A scanning electron microscope study of mycobacterial developmental stages in commercial BCG vaccines

Scanning electron microscopy (SEM) studies were performed on freshly prepared and freeze-dried Tice-substrainMycobacterium bovis BCG vaccine as well as Tice BCG grown on Middlebrook 7H10 agar. Intact colonies of the Tice and Glaxo BCG substrains growing on agar were also examined. The presence of developmental stages of the mycobacterial life cycle previously reported in the literature was confirmed in actively growing BCG and in commercial vaccine preparations. The pleomorphic forms consisted of various size coccal and bacillary cells. Propagation appeared to occur by fission of both forms to produce aggregate bodies and by a coccal-bacillary cycle. Filterable (30–200 nm) granular cocci and coccal microcolonies were also observed in commercially prepared BCG vaccines. The implications of pleomorphism on the biologic activities of various BCG vaccines are discussed.     

The effect of washing on the surface charge of Mycobacterium bovis BCG vaccine, Tice substrain

The zeta potential of three lots of Tice substrain BCG organisms was measured over a pH range of 2.0 to 11.0 at low electrolyte concentration. For two lots, the cells were cationic at pH 4.2-4.4 and anionic above this isoelectric point. Washing the cells twice with water lowered the isoelectric point to 2.7. The cationic/anionic profile was retained in all three lots, although the third lot had an isoelectric point of 3.0 initially. Cells of the Glaxo substrain, on the other hand, were anionic over the entire pH range and are evidently unaffected by the washing process. It appears that cells of the Glaxo strain have only electronegative phosphate groups at their surface whereas the Tice substrain may possess a loosely adhering cell-surface protein.     

Fine structure of cell wall surfaces in the giant-cellular xanthophycean alga <Emphasis Type="Italic">Vaucheria terrestris</Emphasis>

The mechanical strength of cell walls in the tip-growing cells of Vaucheria terrestris is weakened by treatment with proteolytic enzymes. To clarify the morphological characteristics of the components maintaining cell wall strength, the fine structures of the cell walls, with and without protease treatment, were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations indicated that cellulose microfibrils were arranged in random directions and overlapped each other. Most of the microfibrils observed in the inner surface of the cell wall were embedded in amorphous materials, whereas in the outer surface of the cell wall, microfibrils were partially covered by amorphous materials. The matrix components embedding and covering microfibrils were almost completely removed by protease treatment, revealing layers of naked microfibrils deposited deeply in the cell wall. Topographic data taken from AFM observations provided some additional information that could not be obtained by TEM, including more detailed images of the granular surface textures of the matrix components and the detection of microfibrils in the interior of the cell wall. In addition, quantitative AFM data of local surface heights enabled us to draw three-dimensional renderings and to quantitatively estimate the extent of the exposure of microfibrils by the enzymatic treatment.     

Adhesion of Lactobacillus acidophilus to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer)

Lactobacillus acidophilus was isolated from washed and homogenized walls of the crop and caecum of an adult fowl. A strain that adhered well in the Fuller adhesion test was subcultured until colonies on Lactobacillus Selective agar changed from rough to smooth. This coincided with a change from aggregate to planktonic growth in liquid medium and a marked loss of ability to adhere. The ultrastructure of cells from both types of culture was studied by electron microscopy. An S-layer formed the outermost part of the cell wall in the strongly-adherent strain, whereas this layer was covered with polymerized material or was absent in strains that lacked the ability to adhere, or those with reduced adherence.     

Chemical and ultrastructural investigations of Mycobacterium bovis BCG: implications for the molecular structure of the mycobacterial cell envelope

Abstract The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactan-mycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the ‘electron-transparent zone’ (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice^® substrain, revealed an OL bilayer, each of which measured 2–4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized α-glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.     

Electron microscopic examination of capsular material from various serotypes of Actinobacillus pleuropneumoniae.

The capsular material on PPLO broth-grown cells of Actinobacillus pleuropneumoniae representing serotypes 1 to 10 was visualized by transmission electron microscopy after polycationic ferritin labeling and also after stabilization with specific antibodies. All the isolates examined were covered with a layer of capsular material whose thickness varied between 80 to 90 nm and 210 to 230 nm when examined by immunostabilization. We were also able to visualize A. pleuropneumoniae in lungs of infected pigs and to estimate the amount of capsular material covering the cells. Our results indicate that differences in capsular structure exist among the different A. pleuropneumoniae serotypes, and this result may explain in part why the serotypes are not equally virulent.     

