Demonstration of the Glycocalyces Associated with Three Oral Gram-Negative Bacterial Species Using a Modern Acrylic Resin Technique

The complex highly hydrated chemical composition of the bacterial glycocalyx renders it difficult to preserve and visualize at the ultrastructural level. Polyanionic stains such as ruthenium red help to maintain some structural integrity, and other more modern approaches include antibody stabilization, lectins, and the addition of lysine to the primary fixative. It has been suggested that the glycocalyx of certain disease-associated organisms may play a role in the pathogenesis of some microbially based diseases such as periodontitis. New, more adequate, modern methodologies are therefore required for the further study of this structure. In the present study a cold dehydration process in conjunction with LR white acrylic resin has been employed to study the glycocalyces of three oral gram-negative bacterial species reported to be periodontopathogens. When compared with organisms processed conventionally and with ruthenium red, the organisms processed by the cold dehydration and LR white method demonstrated a fibrous matrix that was not seen in the other specimens. These results indicate that a combination of reduced dehydration temperature and cold acrylic resin embedding provides the best methodology for the visualization of the fine structure of the bacterial glycocalyx. This approach may be particularly useful in the study of organisms within specific disease-associated environments such as the periodontal pocket.     

Microscopic examination of natural sessile bacterial populations from an alpine stream.

Natural populations of bacteria assoiciated with the slime on submerged surfaces in a mountain stream were examined by phase-contrast and electron microscopy. The slime contained large numbers of bacteria which were predominantly gram-negative as determined by their cell wall structure. Examination of the in situ distribution of cells revealed that they were enmeshed in an extensive fibrous matrix whose component fibrils were stained with ruthenium red. The arrangement of slime fibrils immediately around individual bacterial cells suggested that this material was produced by these bacteria. This slime facilitated microcolony development and also anchored the bacteria to a particular surface. It is proposed that these slime-enmeshed microcolonies constitute functional communities within which most sessile bacteria live.     

Fixation and Staining of the Bacterial Nucleus

An examination of some early methods in bacterial cytology shows that technics for demonstrating the nucleus of the Eubacteriales were available for at least twenty years before the current era of investigation, which began in 1942. Although the bacterial nucleus reacts in many respects like the chromatin elements of higher plants, it shows certain peculiarities in its staining reactions. For example, hematoxylin, methyl green, and several preparations used to stain chromosomes, apparently do not exhibit the same affinity for the bacterial nucleus that they do for chromosomes in higher plants. Not only is the selection of a fixative important in nuclear studies, but also the manner in which the fixation is obtained. For example, when bacteria are fixed and processed in a completely wet state it is generally impossible to stain their nuclei. None of the special fixatives studied revealed any unusual organization in the bacterial cell or exhibited any advantage over the fixatives now in common use by bacteriologists. In view of the properties of osmium tetroxide vapor, particularly its relative lack of interference with positive nuclear staining, there can be little doubt of its superiority as a nuclear fixative. It appears that the basophilic material removed from the bacterial cell by hydrochloric acid is not only in the cytoplasm, but that a very significant amount of it is in close contact with the cell wall.     

Labeling of binding sites for beta 2-microglobulin (beta 2m) on nonfibrillar surface structures of mutans streptococci by immunogold and beta 2m-gold electron microscopy.

As little detail is known about the surface structure of streptococci in the mutans group and the relationship of surface structure to host ligand-binding functions, the twofold purpose of this investigation was to examine in detail, by a range of electron microscopic techniques, the surface structures of streptococci in the different species of the mutans group and to investigate the distribution of beta 2-microglobulin (beta 2m)-binding sites on such structures. Strains representing Streptococcus mutans, S. cricetus, S. rattus, S. sobrinus, and four fresh isolates were studied by shadowcasting and histochemical staining of whole-mounted cells as well as by ultrathin and thick sectioning of embedded specimens. beta 2m-binding site distribution was visualized by indirect immunogold electron microscopy and by direct bacterial binding of beta 2m-conjugated gold probes. Shadowcast preparations revealed binding of gold probes to the cell surface of known beta 2m-binding strains but not to their polar fibrillar appendages. These long fibrils, common to all strains, were trypsin and sonication sensitive and stained with lead citrate but not with uranyl acetate or ruthenium red. More gold particles were bound by the indirect technique. For grid-mounted bacteria, the gold was mostly bound in clusters at the periphery of the cells. When gold probes were reacted in suspension with bacteria before mounting onto grids, a more even distribution of the gold was seen, but the bacteria were aggregated. Heating the bacteria eliminated beta 2m-gold binding but had no effect on the morphology of the fibrils. Thick sections of embedded bacteria prereacted with beta 2m-conjugated gold probes were analyzed by stereo imaging. A wispy, uranyl acetate-stained fuzzy layer, distinct from the fibrils seen by shadowcasting and extending up to one cell diameter from the cell wall, contained the gold probes. These findings introduce a concept that binding sites for some salivary ligands on mutans streptococci may be clustered on very delicate, nonfibrillar structures extending much further from the cell wall than previously appreciated. As for beta 2m, which composes part of the human histocompatibility antigens, part of the bacterial surface would be coated at a distance from its body with a protein not necessarily recognized as foreign by the host.     

