Electron microscopic study of bacterial development in colonies. The morphology of bacterial colonies

The morphology of colonies of some pathogenic Gram-negative and Gram-positive bacteria has been studied by scanning and transmitted electron microscopy. The presence of covers on the surface of cells in colonies has been revealed. The examination of colony fragments in ultrathin section has revealed that cells exist in associations and the elements of cell covers are differentiated in the form of fibrillar structures in the intracellular space. This investigation has shown that covers in the colonies of the bacteria under study should be regarded as their morphological feature playing an important role in the development of the infectious process.     

Surface morphology of Mycobacterium bovis BCG: relation to mechanisms of cellular aggregation

Growing colonies of Mycobacterium bovis BCG, Tice and Glaxo substrains, and freshly ball milled and freeze-dried Tice BCG vaccines were examined by scanning and transmission electron microscopy (TEM) and by light microscopy after cytochemical staining. BCG organisms in colonies growing on agar were randomly oriented, despite colony morphology, and nearly completely covered by an amorphous material. Aggregates of organisms in vaccine suspensions were also covered with this material, but single cells were not covered. In TEM, the covering material was visualized between groups of cells as an electron-transparent area surrounded by a thin electron-dense layer. This material appeared to originate in the upper cell wall, between the cell wall skeleton and the outer dense layer. Staining of the covering material indicated the presence of protein, carbohydrate and acidic groups, but not exposed lipids. The covering material was absent from the ventral side of colonies, suggesting that its production is oxygen-dependent. These observations suggest that a mycobacterial exudate, previously observed and implicated as a virulence factor, may also bind the cells together, and accounts for the aggregative properties of the organisms in culture.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.     

Parasporal bodies of Bacillus laterosporus sporangia.

Intact colonies of Bacillus laterosporus examined by thin-section transmission electron microscopy revealed sporangia in various stages of development and degeneration as the endospores matured. The sporangia formed a surface layer of hexagonally arranged subunits. The variety of parasporal bodies raised questions of developmental and ecologic utility.     

Surface spreading motility shown by a group of phylogenetically related, rapidly growing pigmented mycobacteria suggests that motility is a common property of mycobacterial species but is restricted to smooth colonies

Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.     

Trafficking and release of mycobacterial lipids from infected macrophages

Analysis of infected macrophages revealed that lipid-containing moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria-containing phagosomes, surface-exposed carbohydrates were labeled with hydrazide-tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria-derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un-infected 'bystander' cells. Their transfer to bystander cells could expand the bacteria's sphere of influence beyond the immediate confines of the host cell.     

Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter

Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.     

Encapsulation of coagulase-negative staphylococci of bovine origin

Capsule expression was assessed in six coagulase-negative staphylococcal strains in serum-soft agar and by india ink and electron microscopy. Classification of strains as encapsulated by serum-soft agar and india ink methods differed. Staphylococcus chromogenes, Staph. hyicus , and Staph. simulans grew as diffuse colonies in serum-soft agar and unstained halos were detected in india ink preparations. Staphylococcus hominis and Staph. simulans grew as diffuse colonies in serum-soft agar but no unstained halo was seen in india ink preparations. Staphylococcus hyicus was the only strain that gave negative results with serum-soft agar and india ink assays. Conventional electron microscopy revealed the presence of capsular polysaccharides on the cell surface of Staph. chromogenes, Staph. hominis and Staph. hyicus. Conventional electron microscopic technique used to examine the surface of cells was detrimental to capsule structure. During dehydration the capsule collapsed and appeared as electron dense aggregates at the surface of cells. To confirm results of conventional electron microscopy and to visualize clearly the cell surface, encapsulated Staph. hyicus and unencapsulated Staph. simulans were observed after freeze-fracture and etching by scanning electron microscopy. The fibrous nature of capsular polysaccharides surrounding cells of Staph. hyicus were distinct and confirmed observation by conventional electron microscopy. A rapid transmission electron microscopic technique is described also for observation of capsule. Results of the rapid TEM method agreed with conventional TEM and SEM. The finding that coagulase-negative staphylococci isolated from bovine milk are capable of capsule production may be important when investigating pathogenicity of these micro-organisms.     

Encapsulation of coagulase-negative staphylococci of bovine origin.

Capsule expression was assessed in six coagulase-negative staphylococcal strains in serum-soft agar and by india ink and electron microscopy. Classification of strains as encapsulated by serum-soft agar and india ink methods differed. Staphylococcus chromogenes, Staph. hyicus, and Staph. simulans grew as diffuse colonies in serum-soft agar and unstained halos were detected in india ink preparations. Staphylococcus hominis and Staph. simulans grew as diffuse colonies in serum-soft agar but no unstained halo was seen in india ink preparations. Staphylococcus hyicus was the only strain that gave negative results with serum-soft agar and india ink assays. Conventional electron microscopy revealed the presence of capsular polysaccharides on the cell surface of Staph. chromogenes, Staph. hominis and Staph. hyicus. Conventional electron microscopic technique used to examine the surface of cells was detrimental to capsule structure. During dehydration the capsule collapsed and appeared as electron dense aggregates at the surface of cells. To confirm results of conventional electron microscopy and to visualize clearly the cell surface, encapsulated Staph. hyicus and unencapsulated Staph. simulans were observed after freeze-fracture and etching by scanning electron microscopy. The fibrous nature of capsular polysaccharides surrounding cells of Staph. hyicus were distinct and confirmed observation by conventional electron microscopy. A rapid transmission electron microscopic technique is described also for observation of capsule. Results of the rapid TEM method agreed with conventional TEM and SEM. The finding that coagulase-negative staphylococci isolated from bovine milk are capable of capsule production may be important when investigating pathogenicity of these micro-organisms.     