Pattern formation and the fine structure of the developing cell wall in colonies of Pediastrum boryanum

A single-layered disc of peripheral pronged cells and central prongless cells impart the typical gear shape to colonies of Pediastrum, while the walls of each cell have a characteristic reticulate triangular pattern. The two-layered wall forms in the cells during colony formation following zoospore aggregation and adhesion. The uniformly thin outer layer reflects contours resulting from differential thickening in the reticulate pattern of the inner, thicker, more fibrillar and granular wall layer. The reticulate pattern thus imparted to the outer wall layer persists in empty zoosporangia following the release of zoospores. Columns of electron-dense material extend through the outer wall layer except at the ridges and centers of the reticulum. Following mitosis and cleavage, the resulting zoospores are extruded within a vesicle membrane consisting of the inner wall layer. Separation of this membrane from the parent cell occurs in material of the inner layer adjacent to the outer wall. Vesicles containing swarming zoospores also contain a granular material which appears to become associated with the aggregating and adhering cells of new colonies. Microtubules occur in zoospores prior to adherence but are absent during wall deposition.     

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.     

Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or α-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.     

Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits.

Cell walls of Clostridium thermohydrosulfuricum and C. thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits. The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits. In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer. During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process. In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer. These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.     

The morphology of colony variants of three species of Candida.

The colonies of 12 isolates of 3 Candida spp. with variant colony forms were studied by scanning and transmission electron microscopy. Small colonies were formed by 4 isolates each of C. albicans and C. parapsilosis and by 1 of C. tropicalis. These had an abnormally high proportion of degenerate yeast cells with an associated increase in granular cytoplasmic material intercellularly. The increased matrix in these small colonies formed a thick superficial coat over the organisms. Rough colonies were formed by 1 isolate each of C. albicans, C. tropicalis and C. parapsilosis. The convoluted regions of these colonies contained many pseudohyphal cells but few degenerate cells and little granular or fibrillar material in their intercellular matrices. The shape of colonies of Candida spp. may be altered by variations in the viability or the morphology of the organisms.     

Seed coat development, anatomy and scanning electron microscopy of Harpagophytum procumbens (Devil's Claw), Pedaliaceae

Seed coat development of Harpagophytum procumbens (Devil's Claw) and the possible role of the mature seed coat in seed dormancy were studied by light microscopy (LM), transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM). Very young ovules of H. procumbens have a single thick integument consisting of densely packed thin-walled parenchyma cells that are uniform in shape and size. During later developmental stages the parenchyma cells differentiate into 4 different zones. Zone 1 is the multi-layered inner epidermis of the single integument that eventually develops into a tough impenetrable covering that tightly encloses the embryo. The inner epidermis is delineated on the inside by a few layers of collapsed remnant endosperm cell wall layers and on the outside by remnant cell wall layers of zone 2, also called the middle layer. Together with the inner epidermis these remnant cell wall layers from collapsed cells may contribute towards seed coat impermeability. Zone 2 underneath the inner epidermis consists of large thin-walled parenchyma cells. Zone 3 is the sub-epidermal layers underneath the outer epidermis referred to as a hypodermis and zone 4 is the single outer seed coat epidermal layer. Both zones 3 and 4 develop unusual secondary wall thickenings. The primary cell walls of the outer epidermis and hypodermis disintegrated during the final stages of seed maturation, leaving only a scaffold of these secondary cell wall thickenings. In the mature seed coat the outer fibrillar seed coat consists of the outer epidermis and hypodermis and separates easily to reveal the dense, smooth inner epidermis of the seed coat. Outer epidermal and hypodermal wall thickenings develop over primary pit fields and arise from the deposition of secondary cell wall material in the form of alternative electron dense and electron lucent layers. ESEM studies showed that the outer epidermal and hypodermal seed coat layers are exceptionally hygroscopic. At 100% relative humidity within the ESEM chamber, drops of water readily condense on the seed surface and react in various ways with the seed coat components, resulting in the swelling and expansion of the wall thickenings. The flexible fibrous outer seed coat epidermis and hypodermis may enhance soil seed contact and retention of water, while the inner seed coat epidermis maintains structural and perhaps chemical seed dormancy due to the possible presence of inhibitors.     

Cell mass of Mycobacterium bovis BCG estimated by gas chromatography

The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.     

