High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

Lanthanum as an electron microscopic stain

Applications of lanthanum as an electron microscopic tracer have been reviewed. This electron-dense trivalent cation, which binds avidly to calcium binding sites, can be used as tracer for delineating extracellular spaces and intercellular junctions. It has served as a basis for classification of junctional structures. It can also be used as a calcium probe, a tracer in studying the permeability of barriers, as an intracellular marker and as an electron microscopic stain for such membrane components as surface glycoprotein. Each of these applications may require a different methodology. Thus methodological considerations in the use of this tracer have also been reviewed. The recent recognition that lanthanum is more than a passive tracer and that by reacting with different cell components may serve as a true stain, will extend the use of lanthanum in electron microscope histochemistry.     

A fast screening method for surface layers on Gram-positive bacteria

A two-step screening method is described to identify regularly arranged surface layers (S layers) on Gram-positive bacterial strains. A non-destructive release of S-layer sheets is achieved by enzymatic hydrolysis of the underlying peptidoglycan using lysozyme. The existence of regular S layers is then directly confirmed by scanning force microscopy or transmission electron microscopy. This method requires a minimal amount of bacterial cells and may be used as a `quick test' for demonstrating the presence of S layers.     

Drosophila melanogaster brain invasion: pathogenic Wolbachia in central nervous system of the fly

The pathogenic Wolbachia strain wMelPop rapidly over‐replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3‐d‐old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbachia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Ultrastructural Study of Plasmodium knowlesi Antigen Used in Vaccination of Rhesus Monkeys*

SYNOPSIS. Material from various steps obtained in the French pressure cell technic of preparing antigen from Plasmodium knowlesi-infected red cells, was examined by electron microscopy. A positively charged colloidal iron solution was used to differentiate between membranes of host red cells and parasites. Red cell membranes take the stain, whereas parasite membranes do not. This antigen which has been used previously to protect monkeys against P. knowlesi appears to consist almost entirely of membrane-bounded vesicles. Some of these vesicles contain a fine granular material, whereas others appear empty. The antigen failed to stain with the positively charged iron solution, which suggests that it is free of contamination by host cell membrane.     

E. coli filament formation induced by heterologous S-layer expression

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 μm x 2.0-6.0?μm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of > 100 μm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM (?) 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process.     

Ruthenium Red and the Bacterial Glycocaly

Ruthenium red, a promising cationic reagent for electron microscopy (EM), has long been an important tool in histology. The reagent was initially used by botanists as a semispecific stain for pectic substances, but it has gradually been embraced by investigators in microbiology and the animal sciences as a stain for anionic glycosylated polymeric substances. Luft developed a reliable method and demonstrated that ruthenium red was a useful reagent for visualizing ultrastructural detail. Many investigators, using modifications of Luft's approach, have identified numerous applications for this important reagent. Ruthenium red has been used to show the ultrastructural detail of bacterial glycocalyces. Strong, sharp and consistent observations of this ultrastructural component of the bacterial cell have given a better understanding its fibrous anionic matrix. Any variations in staining owing to artifactual alteration of the fine delicate ultrastructural features have been overcome by incorporation of diamine lysine into ruthenium red methods, thus providing flexible processing times under less than ideal laboratory sampling conditions. Ruthenium red has broad utility in the biological sciences, and in combination with lysine, it is an excellent EM stain for enhanced visualization of bacterial glycocalyx from culture or from clinical specimens.     

Identifying Assembly-Inhibiting and Assembly-Tolerant Sites in the SbsB S-Layer Protein from Geobacillus stearothermophilus

Surface layer (S-layer) proteins self-assemble into two-dimensional crystalline lattices that cover the cell wall of all archaea and many bacteria. We have generated assembly-negative protein variants of high solubility that will facilitate high-resolution structure determination. Assembly-negative versions of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 were obtained using an insertion mutagenesis screen. The haemagglutinin epitope tag was inserted at 23 amino acid positions known to be located on the monomer protein surface from a previous cysteine accessibility screen. Limited proteolysis, circular dichroism, and fluorescence were used to probe whether the epitope insertion affected the secondary and tertiary structures of the monomer, while electron microscopy and size-exclusion chromatography were employed to examine proteins' ability to self-assemble. The screen not only identified assembly-compromised mutants with native fold but also yielded correctly folded, self-assembling mutants suitable for displaying epitopes for biomedical and biophysical applications, as well as cryo-electron microscopy imaging. Our study marks an important step in the analysis of the S-layer structure. In addition, the approach of concerted insertion and cysteine mutagenesis can likely be applied for other supramolecular assemblies.     