Suppressor T-cell clones derived from pluripotent stem cells (CFU-GEMM) of a patient with Hodgkin's lymphoma

Pluripotent stem cells (CFU-GEMM) give rise to multilineage hemopoietic colonies in culture. The cellular composition revealed that mixed colonies contain cells of different myeloid lineages and mononuclear cells with T-cell surface antigens. T-lymphocytes of primary colonies, replated secondary and tertiary colonies from a patient with Hodgkin's Lymphoma were identified by their reaction with the monoclonal antibody OKT 8. Evidence for a common progenitor of myeloid and lymphoid cells is provided by analysis of individual secondary and tertiary colonies using OKT 3, OKT 4, OKT 8, VIM-D 5, and Ig M + D antibodies for each individual colony. Primary mixed, replated secondary and tertiary colonies revealed OKT 8 positive cells. No reaction with OKT 3, OKT 4, VIM-D 5, or Ig M + D was observed.     

Regulation of human B-cell colony growth

PHA-induced B-cell enriched populations from venous blood of healthy adults developed into B-cell colonies. Analyses of individual colonies revealed that 80-85% of the cells in each colony were surface membrane immunoglobulin positive. Most colonies, 84%, contained surface IgM-bearing cells. Only a few, 16%, were found with surface IgG-bearing cells. Surface IgM- and surface IgG-bearing cells were not observed in the same colony. Thirty-nine percent of the colonies contained cells bearing surface IgD in addition to either surface IgM- or surface IgG-bearing cells. There was no evidence of cytoplasmic immunoglobulin in the colony cells. The development of B-cell colonies was T-cell dependent; it appears that at least two different T-cell subpopulations, one with low density (D = 1.05) and the other with high density (D = 1.08) are responsible for this helper effect. Monocytes were found to inhibit B-cell colony formation; the inhibition was mainly by endogenous prostaglandin E2 (PGE2) synthetized and released by monocytes. The addition of physiological concentrations of synthetic PGE2 to monocyte-depleted B-cell enriched populations inhibited B-cell colony growth, this paralleled the effect of endogenous PGE2 released by monocytes. Indomethacin (10-5 M) obviated the inhibitory effect of monocytes.     

An Azospirillum brasilense Tn5 mutant with modified stress response and impaired in flocculation

The analysis of an A. brasilense Tn5 mutant shows significant phenotypic differences compared to the wild type isogenic strain. The transposon was located disrupting an open reading frame of 840 bp (ORF280) which exhibits similarity to the universal stress protein (USP) family. The USP family encompasses proteins that are expressed as a response to cell growth arrest. The mutant revealed a pleiotrophic phenotype with respect to different stress conditions. The ORF mutation results in an increased sensitivity of cells to carbon starvation and heat-shock treatment. However, the mutant strain displays a higher tolerance to oxidative stress agents. In contrast to the isogenic parent strain, colonies of the mutant are weakly stained by Congo red added to solid media and are impaired in flocculation. Scanning electron micrographs revealed that the mutant lacks part of the surface material present as a thick layer of exopolysaccharides on the surface of the wild type cells. The pleiotrophic phenotype revealed for this mutant and the similarity of the C-terminal region of ORF280 to UspA from E. coli indicates that the A. brasilense ORF280 may be a Usp-like protein. This revised version was published online in June 2006 with corrections to the Cover Date.     

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.     

ELECTRON MICROSCOPE STUDIES OF THE MICROVILLI OF HELA CELLS

Microvilli of HeLa cells cultured in vitro were preserved for electron microscopic examination at different stages of routine cultivation procedures. By a double-embedding technique, vertical sectioning for electron microscopy was possible. It revealed that, although the microvilli were present on all sides of the cell in the dispersed stage and in the attached stage, they were not present on the bottom of the cell when it was stretched on the surface of the dish. When the cells were grown in dense colonies, they were found on top of each other, and microvilli were present on all sides, except on the bottom surface of those cells in contact with the dish. We achieved a more dramatic demonstration of the microvilli by developing a surface-replica technique which retains their spatial arrangement and permits characterization of the distribution of their number, length, and diameter.     

Biofilm growth and detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.     

Retrograde labeling and fine structure of olfactory receptor neurons in cat sharks

Ciliated and microvillar olfactory receptor cells have been reported in many fish species, including teleosts and elasmobranchs. Morphological studies have suggested that microvillar cells are the only olfactory receptor cells in the elasmobranchs; however, there is no direct evidence for this hypothesis. Here we used a cat shark (Scyliorhinus torazame) to determine the cell type of the olfactory receptor cells in elasmobranchs. Retrograde labeling with a fluorescent dye, Dil, labeled only cells in the second layer from the surface of the olfactory epithelium, suggesting that ciliated cells located in the surface layer are not olfactory receptor cells. In addition, electron microscopic observation revealed that the labeled cells in the second layer have a thin dendritic knob extending from the cell body to the free surface of the epithelium. A part of the dendritic knob facing the mucous layer did not have ciliary structures. These results provide evidence that the aciliate cells are the only olfactory receptor cells in the cat shark olfactory organ.