DNA Synthesis, Mitosis and Ploidy of Dividing Cells in Early Crown Gall

DNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with ³H-thymidine in slides in which mitoses had been counted and analyzed before autoradiography. The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two. The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.     

Ultrastructure of complex carbohydrates of rodent and monkey ependymal glycocalyx and meninges

The surfaces of the brain offer metabolic and mechanical support to the underlying parenchyma. Mouse, rat, and monkey brains were fixed by immersion in a glutaraldehyde fixative or glutaraldehyde with cetylpyridinium chloride, followed by block staining for complex carbohydrates using alcian blue with OsO4 postfixation, or OsO4 postfixative solution containing ruthenium red, or alcian blue and then ruthenium red-OsO4 treatment. The ependyma in these species had a glycocalyx extending into the ventricular fluid as a finely filamentous network when stained with alcian blue or with alcian blue followed by ruthenium red-OsO4. Mice in the middle age range had stained material in this glycocalyx resembling the hyaluronic acid reported in the ocular vitreous body. Similar material was seen in the arachnoidal space of these mice and in the inner connective tissue matrix of the dura mater. Both the mouse and monkey had a cell-free zone, termed the inner dural matrix zone, between the thick fibrous dura and its innermost cellular layer. This zone contained filamentous and globular alcian blue-stained material. The complex carbohydrates of the mouse ependymal glycocalyx and inner dural matrix zone underwent changes developmentally. Aged rats were injected intraventricularly with latex beads, which, along with extravasated erythrocytes, were seen to adhere to the ependymal glycocalyx. A similar adhesion of erythrocytes was seen in the mouse and monkey ependymal glycocalyx and in the filamentous network of the mouse and monkey inner dural matrix zone. The ependymal glycocalyx, formed in part of complex carbohydrates, is much thicker than previously demonstrated. Some activities related to the ependymal lining of the ventricles, including the movement of cells or particles, the penetration of metabolites or serum-protein fractions (e.g., immunoglobulins), and cell-surface hydration, probably depend in part on complex carbohydrates that provide a sticky, electrically negative, hydrophilic environment. The complex carbohydrates in the inner dural matrix zone might provide mechanical buffering. Complex carbohydrates in the arachnoidal space may help to maintain a loose tissue that needs not only to be hydrated, but also to be open enough to provide cerebrospinal fluid circulation.     

The cell coat of the sensory and supporting cells of the rainbow trout saccular macula as demonstrated by reaction with ruthenium red and tannic acid.

The surface of most cells is covered by glycoconjugates. The composition and thickness of the surface coat varies among different cell types. The purpose of the present study was to demonstrate the presence of and to characterize the cell coat surrounding the cells in the saccular macula of the rainbow trout. Tissues were fixed in Karnovsky's fixative containing either ruthenium red (0.5, 1, or 2%) or tannic acid (1, 2, or 4%). The apical surface of the sensory and supporting cells reacted with both agents. Varying the concentration of the compounds within a certain range did not significantly affect the degree of tissue staining. Whereas ruthenium red staining was distributed evenly along the luminal surface of the epithelium and along the length of the stereocilia, tannic acid formed electron-dense clumps on the luminal surface of sensory and non-sensory cells and in the basal region of the macular epithelium. The stereocilia of the sensory cells also exhibited tannic acid-positive, electrondense precipitate, particularly near the distal ends of these processes, while uniform staining of the plasma membrane was seen along their lengths. The results of this study suggest that the trout saccular macula is provided with extracellular microenvironments which may be necessary for functional integrity.     