Active specific immunotherapy of residual micrometastasis

Summary A vaccine of Bacillus Calmette-Guérin (BCG) admixed with tumor cells induced systemic immunity and had a therapeutic effect on subclinical, disseminated micrometastasis. Inbred strain-2 guinea-pigs given IV injections of 5×10³ to 10⁶ syngeneic L10 hepatocarcinoma cells were vaccinated after metastatic foci were established in the lung parenchyma. The purpose of this study was to establish the variables that can be manipulated to assure optimal immunotherapy while minimizing deleterious side effects of the BCG. In the present study we examined the variables of source, dose, and ratio of BCG to tumor cells. Four BCG sources (lyophilized Tice and Connaught; fresh-frozen Phipps and Tice) were compared. No significant differences among these BCG preparations could be detected with respect to adjuvant potential when they were admixed with attenuated tumor cells in a vaccine. The dose study clearly demonstrated that a BCG dose dependency exists with relation to induction of effective cell-mediated immunity or survival from disseminated micrometastatic disease. Furthermore, evaluations of dose versus ratio of BCG to tumor cells also supported a BCG dose dependency, with the lowest effective BCG dose being directly influenced by tumor burden of the host. Cutaneous reactivity and hypersensitivity of the primary and secondary immunization sites of tumor-bearing animals treated with effective and ineffective vaccines supported the direct association of reaction to BCG and specific tumor immunity. However, when an in vitro leukocyte migration inhibition assay was used, the degree of reactivity to BCG could not be exploited as a quantitative, diagnostic monitor of effective systemic tumor immunity.     

Intercellular relationships in the external glial limiting membrane of the neocortex of the cat and rat.

The external glial limiting membrane of the cerebral cortex appears to be a complete astrocytic mantle covering the pial surface of the molecular layer. It consists of flattened cell bodies arranged singly or in small groups spaced about 100 mu apart and multitudes of interdigitating processes arrayed in layers. The glial mantle is thicker in the sulci than on the gyri. It is covered externally by a basal lamina which is associated with collagenous fibrils and cells of the pia mater. The extracellular space in aldehyde-perfused material appears as a regular, electron-lucent interval 150 A wide between adjacent cell membranes. Gap junctions are frequently encountered in the external glial limiting membrane; desmosomes are present between astrocytic processes but are seen much less often.     

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: A combined freeze-etch electron microscopy and biochemical study

The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimension as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced the ordered fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis bacillus Calmette–Guérin (BCG) substrains

During the serial passage of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. 'Early-shared strains' of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (α, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1β (IL-1β), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-α, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-γ (IFN-γ) than did 'late-shared strains' of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-α in the presence of IFN-γ was also observed by trehalose 6,6'-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that 'early-shared strains' are more potent immunostimulating agents than 'late-shared strains', which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.     

家兔囊胚在饲养层上的生长行为

研究了家兔囊胚在饲养层上的生长行为。结果表明,滋养层细胞铺展后,形成一层透明的膜状结构。覆盖在内细胞团之上。内细胞团在４８小时后,可见到一定程度的增殖,第３天增殖速度加快。生长的内细胞团可呈团状、条状、网状和混合型四种形态。混合型内细胞团分化出现较早,呈团状、条状和网状生长的内细胞团分化较晚。团状内细胞团一般在原位生长,而呈条状和网状生长的内细胞团增殖很快并迅速扩展到各处。滋养层细胞覆盖于未分化的     

CELL DIVISION AND THE ROLE OF THE PRIMARY WALL IN THE FILAMENTOUS DESMID ONYCHONEMA LAEVE (CHLOROPHYTA)1

Cell division and the role of the primary wall in filament formation in the desmid Onychonema laeve Nordst. were investigated by transmission and scanning electron microscopy. In addition, sequential chemical extractions and enzyme treatments were performed, on cell walls of intact filaments. Interphase cells are deeply constricted, consisting of two semicells, each elliptical in front view and circular in side view. In addition to two short lateral spines, each semicell has two apical processes that originate on opposite sides at an angle of about 15° from the central axis and overlap the adjacent cell. Division is initiated as in other desmids by a slight separation of the semicells and development of a girdle of primary wall material at the isthmus. In O. laeve the girdle of primary wall expands to form a spherical vesicle (termed a division vesicle) between the separating semicells. Nuclear division and septum formation occur in this vesicle when it is nearly the full diameter of the filament. Morphogenesis of the apical processes begins with completion of the septum, before the secondary wall appears. At maturity each apical process is surrounded by a thick layer of both secondary and primary wall, except that its capitate tip protrudes through the shroud of primary wall. Sequential treatment with hot ammonium oxalate, 4% NaOH, 17.5% NaOH and 10% chromic acid or various enzyme solutions did not cause filament breakage. SEM and TEM views of O. laeve after these treatments show intact secondary walls and intact primary wall material covering and connecting the apical processes of adjacent cells. It is the persistence of the primary wall between cells and around the apical processes that maintains the long, unbranched filamentous morphology of Onychonema laeve.