHODS AND RESULTS: Spores at different stages of germination were stained with modified spore stain (Wirtz-Conklin) and evaluated for staining properties. Bacillus spores suspended in deionized water, which does not support germination, stained greenish-blue. Spores suspended in germination enhancers that did not form bacilli stained pink, indicating the initiation of germination. Spores suspended in culture media, which promotes bacterial outgrowth, formed bacilli and were also stained pink. CONCLUSIONS: Modified spore stain (Wirtz-Conklin) was found to be useful to detect the initiation of spore germination as early as 30 min following incubation in a germination environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple staining procedure is useful in detecting the initiation of germination of bacterial spores.     

Comparison of Acridine Orange, Acriflavine, and Bisbenzimide Stains for Enumeration of Bacteria in Clear and Humic Waters

In highly humic water, acridine orange precipitated with dissolved humic matter, resulting in such bright background fluorescence that no bacteria could be seen. With bisbenzimide staining, a similar precipitate was nonfluorescent but obscured many cells. An acriflavine staining method proved useful and reproducible both in clear and in humic waters. Fading of fluorescence was not a problem, and stained samples could be stored after preparation. The fluorescence of cells stained with acriflavine was weaker than that with acridine orange, making counting extremely small cells slightly more difficult with the former stain.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

缩球囊霉孢子萌发及细胞核在孢子和芽管中分布的研究*

本文研究了从本校分离的丛枝菌根真菌缩球囊霉Glomus constrictum的孢子萌发和萌发孢子的细胞核DAPI(46-diamidino-2-phenylindol)染色。结果显示,G. constrictum孢子直径为179.5-198.7μm ,顶生于产孢菌丝上。经表面消毒处理后,孢子在水琼脂平板上7天开始萌发。DAPI染色后,经稀释荧光计数,单个孢子细胞核数目约为5300,在孢子中无序分布, 细胞核直径约为9.9-11.2μm 。孢子萌发过程中,细胞核总数无明显变化,只是部分细胞核从孢子流向了萌发伸长的菌丝。     

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.     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Peroxidase Staining Combined with Autoradiography for Study of Eosinophilic Granules

Giemsa staining and a peroxidase reaction were applied to blood films in conjunction with autoradiography to establish the types of granulocytes that stain differentially with the benzidine-peroxidase reaction. Differential counts made on Ciemsa-stained and peroxidase-stained autoradiograms were compared. In T. spiralis-infected rats with an elevated eosinophil count, as judged by Giemsa staining, the percentage of granulocytes that stained more intensely with peroxidase was increased. The results suggested that the eosinophils were the intensely peroxidase-positive cells. Blood smears were stained for peroxidase before being coated with NTB₂ liquid emulsion. Although the blue color of the peroxidase reaction faded during photographic development, the color redeveloped when peroxidase-stained autoradiograms were stained once again after photographic development. It was found necessary to stain for peroxidase both before and after autoradiography. The correlation of Giemsa-stained and peroxidase-stained autoradiograms indicated that the peroxidase stain can be combined with autoradiography to obtain authentic results.     

Staining Glycol Methacrylate Embedded Cartilage with Triethyl-Carbocyanin Dbtc (“Ethyl-Stains All”) With Special Reference to the Interlacunar Network

The dye, triethyl-carbocyanin DBTC, was tested for differential staining of cartilage structures. Femoral head articular cartilage from neonatal rats was processed for histology to demonstrate the interlacunar network. Sections of glycol methacrylate (GMA) embedded cartilage were stained at pH 2.8, 5.4, 6.1 and 8.0 to determine the optimal staining conditions. Only at pH 6.1 were all cartilage structures stained and the best contrast achieved. Streptomyces hyaluronidase, chondroitinase ABC, pepsin, trypsin, and pronase digestions were carried out prior to staining at pH 6.1 to evaluate the selectivity of the stain. Undigested chondrocyte nuclear chromatin stained dark purple; staining intensity was reduced slightly by pepsin or trypsin digestion. Undigested chondrocyte cytoplasm stained light blue but stained purple after hyaluronidase digestion. Undigested extracellular matrix stained light violet; staining was almost entirely eliminated by chondroitinase ABC digestion, was unaffected by hyaluronidase, and was either unaffected or increased after proteinase digestion. Staining of a narrow zone of matrix adjacent to the network was prevented by proteinase digestion while the network element appeared as a thin dark line. The network appears to be a trilaminar structure; a core element of hyaluronic acid and protein surrounded by a protein sheath. Triethyl-carbocyanin DBTC staining of cartilage offers slightly more selectivity and contrast than methylene blue, toluidine blue or safranin O. At pH 6.1, DNA, perhaps RNA, and hyaluronic acid stained deep purple; chondroitin sulfate, light violet; protein (collagen), stained very light violet if at all.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Stereum sanguinolentum: Is it an amphithallic basidiomycete?

Monobasidiospore isolates were prepared from basidiocarps of Stereum sanguinolentum. Five isolates per basidiome were paired with each other and with isolates from the trama. Interbasidiome pairings of the trama isolates and of a selection of single-spore isolates also were performed. Thin sections of the hymenium were stained with DAPI and examined by fluorescence microscopy to study the nuclei in the basidia. Spore prints were stained with DAPI to count the number of nuclei per spore. SEM was used to determine the number of basidiospores per basidium. All intrabasidiome pairings were compatible. In contrast, interbasidiome pairings, except one, were incompatible, independent of whether single-spore or trama isolates were paired. Fertile basidiomes were formed in single-basidiospore cultures. Basidia were regularly four-spored. On average, 5% of the basidiospores possessed one nucleus, 82% two, 2% three and 1% four nuclei. Ten percent of the spores appeared to be empty. Karyogamy, meiosis and postmeiotic mitosis were observed in the basidia. Nuclei resulting directly from meiosis, i.e., without having undergone postmeiotic mitosis, sometimes were observed in the sterigmata or spore primordia. The high number of vegetative compatibility groups (VCG) of S. sanguinolentum observed in this study and earlier studies is difficult to explain without sexual or parasexual recombination. We suppose that the majority of spores with ≥2 nuclei are amphithallic, possessing at least one nucleus of each mating type. Recombination could occur by exchange of nuclei among VCGs via anastomoses between homothallic compartments. Transfer of nuclei from heterothallic to homothallic mycelia or matings between homothallic mycelia, which originate from monokaryotic spores, might be other paths for gene exchange.     

Studies on Cell Number and Nuclei in Spores and on Ploidy Level in Ascochyta rabiei Isolates

Isolates of the phytopathogenic ascomycete Ascochyta rabiei (Pass.) Labr. were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and compared for differences in number of nuclei per pycnidiospore and the ploidy level. Microscopic analyses revealed that within the examined isolates five different combinations of cell number and number of nuclei in spores exist. A one-celled spore may contain one, two and four nuclei, respectively, and in the case of two-celled spores there exist types with one and two nuclei in one cell. Microfluorometric analyses of wild types and benomyl-treated isolates revealed differences in ploidy level among the wild types.     

Persistence of Nosema locustae Spores in Soil as Determined by Fluorescence Microscopy

Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5°C but exhibited a 1,000-fold decrease after 1 week of incubation at 27°C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27°C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.     

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.