Morphological observation and characterization of the Pseudoregma bambucicola with the scanning electron microscope

Both leica microscopic camera system and scanning electron microscopy was used to observe and characterize the feet, back, abdomen, antennae and mouthparts of the Pseudoregma bambucicola from the bamboo, Bambusa multiplex. The possible functions of all the external morphological characteristics of the P. bambucicola were described and discussed in detail, which offers a basis for further enriching the biology, phylogeny and ecological niche of the P. bambucicola. Moreover, the morphological results should contribute to morphological identification and differentiation of the P. bambucicola from other aphids in the same family.     

Digestion of prey in foraminifera is not anomalous: A correlation of light microscopic, cytochemical, and hvem technics to study phagotrophy in two allogromiids

Correlative light, high-voltage electron and conventional electron microscopic methods were used to investigate digestion in two allogromiid foraminiferans, Allogromia sp., strain NF, and A. laticollaris Arnold. Microscopic observations showed that bacterial prey are phagocytosed by reticulopodia and are transported to the allogromiid cell body within blister-like phagosomes. Larger prey (algae, diatoms) are transported along the reticulopodial surface and are either stored extrathalamously or phagocytosed at the oral opening (peduncle). Studies of allogromiids optimally fixed and labeled with an extracellular-space label (colloidal thorium) showed that phagocytosed prey are completely enclosed by a plasma membrane envelope; this finding was corroborated by a serial-section three-dimensional reconstruction of the oral zone of one allogromiid. Cytochemical staining for acid phosphatase showed that lysosomes are absent from reticulopods but abundant in the cell body, particularly in the oral zone cytoplasm. We conclude that digestion in allogromiid foraminiferans is accomplished by a vacuole-based digestive apparatus and not by extracellular digestion within a lacunary system, as has been suggested in earlier studies.     

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3′-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.     

Pinacyanol Erythrosinate as A Stain for Mast Cells

An alcoholic solution of the compound dye, pina-cyanol erythrosinate when diluted to the optimum dissociation point is a differential tissue stain which, in addition, selectively stains and differentiates mast cells. It can be made up and used like any other compound dye (e.g., Bowie's stain, neutral gentian, etc. or like a blood stain). It can be used after any of the common fixatives and has the advantage of selectively staining all types of mast cells in their various functional phases, even in those species (notably rabbit and man) in which they may be difficult to demonstrate with other mast cell stains after aqueous fixatives.     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.     

The regular surface layer of Acetogenium kivui: some structural, developmental and evolutionary aspects

Abstract The structure of the regular surface layer of Acetogenium kivui has been investigated by electron microscopy in conjunction with image processing techniques. From the averaged unit cell structure a tentative model of the subunit organization is derived. The occurrence of mono- and polycrystalline S-layer sheets is interpreted in terms of transitions related to the growth cycle of cell. The question is raised as to whether the striking structural similarity of S-layers, even from evolutionary rather distant species, is due to divergent or convergent evolution.     

鲤鳃表面结构扫描电镜研究

鲤的鳃丝表面显示了由三边形一六边形不等的表皮细胞,呈现出各种图案式的结构。     

Fluorescence Microscopic Study of Microorganisms Treated with Chaotropic Agents

The yeasts Saccharomyces cerevisiae and Pichia pastoris and the bacteria Micrococcus luteus, Bacillus subtilis, and Anaerobacter polyendosporus have been treated with the chaotropic agents guanidine hydrochloride and guanidine thiocyanate and certain detergents and studied using fluorescence microscopy. Studies with the use of fluorochromes that can selectively stain nucleic acids (diamidino-2-phenylindole (DAPI), propidium iodide, and acridine orange) show that treatment of the bacterial and yeast cells at 37°C for 3–5 h induces a release of DNA from the cytoplasm and its accumulation in the cellular zone, known as ectoplasm, located between the cell wall and the remainder of the cytoplasm (called endoplasm) in the form of one or several large granules. After treating the cells with the chaotropic agents at 100°C for 5–6 min, the DNA is diffusively distributed over the ectoplasm. The fluorochromes used do not allow the detection of RNA. These findings are in agreement with previous data obtained from electron microscopic study of thin cell sections. After 33 PCR cycles, a considerable portion of DNA leaves the cells; as a result, they show a low level of diffusive fluorescence when stained with DAPI. When endospores of B. subtilis are treated with the chaotropic agents, they become highly permeable to the fluorochromes. Fluorescence microscopic study of such endospores shows that they contain DNA in the central part of their cores.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 505–510.Original Russian Text Copyright © 2005 by Duda, Danilevich, Akimov, Suzina, Dmitriev, Shorokhova.