Formation of Bacterial Microcolonies on Feed Particles in the Rumen

Examination of particulate feed that had been digested in vivo in the rumen, and of the leaves of specific legumes that had been digested in vitro by a mixed population of rumen bacteria, showed that very extensive glycocalyx-enclosed bacterial microcolonies developed on many of the available surfaces. Some of these adherent bacteria colonized a surface almost exclusively and attracted another specific type of bacteria as the second members of a distinct morphological consortium. The true extent of the exopolysaccharide glycocalyces of these adherent rumen bacteria was seen in cases where the fibers were attached at multiple points, and their role in microcolony formation and adhesion could be unequivocally ascribed.     

A plate assay method for the detection of fungal polygalacturonase secretion

Abstract A method for the detection of polygalacturonase activity has been developed using ruthenium red staining of fungal colonies on polygalacturonate- agarose plates. Ruthenium red was shown to penetrate beneath the surface layers of the gel, in the regions surrounding a fungal colony where degradation of polygalacturonate had occurred. Without degradation of polygalacturonate ruthenium red did not penetrate the medium, was restricted to binding to the surface layers and was easily washed off. The medium containing undegraded polygalacturonate was a colourless clear background and areas of polygalacturonate degradation around the colonies were visualised as an intense purple-red halo. The method has been used to screen yeasts and filamentous fungi for polygalacturonase secretion.     

Mise en évidence par immunofluorescence des cellules sécrétrices de glucagon dans le pancréas endocrine du poisson téléostéen Xiphophorus helleri H.

Summary The ruthenium red staining of the surface coats was studied in the adrenal medullary cells of golden hamster. Both immersion and perfusion fixation was used with the ruthenium red containing fixative, however, only the perfusion fixation gave positive results. A rather thick electron dense ruthenium red positive layer was found on the plasma membrane of the endothelial cells, around the capillaries in the basal lamina, in the basement membrane of the chromaffin cells as well as on the apical and lateral cell surfaces of the adrenomedullary cells. Coated pits and coated vesicles usually showed an intensive ruthenium red staining, but the other cell components in the cytoplasm did not. On the basis of these observations author suggests that the ruthenium red positive material corresponds to acidic mucopolysaccharides in the hamster adrenal medulla, and its wide-ranging occurrence is indicative of its significance in the secretion process of catecholamines.Wellcome Research Fellow.     

An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.     

Role of estrogen in controlling the genital microflora of female rats

Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17beta-estradiol (1,3,5(10)-estratriene-3,17beta-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen.     

Role of estrogen in controlling the genital microflora of female rats.

Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17beta-estradiol (1,3,5(10)-estratriene-3,17beta-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen.     

The ultrastructure of Candida albicans infections

Scrapings of Candida albicans plaques from the tongue and buccal mucosa of patients with oral candidiasis were examined by electron microscopy. In addition, urine sediment from patients with infection of their catheterized urinary tracts was similarly examined. Three types of C. albicans-oral epithelial cell interactions were noted: a loose adherence apparently mediated by a ruthenium red positive matrix, a "tight" adherence where no space could be seen between the host and yeast cell. and invasion of host cells by yeast hyphal elements. Adhesion of Candida blastospores to hyphal elements and adhesion of bacteria to Candida cells was also frequently observed. Urine sediments from patients with mixed bacteria-yeast infections demonstrated adhesion of the bacteria to the yeast cells. This phenomenon was also demonstrated in in vitro experiments and fibrous ruthenium red material invariably occupied the zone of adhesion. Phagocytosis of yeast by polymorphonuclear leukocytes was found in urinary, but not in oral. candidiasis. Our in vivo and in vitro observations indicate that a ruthenium red positive matrix covers the surfaces involved in the yeast to yeast, yeast to host, and yeast to bacteria adhesion.     

A bioinformatic approach to the identification of bacterial proteins interacting with Toll-interleukin 1 receptor-resistance (TIR) homology domains

Members of the Toll-like receptor (TLR) family are currently under intense scrutiny for their role in the sampling and recognition of pathogens. It has already been reported that both vaccinia virus and Yersinia spp. express proteins that help them evade the TLR mediated immune response, acting through the Toll-interleukin-1 receptor-resistance (TIR) domain and leucine-rich repeat region of the host TLRs respectively. The TIR domain is involved in the dimerisation of the TLRs and their complexation with their adapter molecules. We tested here the hypothesis that bacteria have the ability to secrete proteins containing similar motifs to the intracellular TIR domains that are involved in the TIR-TIR interaction necessary for the subsequent signal transmission. Based upon their sequence homology, proteins expressing TIRs have been divided into three sub-classes, based around the TLRs, the TLR adapter proteins, and the interleukin-1 and -18 adapter proteins. The highly conserved regions from these separate sub-families were then used to identify similar bacterial proteins. The bacterial proteins identified were then included in an iterative MEME-BLAST process to broaden the search. Tollip, a known TLR antagonist and adapter protein, was included in this investigation although it does not fit into any of the three sub-classes outlined above. If suitable bacterial proteins had been identified, it would signify that certain bacteria had evolved a mechanism to aid them in avoiding detection by the innate immune system acting through the TIR domains. At this stage one has to conclude that there is no evidence currently available suggesting such a mechanism, when using the strategy applied here.     

豌豆根瘤中细菌的扫描电镜观察

用扫描电镜观察了豌豆根瘤的侵染细胞.结果表明,在这些细胞中有大量的细菌,它们主要是杆状细菌,其次是球形、Y形和T形细菌,其它形状的细菌很少.除了细菌形状不同外,还有一些细菌比较特殊,如有的细菌较长,菌体出现部分收缩并形成一个或一个以上的收缩环,其形状类似一条莲根;有的细菌很大,它的体积是普通细菌的2倍或2倍以上;有的细菌粗细不均匀,端部膨大,呈棒槌状.侵染细胞中有许多小泡,它们大小不同,呈球形.它们存于细菌之间,其中一些小泡还位于细菌的表面上,而且附近细菌的表面有时还有各种隆起.     

The functional morphology of the organs of feeding and digestion of the hydrothermal vent bivalve Calyptogena magnifica (Vesicomyidae)

The discovery, around Galapagos Rift hydrothermal vents, of an unique community of animals dependent upon the chemoautotrophic oxidation of hydrogen sulphide by bacteria, has aroused wide interest. In the gutless pogonophoran Riftia pachyptila. trophosomal symbiotic bacteria are thought to be principally responsible for this unique form of nutrition. Similar symbiotic bacteria have been postulated for the ctenidia of the Rift clam Calyptogena magnifica. Such a mode of nutrition was deemed necessary since Calyptogena was thought not to possess ctenidial food grooves, thereby making normal filter-feeding impossible. This study reports upon the anatomy of a specimen of C. magnifica and demonstrates the presence of narrow ctenidial food grooves and the normal bivalve complement of feeding and digestive organs. Using a variety of general bacterial and DNA specific stains, no evidence of symbiotic intracellular bacteria has been found in the ctenidia (or any other tissues). It is concluded that C. magnifica is principally a filter feeder, albeit with modification for collecting and processing a diet of bacterial cells, with the possibility (as in all bivalves) of direct absorption. High chemoautotrophic activity levels in the ctenidia probably result from entrapment of vent water bacteria collected during filter feeding.     

Correlation between humification and the oxidase activity of the causative micro-organisms

Summary Decomposition of finely ground barley straw at 28°C was followed for 250 days in vessels which had been inoculated with mixed bacterial isolates of known oxidase activity derived from a sample of barley field soil. The artificial bacterial populations selected consisted of either 1) isolates which reacted positively in a number of oxidase tests, 2) isolates which gave negative responses in the same tests, or 3) these two groups in combination. For comparison, a suspension of the soil from which the bacterial isolates were derived was also used as an alternative inoculum. It was shown that the oxidase-negative bacteria were more effective than the oxidase-positive ones in the production of humic substance, and in decreasing the carbohydrate content of the straw.     

具有群体感应抑制活性的海洋细菌的筛选

【目的】从海洋环境中筛选出能有效抑制细菌群体感应的活性菌株,为以致病菌群体感应为靶点的新型疗法提供新的天然产物资源。【方法】以紫色杆菌(Chromobacteriumviolaceum)为报告菌,采用滤纸片法和双层软琼脂法相结合的筛选模型进行群体感应抑制活性菌的筛选。【结果】通过对美国圣璜岛海域海绵中分离出来的272株海洋细菌群体感应抑制活性的筛选,得到了具有抑制紫色杆菌素产生的细菌51株,其中74号菌株抑制效果最好,具有进一步研究的价值。【结论】海洋细菌中有很多具有抑制细菌群体感应效应的菌株,是天然群体感应抑制剂的潜在来源。     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.